Inger Damon 1 , Peter Jahrling and James LeDuc

1 2 Inger Damon 1 , Peter Jahrling and James LeDuc

1 Centers for Disease Control and Prevention, Atlanta, GA, USA; 2 USAMRIID, Maryland, USA

INTRODUCTION help the virus to evade the host’s defence mechanisms; the use of poxviruses as vaccine vectors; the natural

This chapter deals only with those poxviruses that evolution of diseases such as myxomatosis; the cause human infection. The last naturally occurring

maintenance of diseases such as monkeypox, cowpox case of smallpox occurred in 1977; the last cases as a

and possibly buﬀalopox in wildlife populations; the result of a laboratory exposure occurred in 1978.

possible eﬀects of such viruses on their wildlife Concern for the potential malevolent use of variola has

reservoirs; evaluation of the importance of human led to increased eﬀorts in clinical education for the

monkeypox; and the development of improved vac- recognition of smallpox and other poxvirus infections

cines for animal poxvirus infections (Moss, 2001; in general. The remaining human poxvirus infections

Baxby, 1998). Although perhaps less important than are largely less clinically signiﬁcant, although occa-

foot and mouth disease and rinderpest, poxviruses of domestic animals, such as sheeppox, camelpox and

sional severe infection and even death may occur. With avianpox, can cause considerable problems for com- the exception of molluscum contagiosum, existing munities dependent on these animals (Baxby, 1988, human poxvirus infections are acquired from animals.

1998).

This, and the fact that some are restricted geographically, is of value when possible cases are being investigated (Table 15.1). However, increased global travel and commerce will require enhanced clinical

THE VIRUSES vigilance and knowledge of the appearance of poxvirus

infections in previously unanticipated geographical The poxviruses of vertebrates are separated into eight locations, e.g. the recent occurrence of monkeypox in

genera, and species within each genus are very closely the USA (MMWR, 2003).

related. Some poxviruses have yet to be assigned to Because of the concern for emerging and re-

genera, and the relationships of some viruses within emerging infectious diseases, there is still considerable

genera need further clariﬁcation (Moyer et al., 2000; interest in poxviruses, despite the eradication of

Baxby, 1998).

smallpox. Fundamental and applied work with these Genetic hybridisation can occur within a genus, and viruses generates additional knowledge of them and

the serological relationship between species is very opens up opportunities for their utilisation, including:

close. Traditionally, virus isolates have been identiﬁed studies on gene expression, including the expression of

on the results of biological tests. These methods are foreign genes and particularly gene products that may

still used for presumptive identiﬁcation, but increasing use is being made of nucleic acid signature detection and genome analysis. Fortunately, the validity of

*Contains signiﬁcant material from the 4th edition chapter on species established on biological criteria has in general Poxviruses by Derrick Baxby, University of Liverpool, UK.

been endorsed by genome analysis. The human

pathogens are reasonably well-characterised species and are distributed among four genera (Table 15.1) (Moyer et al., 2000).

Structure and Replication Morphology

In general, a poxvirus may be recognised as such by its large size and brick-shaped morphology. Most work has been done on vaccinia, which can be taken as representative of orthopoxviruses, molluscum and tanapox viruses. Virions are 200–2506250–300 nm, basically brick-shaped but somewhat pleomorphic; parapoxviruses are slightly smaller and somewhat narrower (c. 160 nm) (Figure 15.1a, b). Virions released naturally from cells (‘extracellular enveloped virus’; EEV) have an outer envelope which is soon lost on manipulation and not found on virions released artiﬁcially (‘intracellular mature virus’; IMV) (Figures 15.1a,b and 15.2b). A small portion of IMVs may be further processed to acquire a bilayer envelope of Golgi intermediate compartment membrane that contains speciﬁc viral proteins. The intracellular enveloped IMV (IEV) then moves along cellular microtubules to the cell surface, where actin polymerises behind IEV. IEVs exit the cell via distinctive microvilli by fusing the outermost lipoprotein layer with the plasma membrane, thereby releasing the IMV within the inner

lipoprotein layer. The released particle is the extracellular enveloped virion (EEV); EEVs that stay attached to the outer surface of the cell are termed cell-associated EEVs (CEVs). IMVs, EEVs and CEVs are mature infectious particles, each with distinct surface antigenic properties. The extra envelope has antigens not present in IMV, and EEV plays an important role in pathogenesis (Smith et al., 1997, 2003; Payne, 1980). The appearance of both EEV and IMV in the electron microscope is aﬀected by stain penetration. The majority of virions have short surface tubules c. 10 nm in diameter and are referred to as M (‘mulberry’) forms (Figure 15.1b); a minority, slightly larger and electron-dense, appear to have a thick (c. 20–25 nm) membrane or capsule (hence ‘C’ forms) (Figure 15.1c). The M form of parapoxvirus is covered by one long tubule which winds round the virion (Figure 15.1a). This gives the characteristic criss-cross eﬀect due to superimposition of the images of the top and bottom surfaces of the virion when seen in the electron microscope. All the forms described (EEV-M, EEV-C, IMV-M, IMV-C) are infectious.

Thin sections show a central dumbbell-shaped core or nucleoid, ﬂanked by two ‘lateral bodies’. Inside the cell, virions are often bounded by double or multiple membranes (Figure 15.2a). When released naturally, the outer membranes fuse with the cell or Golgi membranes and the virion is extruded from the cell, invested by the envelope described above (Figure 15.2b).

492

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

Table 15.1 Poxviruses pathogenic for humans Genus

Virus

Reservoir host Animals naturally

infected

Geographical distribution

Comment

Orthopoxvirus Smallpox

Humans

Formerly worldwide Last endemic case 1977.

Eradication conﬁrmed 1979 Monkeypox

Squirrels?

Squirrels African rodent

species Monkeys

West and Central Africa

Rare zoonosis. Overall mortality 10%. Limited case-to-case spread. Recent importation to non-endemic area (USA)

Cowpox

Rodents

Cats Cattle

Europe, West CIS (USSR)

Rare zoonosis. Contact with cattle unusual Vaccinia (buﬀalopox)

Buﬀalo? Rodents?

Buﬀalo

India Variant of vaccinia. Established in nature Parapoxvirus

Orf Pseudocowpox

Sheep Goats Cattle

Worldwide Common trivial zoonoses Occupational hazards

Molluscipoxvirus Molluscum

Humans

Worldwide Trivial infections. Often sexually transmitted Yatapoxvirus

Kenya, Zaire, (Democratic Republic of Congo)

Rare trivial zoonosis. Obtain travel history

POXVIRUSES

Figure 15.1 (a) Mulberry (M) form of non-enveloped parapox virion; (b) naturally-released (extracellular) M form of Vaccinia virus ; (c) capsule (C) form of Vaccinia virus (negative stain; bar=150 nm)

Chemical Structure

Antigenic Structure

The genome of poxviruses is one long piece of double- Poxviruses are antigenically complex. Extracts of stranded DNA which varies in size from 130 kb

infected tissue contain a number of precipitating (parapoxvirus) to 260 kb (fowlpox virus). The mole-

antigens, the precise number of which is unknown. cule has cross-linked inverted terminal segments and,

Such extracts will ﬁx complement and react in ELISA on denaturation, forms a closed circle of single-

and RIA tests, but it is not known how many antigens stranded DNA (Moss, 2001). The central portion is

take part in these reactions. Virus neutralisation is a highly conserved but diﬀerences in the terminal regions

complicated process: analysis with monoclonal anti- of diﬀerent species provide a means for separating

bodies has detected nine neutralising epitopes them (Esposito and Knight, 1985). The complete

distributed among the IMV of the diﬀerent species of sequences of some strains of vaccinia and smallpox

orthopoxviruses (Czerny et al., 1994). Extra antigens virus and considerable sequences of other poxviruses

are present on the outer envelope of EEV, although for have been determined, and the location and function

technical reasons progress here has been less rapid of the genes is usually related to the HindIII restriction

(Vanderplasschen et al., 1997; Baxby, 1998). Ortho- map of the Copenhagen strain of vaccinia virus (Figure

poxviruses produce a haemagglutinin antigen which

15.3) (Johnson et al., 1993). The DNA is not infectious reacts with erythrocytes from certain fowls. This per se . The virion contains a number of virus-coded

antigen is present on the envelope of EEV and can enzymes, in particular a DNA-dependent RNA poly-

also be detected in virus-free supernatants. merase, which transcribe the viral genome (Moss,

2001). The virions contain many polypeptides; over 100

Replication and Cultivation have been detected by two-dimensional polyacrylamide electrophoresis, and there is coding potential for

Only the briefest summary can be given here but an

c. 200. Some are glycolysated and some of these are authoritative and extensively referenced account has found in the envelope of EEV (Moss, 2001).

been published recently (Moss, 2001).

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

Virions are taken into the cell by pinocytosis and/or phagocytosis, and there is increasing evidence that IMV and EEV attach to diﬀerent receptors (Vander- plasschen and Smith, 1997). Constitutive cellular enzymes initiate virus uncoating, but virion-coded enzymes are essential for the ﬁnal stages.

Transcription and translation are under close control; poxvirus genes cannot be controlled by mammalian promoters and there are differences between early, intermediate and late poxvirus-specific promoters. Many gene products are subject to post-transcriptional modification, e.g. by glycosylation and proteolytic cleavage.

Replication takes place in cytoplasmic factories referred to as B-type inclusions, in which virions at various stages of assembly are seen. Cells infected with some poxviruses (e.g. cowpox, avian poxviruses) also contain electron-dense A-type inclusions, usually containing mature virions; A-type inclusions are easily seen by light microscopy (Figure 15.4).

Molluscum contagiosum virus (MCV) has never been cultivated in vitro, although some early antigens and a non-transmissible cytopathic eﬀect may be detected in cell culture (Birthistle and Carrington, 1997). DNA and antigens may persist in a primary human ﬁbroblast system for long periods of time (Bugert et al., 2001). The remaining human pathogens can usually be cultivated in easily obtained cell cultures (including Vero, other monkey kidney cell lines, A549 and others), and orthopoxviruses will produce pocks on the chorioallantoic membrane (CAM) of 12 day-old

Figure 15.2 Thin sections of Vaccinia virus. (a) Virion within cell, invested with double membrane; (b) extracellular

chick embryos. Methods for isolation and identiﬁca- virion showing outer envelope, partly detached. D, double

tion of individual virus species have been reviewed membrane; E, outer envelope; I, inner membrane; L, lateral

recently (Damon and Esposito, 2002; Ropp et al., body; N, nucleoid or core (bar=100 nm)

1995; Meyer et al., 1997, 1998).

Figure 15.3 Representation of the genome of Copenhagen vaccinia showing HindIII restriction fragments labelled A–P according to decreasing size, inverted terminal repeats (boxes) and two large non-essential regions (horizontal bars) are indicated. Redrawn

from Baxby (1998). Published by Edward Arnold (Publishers) Ltd

POXVIRUSES

495 Table 15.2 Examples of non-essential poxvirus gene

products that contribute to ‘virulence’ Gene a Product b Comment

C3L

C4b receptor

Binds C4b. Negative mutants attenuated

C11L

EGF

Promotes cell proliferation. Inactivation attenuates

(C22L)

TNF receptor

Binds TNF, contributes to virulence (disrupted in CopVac)

B5R

EEV antigen

Essential for EEV production

B8R

INF-g receptor Bind IFN-g. Blocks host defences

(B13/14R)

Serpin

Inhibits inﬂammation (non- functional in CopVac)

Figure 15.4 Section of cowpox-infected cat skin showing

Binds IL-1b. Inhibits febrile hypertrophy,

B16R

IL-1b

response conspicuous intracytoplasmic A-type inclusions (some

hyperplasia,

leukocyte

inﬁltration

and

Host range gene in cowpox. indicated). Reproduced from Baxby et al. (1994) published

(CHO)

Host range

Non-expression causes by Blackwell Science Ltd

apoptosis in CHO cells a With reference to the genome of Copenhagen vaccinia [CopVac].

Initial letter denotes Hind III fragment, number and ﬁnal letter

Poxvirus Gene Products and Pathogenesis

indicate gene number within fragment and direction of transcription. CHO, Chinese hamster ovary. Bracketed entries, gene non-functional or not present in CopVac (see column 3).

There has always been interest in the ‘virulence’ (i.e.

b C4b, complement component; EGF, epidermal growth factor; TNF,

safety) of vaccinia virus, particularly given its close

tumour necrosis factor; EEV, extracellular enveloped virus; INF,

relationship to variola. Although some empirical interferon; Serpin, serine protease inhibitor; IL, interleukin. studies showed that, for example, TK minus mutants

were attenuated for some hosts, it was apparent that factors are involved in EEV dissemination (Smith et al., virulence was a complex multifactorial system. Analy-

sis of the genome has identiﬁed genes whose expression is not necessary for replication, and strains which do or do not express some of these genes have been compared in vivo and in vitro. The nucleotide and

SMALLPOX amino acid sequences and the observed or predicted

properties of these gene products provide evidence for In 1980, the WHO General Assembly accepted the their demonstrated or predicted role in pathogenesis.

conclusion of an independent commission that small- Some examples are given in Table 15.2, and further

pox had been eradicated; destruction of the remaining details are provided elsewhere (Smith et al., 1997;

virus stocks, scheduled for 1999 and then for 2002, has Johnston and McFadden, 2003; Seet et al., 2003). Gene

been delayed in an eﬀort to permit research for products identiﬁed include some which help the virus

improved preparedness in the event that smallpox to evade the immune system by binding components

recurs as the result of the malevolent use of variola of, for example, complement (gene C3L), interferon

virus. The importance of smallpox should not be (B8R) or interleukins (B16R), or which inhibit inﬂam-

forgotten. It had a measurable impact on the develop- mation (B13/14R). Other products determine host

ment of civilisation, was the ﬁrst virus disease for range and control apoptosis (gene CHO) or promote

which vaccination became available, and was the ﬁrst cell proliferation (C11L). The febrile response to

and only disease to be eradicated. Authoritative infection is mediated by interleukin 1b and is inhibited

information on all aspects of smallpox is available in by a virus-coded gene product (B16R), which binds

the comprehensive account by Fenner et al. (1988). this cytokine. The dissemination of naturally-released

Naturally acquired variola virus infection causes a virions (EEV) is an important aspect of pathogenesis

systemic febrile rash illness. For ordinary smallpox, the and genes have been identiﬁed (e.g. B5R) which code

most common clinical presentation, after an asympto- for proteins necessary for the proper development of

matic incubation period of 10–14 days (range 7–17 EEV and expression of virulence. In addition, host cell

days), was fever, quickly rising to about 1038F, days), was fever, quickly rising to about 1038F,

Variola major smallpox was differentiated into four main clinical types: (a) ordinary smallpox (*90% of cases) produced viraemia, fever, prostration, and rash; mortality rates were generally proportionate with the extent of rash and, using the WHO classification, ranged from <10% for ordinary discrete to 50–75% for the rarer ordinary confluent presentation; (b) (vaccine)-modified smallpox (5% of cases) produced

a mild prodrome with few skin lesions in previously vaccinated people and a mortality rate well under 10%; (c) ﬂat smallpox (5% of cases) produced slowly developing focal lesions with generalised infection and

a *50% fatality rate; and (d) haemorrhagic smallpox (<1% of cases) induced bleeding into the skin and the mucous membranes and was invariably fatal within a week of onset. A discrete type of the ordinary form, with typical febrile prodrome and rash, resulted from alastrim variola minor infection (Fenner et al., 1988). The WHO established a classification system for smallpox case types based on disease presentation and rash burden. Haemorrhagic and flat smallpox are briefly described below. Ordinary smallpox was sub- grouped into three categories, based on the extent of rash on the face and the body. In ordinary confluent smallpox, no area of skin was visible between the vesiculo-pustular rash lesions on the trunk or the face. Patches of normal skin were visible between rash lesions on the trunk in ordinary-semi-confluent disease, as well as on the face in ordinary discrete disease. (Vaccine)-modified disease presented with sparse numbers of lesions.

Prior to its eradication, smallpox as a clinical entity was relatively easy to recognise, but other exanthema- tous illnesses were mistaken for this disease (Fenner et al. , 1988) e.g. the rash of severe chickenpox, caused by

varicella-zoster virus, was often misdiagnosed as that of smallpox. However, chickenpox produces a centri- petally distributed rash and rarely appears on the palms and soles. In addition, in the case of chickenpox, prodromal fever and systemic manifestations are mild, if manifest at all; the lesions are superﬁcial in nature; and lesions in diﬀerent developmental stages may present in the same area of the body. Other diseases confused with vesicular-stage smallpox included monkeypox, generalised vaccinia, disseminated herpes zoster, disseminated herpes simplex virus infection, drug reactions (eruptions), erythema multiforme, enteroviral infections, scabies, insect bites, impetigo and molluscum contagiosum. Diseases confused with haemorrhagic smallpox included acute leukaemia, meningococcaemia and idiopathic thrombocytopenic purpura. The Centers for Disease Control and Pre- vention (CDC), in collaboration with numerous professional organisations, has developed an algorithm for evaluating patients for smallpox. The algorithm and additional information are available at http:// www.bt.cdc.gov/agent/smallpox/diagnosis/evalposter.asp and at www.bt.cdc.gov/EmContact/index.asp.

MONKEYPOX

Monkeypox virus was so-named because it was first detected in captive Asiatic monkeys; however, the virus has only been found naturally in Africa and evidence points to squirrels (Funisciurus spp., Heliosciurus spp.) as important reservoir hosts. Recently, a Gambian rat (Cricetomys gambiensis), rope squirrel (Funisciuris spp.) and dormouse (Graphiuris spp.) from the African shipment originating in Ghana and implicated in the US monkeypox outbreak were found to be infected with monkeypox by viral isolation and nucleic acid detection (PCR) (MMWR, 2003). Initial surveys in Zaire (now Democratic Republic of Congo) detected monkeypox-specific antibodies in 85/347 (25%) squirrels sampled but from none of 233 terrestrial rodents. Monkeypox-specific antibody has been detected in very few monkeys, which, like humans are probably only occasional hosts (Khodakevich et al., 1988). Subsequent work (Hutin et al., 2001) in the Demo- cratic Republic of Congo found evidence of orthopoxvirus seroreactivity in some terrestrial rodents tested, including Gambian rats (Cricetomys emini) and elephant shrews (Petrodromus tetradactylus).

Particular attention was focused on monkeypox from 1970, when smallpox surveillance activities in Africa revealed cases of human monkeypox, clinically indistinguishable from smallpox, particularly in Zaire.

496

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

POXVIRUSES

497 exanthem with an incubation period of about 12 days.

During the incubation period virus is distributed initially to internal organs and then to the skin (Fenner et al. , 1988; Jezek and Fenner, 1988). The main diﬀerences are a greater degree of lymphadenopathy and a lower capacity for case-to-case spread. The major problems concern the source of infection and the mode of transmission.

Clinical Features In general the clinical features of disease as seen in

Central Africa are those of a classical or modiﬁed case of smallpox. The most obvious diﬀerence is the pronounced lymphadenopathy, which involves the submandibular, cervical and sublingual regions (Figure 15.5).

Most cases occur in unvaccinated children. In Zaire during 1981–1986, 291 cases (86%) occurred in children <10 years old and only 12 (4%) of these had vaccination scars. The illness lasts 2–4 weeks. Of 292 unvaccinated patients, 22 (7.5%) had a mild illness with <25 skin lesions and were not incapacitated; 55 (19%) had 25–99 lesions, were incapable of most physical activity and required nursing; and 218 (75%) had >100 lesions, were totally incapacitated and required intensive nursing. Complications occurred in about 40% of patients: the most common were bacterial skin infections (16%) and respiratory (12%) and gastrointestinal (5%) disorders. The overall mortality was approximately 10%; however, all the deaths occurred in unvaccinated children, in which group mortality is around 15% (Jezek and Fenner, 1988; Jezek

Figure 15.5 Human monkeypox on the 8th day of rash. et al. , 1988). Disease in the 2003 outbreak in the USA Note the submaxillary and inguinal lymphadenopathy.

Reproduced from Fenner et al. (1988) by permission of WHO appears to have been milder, and only two of the 37

laboratory cases had complications, one with keratitis and one with encephalopathy (MMWR, 2003).

Sporadic outbreaks continue to occur and cause concern (Heymann et al., 1998; Bremen, 2000), but most information is available about cases that

Diagnosis occurred prior to 1988. More recently, monkeypox infection of humans was identiﬁed in the USA, as a

Until 2003, human monkeypox had not been detected result of exposure to ill prairie dogs, probably infected

outside Africa. Clinical diagnosis may present a after exposure to infected West African small mam-

problem because fewer physicians now have experience mals imported as exotic pets (MMWR, 2003).

with smallpox, which human monkeypox closely resembles. Access to a virus diagnostic laboratory should permit detection of virus by electron microscopy and molecular methods, and this provides a

Pathogenesis sensitive presumptive diagnosis. In some circumstances it may be important to distinguish between monkey-

The pathogenesis of human monkeypox is essentially pox and tanapox (see below). In the past it was the same as that of smallpox, i.e. an acute febrile

essential to diﬀerentiate between monkeypox and

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

variola, which could be done by examination of pock occurred more frequently during 1996–1997 than appearance (haemorrhagic or not) and presence of

earlier (Heymann et al., 1998). Monkeypox continues pocks produced on the CAM at 398C, a temperature

to be sporadically reported (Meyer et al., 2002). which inhibits smallpox. Currently, an array of

Comparison of the genomes of smallpox virus and molecular diagnostic techniques permits the speciation

monkeypox virus strains isolated up to 1986 suggested of these viruses (Ibrahim, 2003; Meyer et al., 1997,

that they have evolved separately (Douglas and 1998; Ropp et al., 1995; Loparev et al., 2001).

Dumbell, 1992) and the results of complete genome At the same time, detailed analysis of monkeypox

analysis (Shchelkunov et al., 2002) confirm this DNA has detected minor differences among different

observation.

strains which may be of value in epidemiological Laboratory workers studying monkeypox virus investigations (Douglas et al., 1994). The close

should be vaccinated with vaccinia and handle the serological relationships among orthopoxviruses

virus in safety cabinets. BSL-2 containment, per the makes detection of monkeypox-speciﬁc antigens diﬃ-

BMBL deﬁnition, should at a minimum be used. BSL- cult but methods are available and being reﬁned which

3 laboratory practices provide additional biosafety. may be of value, particularly for epidemiological studies of monkeypox virus in its natural reservoirs (Khodacevich et al., 1988).

VACCINIA Of human cases in the 1981–1986 Zaire survey, 74%

were conﬁrmed by detection of virus by electron Vaccination microscopy and virus isolation, and in another 22%

retrospectively by serology. Smallpox vaccination, although an eﬃcient prophylactic against smallpox, was not without well-documented

Epidemiology and Control risks. These ranged from rare but severe complications, such as generalised vaccinia, which occurred in about

Management of individual cases is supportive, with 200/million primary vaccinees, to relatively mild but case-to-case spread reduced by isolation and, if

still troublesome satellite lesions or nondescript rashes, available, the use of smallpox vaccine for contacts.

which occurred in about 8% of vaccinees (Baxby, Potential use of newer therapeutics will be discussed at

1993; Fulginetti et al., 2003). Routine use of smallpox the end of this chapter.

vaccine is now discontinued; however, to enhance Human cases in Africa occur in villages in the

response preparedness to the potential malevolent rainforests where a variety of animals are captured for

reintroduction of smallpox, a number of public health food. Infection of children might be explained by their

personnel have recently been vaccinated. Policies for playing with carcasses. The results of the comprehen-

vaccine use in laboratory personnel working with sive surveys carried out in the 1980s indicated that

orthopoxviruses varies internationally. It is necessary those infected were principally unvaccinated children

for those working with monkeypox virus, but policies and that case-to-case spread was unusual. Control

about its use for those working with cowpox virus and measures are based in interposing a buﬀer zone of

vaccinia virus vary (Baxby, 1993). Vaccinia immune cleared land between the arboreal reservoir and

globulin (Cono et al., 2003) is recommended for use in cultivated land, the development of animal husbandry

certain instances of adverse events associated with as a source of meat, and on education in the handling

vaccination.

of wildlife, with emphasis on any trapping done by those previously vaccinated; continued vaccination

Buffalopox was not thought necessary (Khodakevich et al., 1988). Only occasional human cases were reported from

Attention has been drawn to ‘buﬀalopox’ in water Central Africa following cessation of routine surveil-

buﬀaloes and their handlers, particularly in India. lance activities post-smallpox eradication, but there

Some outbreaks have certainly been caused by typical was a resurgence in 1996–1997 which has not yet been

strains of vaccinia virus. However, studies on isolates fully explained. Increased political unrest would lead

obtained after the cessation of routine vaccination to population displacement and breakdown of routine

suggest that cases continue to be caused by a virus control measures, and the levels of vaccine-induced

diﬀerent from vaccinia (Dumbell and Richardson, immunity will decline with time. A potentially serious

1993). These isolates, unlike vaccinia, do not produce ﬁnding which requires clariﬁcation is the observation

pocks on the CAM above 398C. Their DNA is that case-to-case transmission appears to have

suﬃciently similar to that of vaccinia virus to regard

499 them as variants or subspecies of it (Dumbell and

POXVIRUSES

its epidemiology. Despite its name, cowpox virus is not Richardson, 1993; Moyer et al., 2000). However, at

enzootic in cattle. The virus is maintained in a variety present it is not known whether buﬀaloes are the

of European rodents, and the most commonly reservoir host or whether wildlife reservoirs are

reported victim is the domestic cat, from which source involved, as in the case of cowpox (see below).

human infections are acquired (Baxby and Bennett, 1997b).

Recombinant Poxvirus Vaccines ‘Foreign’ genes have been inserted into poxviruses,

Pathogenesis particularly vaccinia. The resultant recombinant

Most information is available about cowpox in the strains retain their infectivity and, in general, the domestic cat, an accidental host in which a relatively inserted genes are expressed properly. Thus, the use of mild generalised disease occurs following primary such recombinants as vaccines has been advocated. infection via a bite or scratch (Bennett et al., 1990). Most work has been done on vaccinia as a vector, and Human infection, similarly acquired, usually vaccinia recombinant vaccines have been used to remains localised and is characterised by a marked control wildlife rabies in Europe (Pastoret and inﬂammatory and erythematous response (Baxby et al., Brochier, 1996), and in the USA (Rupprecht, 2001).

Human infection with such vectored vaccines has been 1994). The bulk of the lesion is caused by hypertrophy rarely reported (Rupprecht, 2001). Such concerns have

and proliferation of the basal cell layer of the led to the development of severely attenuated vaccinia

epidermis, together with massive inﬂammatory inﬁl- vectors (Staib and Sutter, 2003; Smith et al., 1997).

tration. Infection usually spreads into follicles and Other poxviruses are being considered as vaccine

typical A-type inclusions are usually seen (Figure vectors, in particular, the use of avian poxviruses as

15.4). By analogy with smallpox vaccination, a vectors for mammalian vaccines. These are of interest

transient viraemia might be expected. because avian poxviruses induce a good immune

response to the foreign gene product without initiating Clinical Features productive infection in mammals (Limbach and

Paoletti, 1996). Most information is available from a detailed analysis of 54 human cases investigated during 1969–1993 (Baxby et al., 1994). Lesions are generally restricted to

COWPOX the hands and face and most patients (72%) have only one lesion. Multiple lesions may be caused by

Cowpox is a relatively unimportant zoonosis, of multiple primary inoculations, autoinoculation, and interest principally because of recent re-evaluation of

very occasionally by lymphatic or viraemic spread.

Figure 15.6 Human cowpox. (a) Primary and secondary lesions; the former at the early eschar stage, the latter at the early vesicular stage; (b) at the late vesicular–early pustular stage showing a haemorrhage lesion with marked oedema and erythema. (a) Reproduced from Baxby et al. (1994) published by Blackwell Science Ltd. (b) From Baxby D (1982) The natural history of cowpox. Bristol Medico-Chirurgical Journal , 97, 12; reproduced by permission of the Editor

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

Occasionally, a very severe infection and death may inclusions is diagnostic, as it would be if found in occur, usually in immunosuppressed individuals.

biopsy material. Not all strains of cowpox are identical The lesion passes through macular, papular, vesicular

and genome analysis may show diﬀerences of epide- and pustular stages before forming a hard black crust

miological value.

(Figure 15.6a). The lesion is usually very painful and erythema and oedema are common at the late vesicular and pustular stages (Figure 15.6b). There is usually

Epidemiology lymphadenitis, fever and general malaise, often

referred to as ‘ﬂu-like’. These features are usually As indicated above, cowpox is maintained in rodents; severe in children, and absence from school or work is

in the UK these are bank voles and woodmice (Crouch common; 16/54 patients (30%) were hospitalised.

et al. , 1995; Baxby and Bennett, 1997b). The domestic Most cases take 6–8 weeks to recover; in some cases

cat is the most common source of human infection and it may take 412 weeks. Scarring is usually permanent.

this probably explains the occurrence of cases in children; 26% of 54 cases were in children <12 years. Most feline and human cases occur between July

Diagnosis and October, with only occasional cases between Clinical January and June. Human cases occur in which no source is traced, but despite detailed enquiries only The main drawback to clinical diagnosis of cowpox is

three human cases in the UK since 1968 have been its rarity and the failure to appreciate important

traced to a bovine source, and no case of bovine epidemiological information (see below). Cowpox,

cowpox has been detected since 1976. which is restricted geographically (Table 15.1), should

Cowpox virus has a wide host range, and an

be considered in anyone, including young children, interesting ﬁnding has been the occurrence of cowpox presenting with a painful haemorrhagic lesion or black

in a variety of captive exotic species in European zoos. eschar, with or without erythema and oedema,

Victims have included cheetahs, lions, anteaters, accompanied by lymphadenopathy and a systemic

rhinoceros, elephants and okapi, and infection has ‘ﬂu-like’ illness. This applies particularly to patients

occasionally been transmitted to animal handlers seen in July–October and/or who have had contact

(Pilaski and Ro¨sen-Wolﬀ, 1988; Baxby and Bennett, with cats (see below). Attempts to establish contact

1997b).

with cattle are usually counterproductive. Diﬀerential diagnoses include parapoxvirus (see below), herpes and anthrax. Colour illustrations of all these lesions are

Control available (Baxby et al., 1994), which, together with a

properly taken history, should help clinical diagnosis. Much evidence, albeit circumstantial, suggests that cow- Unfortunately, the rarity of human cowpox means that

pox virus is of low infectivity for humans (Baxby and general practitioners or even consultants seldom have

Bennett, 1997b). Careful handling of infected cats an opportunity to investigate a second case.

prevents cat-to-human transmission and no case has occurred in a handler after diagnosis of the feline case. Person-to-person transmission has not been reported.

Laboratory Management is supportive, with antibiotics to control any bacterial superinfection. Serious infections could be

Electron microscopy of vesicle ﬂuid or extracts of treated with vaccinia immune-globulin, if available. crusts is particularly valuable because it will diﬀer-

Aciclovir has no activity against poxviruses. Corticoster- entiate between parapox, herpes and presumptive

oids are contraindicated: when used they have cowpox infections. Of 24 cases of cowpox where

exacerbated infection and delayed recovery. Therapeutics adequate material was available, electron microscopy

are discussed in more depth at the end of this chapter. was successful in 23. Molecular diagnostics (Meyer et

al. , 1997, 1998; Ropp et al., 1995; Loparev et al., 2001) may also be used to identify cowpox.

If necessary, virus may be isolated on the CAM, PARAPOXVIRUS where the production of intensely haemorrhagic pocks is diagnostic. Cytopathic eﬀect occurs in many cell

Parapoxvirus infections are widespread in sheep, goats lines (Vero, MRC-5, RK13) and detection of A-type

and cattle. Human infections from these sources are a

501 common occupational hazard for those in contact with

POXVIRUSES

infected animals. Parapoxvirus infection in sheep and goats is usually referred to as ‘contagious pustular dermatitis’ or ‘orf’, and the corresponding human infection as ‘orf’. Parapoxvirus infection of cattle is usually referred to as ‘paravaccinia’, ‘pseudocowpox’ or ‘ring sores’, and the human equivalent as ‘paravaccinia’, ‘pseudocowpox’ or ‘milker’s nodes’.

Pathogenesis Infection occurs via cuts and scratches and usually

remains localised. Lesions are produced by hypertrophy and proliferation of epidermal cells, often marked, and leukocyte inﬁltration. Histological examination shows many small multilocular vesicles within the dermis; true macrovesicles rarely occur (Johanneson et al. , 1975; Yirrell and Vestey, 1994). Lymphadeno- pathy, malaise and generalised lesions are relatively uncommon and the immune response is poor (Leavell et al. , 1968; Yirrell et al., 1994).

Clinical Features The progressive stages of human infection have been

described in detail (Leavell et al., 1968; Johanneson et al. , 1975; Yirrell and Vestey, 1994) and illustrations provided (Baxby et al., 1994; Diven, 2001). Lesions start as erythematous papules and progress to a ‘target’ stage (Figure 15.7a). This, seen 1–2 weeks after infection, has a red centre surrounded by a white halo and an outer inﬂamed halo. This progresses to a nodular, then papillomatous stage, which often has a ‘weeping’ surface. In some patients this may enlarge and persist for some weeks before resolving (Figure 15.7b), and may cause some concern (see Diagnosis, below). The lesion resolves via a crusting stage (Figure 15.7c), which may last some weeks (Johanneson et al.,

Figure 15.7 Parapoxvirus infection showing lesions in 1975; Yirrell and Vestey, 1994). Occasionally very diﬀerent patients. (a) At the ‘larger’ stage; (b) an ulcerated

granulomatous lesion which could be misdiagnosed as a large granulomatous lesions occur which may need

malignancy; (c) crusting lesion. (a) From Baxby D (1982) The surgical removal (Pether et al., 1986).

natural history of cowpox. Bristol Medico-Chirurgical Most patients have only one lesion, but multiple

Journal , 97, 12; reproduced by permission of the Editor. (b) primary lesions may occur. Systemic reaction is

Kindly supplied by Dr M S Lewis-Jones. (c) Reproduced from relatively uncommon and the lesion is often not

Baxby D et al. (1994) published by Blackwell Science Ltd particularly painful. Attention has been drawn to

erythema multiforme as a common complication of orf but short-lived cell-mediated response and a relatively but, because most ordinary cases go unreported, the

poor and short-lived humoral response. This is actual incidence of erythema is probably low. The

consistent with the occurrence of second attacks in immune response in natural human infection has been

8–12% of individuals (Robinson and Peterson, 1983; investigated (Yirrel et al., 1994). There is a vigorous

Yirrell and Vestey, 1994).

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

Diagnosis infection in the farming and meat industries occasionally causes concern, and has led to industrial disputes

Clinical (Johanneson et al., 1975; Robinson and Petersen, 1983). Individuals should take care not to spread

The viruses which cause orf and paravaccinia are infection by autoinoculation or to contacts, including closely related (Moyer et al., 2000; Mercer et al., 1997)

animals. The vaccine used to control orf in sheep is and in the UK human cases are reported as ‘orf/

fully virulent and has caused human infection. paravaccinia’, whatever the animal source (Baxby and

Bennett, 1997a). Clinical diagnosis of uncomplicated cases in patients with a known animal contact should

MOLLUSCUM CONTAGIOSUM not cause diﬃculties. However, farm workers, etc.

recognise the infection and tend not to seek medical Although lesions resembling molluscum and contain- attention for routine cases. Consequently, a dispro-

ing poxvirions have been detected in, for example, portionately large number of reported cases have no

horses, human molluscum contagiosum is regarded as known contact with infected animals. Of approxi-

a speciﬁcally human infection and there is no evidence mately 500 cases surveyed during 1978–1995, some

of transmission between humans and other animals. 45% had no such contact. Clinical diagnosis of such

Molluscum contagiosum is a benign skin tumour cases, particularly if severe or prolonged, may cause

which occurs worldwide. It has been the subject of a diﬃculties. In particular, large weeping granulomatous

comprehensive recent review (Birthistle and Carrington, or papillomatous lesions may be misdiagnosed as

malignancies, resulting in one case in unnecessary amputation (Johanneson et al., 1975).

Pathogenesis Laboratory

After a variable, sometimes lengthy, incubation period, papules develop, formed by epidermal hypertrophy.

Virions with the characteristic morphology of para- This produces a nodule and also extends the dermis poxviruses are usually easily seen by EM in lesion

downwards, but the basement membrane usually extracts, and this provides a rapid, certain diagnosis. The

remains intact. Characteristic inclusions (Henderson– virus can be grown in cell culture but this is not

Paterson bodies) are formed in the prickle cell layer attempted routinely. Some nucleic acid detection tech-

and gradually enlarge as the cells age and migrate to niques are published (Torfason and Gunadottir, 2002).

the surface. These cells are replaced by hyperplasia of the basal cell layer. The inclusion is a well-deﬁned sac packed with virions (Shelly and Burmeister, 1986). The

Epidemiology lesion is circumscribed by a connective tissue capsule and the dermis, apart from distortion, remains

Human infection is an occupational hazard of farm- essentially normal. Occasionally an inﬂammatory workers, abattoir workers, veterinary surgeons and

inﬁltration of the dermis may occur (Brown et al., students and others with frequent exposure to sheep,

cattle or goats. It is most common in the lambing and calving seasons, and more commonly reported in sheep workers than cattle workers; this probably reﬂects

Clinical Features diﬀerences in animal husbandry. Of 191 cases with a known source surveyed during 1978–1995, 84% had an

Infection is via trauma to the skin. The characteristic ovine source and 16% were from cattle. During the

lesion begins as a small papule and, when mature, is a same period 32 cases occurred in abattoir workers

discrete, waxy, smooth, dome-shaped pearly or ﬂesh- (Baxby and Bennett, 1997a).

coloured nodule, often umbilicated (Figure 15.8). There are usually 1–20 lesions but occasionally there may be hundreds. They may become conﬂuent along

Control the line of a scratch and satellite lesions are occasionally seen.

Most workers at risk get infected at some stage and In children, lesions occur mainly on the trunk and reinfection is not uncommon. The impact of human

proximal extremities. In adults they tend to occur on

POXVIRUSES

503 of transmission are associated with mild skin trauma,

such as contact sports and shared towels; however, there is increasing evidence that the disease is sexually transmitted and that genital lesions are more common (Birthistle and Carrington, 1997).

Control Infection is benign and recovery usually spontaneous,

but treatment may be sought for cosmetic reasons, particularly for facial or multiple lesions. Various treatments have been tried (Birthistle and Carrington, 1997). Chemical treatments include phenolics, silver

Figure 15.8 Molluscum contagiosum

nitrate, trichloroacetic and glacial acetic acid. Physical umbilication. From a transparency kindly supplied by Dr J

lesions showing

methods include curettage and cryotherapy. Mild Verbov

trauma may induce a cure, which may be due to release of virus-infected cells accessible to the immune system.

Prevention in developed countries is based on the trunk, pubic area and thighs, but in all cases

attention to personal hygiene, and in developing infection may be transmitted to other parts by countries to this and to general improvements in autoinoculation (Brown et al., 1981). Individual lesions

living conditions. Although relatively unimportant per last for about 2 months but the disease usually lasts 6–9

se , the possibility that molluscum may act as a months (Steﬀen and Markman, 1989). More severe

marker for more serious conditions has been raised and prolonged infection tends to occur in individuals

(Oriel, 1987).

with impaired cell-mediated immunity, including human immunodeﬁciency virus (HIV) infection (Birthistle and Carrington, 1997).

TANAPOX Human infection with tanapox virus was ﬁrst recog-

Diagnosis nised in the Lake Tana area of Kenya in 1957, and particular attention was paid to it during post-

The appearance of lesions in normal cases is generally eradication smallpox surveillance. An account of 264 suﬃciently characteristic to permit clinical diagnosis.

laboratory-conﬁrmed cases from Zaire (Democratic Virions can usually be seen in large numbers if

Republic of Congo), with colour illustrations, is material expressed from the lesion is examined by

available (Jezek et al., 1985), as is information on the electron microscopy. The lack of a marked inﬂam-

virus itself (Knight et al., 1989). Recent anecdotal matory response and failure to isolate an agent in cell

reports of human disease outside Africa have been culture or CAM should eliminate other poxvirus

published and illustrate the need to consider poxvirus infections.

aetiologies of illness in travellers returning from and emigrants from endemic areas (Croitoru et al., 2002; Stich et al., 2002).

Epidemiology Restriction endonuclease analysis of molluscum virus

Pathogenesis and Clinical Features DNA has detected three main subtypes. Their incidence varies from 80–90% for MCV-I to ca. 1% for

Infection is via the skin. The lesion is characterised by MCV-III but all subtypes cause similar lesions and

pronounced epidermal hyperplasia with little involve- infect the same anatomical sites (Scholtz et al., 1989;

ment of the dermis. There is a short prodromal illness Porter et al., 1992).

with fever and malaise. The lesion starts as a macule The virus occurs worldwide and tends to be more

and progresses to a raised nodule, which becomes common in socially deprived areas. Traditional modes

umbilicated (Figure 15.9). The lesions are relatively

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

large (*10 mm) and usually break down to form ulcers (Figure 15.9b). There is usually erythema and oedema and lymphadenopathy is common. The lesions generally disappear within 6 weeks. Most (78%) patients have only one lesion, and very few have more than two. They may occur on any exposed area but the head tends to be spared.

Diagnosis For diagnosis of tanapox the limited geographical

distribution should be considered, as well as travel history. The solid nodular/ulcerated lesions are larger and develop more slowly than those of monkeypox, but are smaller and develop more rapidly than those of tropical ulcers.

Virus can be detected by electron microscopy but this would not exclude morphologically similar viruses; nucleic acid tests (Stich et al., 2002) or diagnostic serological tests on lesion extract would do this. Tanapox virus grows in a number of cell lines (e.g. owl monkey kidney, Vero, MRC-5, BSC-1) but not on CAM.

Epidemiology and Control The virus probably has a simian reservoir and is

restricted to Africa, principally Kenya and Zaire (Democratic Republic of Congo). Human-to-human transmission does not occur naturally, and it is thought that transmission from monkeys occurs mainly due to overcrowding during ﬂooding, unrest, etc. With the exception of vaccination, measures for the prevention of monkeypox would be applicable to tanapox; however, the mild and sporadic nature of the

Figure 15.9 Human tanapox lesions. (a) After 10 days, showing oedema and umbilication; (b) after 26 days, showing

infection probably means that speciﬁc measures are ulcer formation. Reproduced from Jezek et al. (1985) by unnecessary.

permission of WHO

DIAGNOSIS Summary of Diagnostic Approaches When considering a diagnosis of poxvirus infection,

Electron microscopy is important in rapid diagnosis the restricted geographical distribution and potential

and will conﬁrm parapox infection or exclude pox animal source are important features to consider. In

infection altogether if herpes virus is seen. Further many cases the diﬀerential diagnosis is between a

information, if required, can be obtained by virus particular poxvirus infection and other infections, e.g.

isolation in cell culture and/or CAM, often making use herpes, tropical ulcers, anthrax, bacterial abscess,

of eﬃciency of growth at elevated temperatures. Virus- rather than between two diﬀerent poxviruses.

speciﬁc antigens may be detected by a variety of

505 techniques, and nucleic acid detection tests for virus-

POXVIRUSES

Baxby D (1993) Indications for smallpox vaccination: policies speciﬁc gene products by polymerase chain reaction

still diﬀer. Vaccine, 11, 395–399. (PCR) are now widely used (Ibrahim et al., 2003;

Baxby D (1998) Poxviruses. In Topley and Wilson’s Meyer et al., 1997, 1998; Ropp et al., 1995). , vol 1 (eds Collier Microbiology and Microbial Infections LH, Balows A and Sussman M), pp 367–383. Edward Poxviruses will remain infective at ambient tempera-

Arnold, London.

tures, particularly if kept dry. If specimens cannot be Baxby D and Bennett M (1997a) Poxvirus zoonoses. J Med tested ‘on the spot’, infectivity is retained during

Microbiol , 46, 17–20.

transport by ﬁrst class mail without the need for Baxby D and Bennett M (1997b) Cowpox: a re-evaluation of special transport medium. Vesicle ﬂuid should be

the risks of human infection based on new epidemiological information. Arch Virol, 13(suppl), 1–12.

smeared on a slide and air-dried. On receipt the Baxby D, Bennett M and Getty B (1994) Human cowpox material can be reconstituted in buﬀer. Scrapings from

1969–93; a review based on 54 cases. Br J Dermatol, 131, molluscum and parapox lesions can be treated

similarly. The infectivity of virus in dried crusts is Bennett M, Gaskell CJ, Baxby D et al. (1990) Feline cowpox retained for long periods. Virus may be extracted from

infection. J Small Animal Pract, 31, 167–173. such material by freeze–thawing and ultrasonic treat-

Birthistle K and Carrington D (1997) Molluscum contagio- ment. However, if the diﬀerential diagnosis includes sum virus. J Infect, 34, 21–28. Bray M, Martinez M, Smee DF et al. (2000) Cidofovir

pathogens less resistant than poxviruses, greater care protects mice against lethal aerosol or intranasal cowpox should be taken and appropriate transport medium,

virus challenge. J Infect Dis, 181, 10–19. etc. used.

Bremen JG (2000) Monkeypox: an emerging infection for humans? In Emerging Infections, vol 4 (eds Scheld WM, Craig WA and Hughes JM), pp 45–67. ASM Press, Washington, DC.

Therapeutics

Brown ST, Nalley JF and Kraus SJ (1981) Molluscum contagiosum. Sex Transm Dis, 8, 227–234.

An active area of research involves the development of Bugert JJ, Melquiot N and Kehm R (2001) Molluscum and evaluation of therapeutics for orthopoxvirus contagiosum virus expresses late genes in primary human ﬁbroblasts but does not produce infectious progeny. Virus infections. A comprehensive review of the history

Genes , 22, 27–33.

and recent developments in the development of Cono J, Casey C and Ball D (2003) Smallpox vaccination and poxvirus antivirals has been recently published

adverse reactions. Morbid Mortal Wkly Rep, 52, RR04. (Neyts and Clercq, 2003). In addition, work testing

Croitoru AG, Birge MB, Rudikoﬀ D et al. (2002) Tanapox such compounds on experimental models of variola-

virus infection. Skinmed, 1, 156. infected non-human primates is ongoing (LeDuc et al., Crouch AC, Baxby D, McCracken CM et al. (1995) Serological evidence for the reservoir hosts of cowpox

2002), in addition to other animal models of ortho- virus in British wildlife. Epidemiol Infect, 115, 185–191. poxvirus infections (Bray et al., 2000). Cidofovir (and

Czerny C-P, Johann S, Ho¨lzle L et al. (1994) Epitope potentially its derivatives), an antiviral in use for

detection in the envelope of intracellular mature orthopox treatment of cytomegalovirus infections in immuno-

viruses and identiﬁcation of encoding genes. Virology, 200, suppressed patients, appears to show promise as an 764–777. Damon IK and Esposito JJ (2002) Poxvirus infections in

antiviral for treatment of some poxvirus infections humans. In Manual of Clinical Microbiology, 8th edn (eds (Kern et al., 2002). The use of vaccinia-immune

Murray PR, Tenover F, Baron EJ et al.), pp 1583–1591. globulin (VIG) is best described for treatment of certain

American Society for Microbiology, Washington, DC. complications of vaccinia (smallpox) vaccine adminis-

Diven DG (2001) An overview of poxviruses. J Am Acad tration (Cono et al., 2003), but may have utility in the

Dermatol , 44, 1–16.

treatment of certain other orthopoxvirus infections. Douglas NJ and Dumbell KR (1992) Independent evolution of monkeypox and variola viruses. J Virol, 66, 7565–7567. No clear beneﬁt has been shown for this product alone

Douglas NJ, Richardson M and Dumbell KR (1994) in the treatment of smallpox (Fenner et al., 1988).

Evidence for recent genetic variation in monkeypox viruses. J Gen Virol , 75, 1303–1309.

Dumbell KR and Richardson M (1993) Virological investigations of specimens from buffaloes affected by buffalopox in

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16 Alphaviruses

Graham Lloyd

Health Protection Agency, Porton Down, Salisbury, UK

INTRODUCTION Forest (with the exception of Mayaro and Una), Barmah Forest, Middelburg and Ndumu lineages

In 1967 the World Health Organization (WHO, 1967) occur in the Old World. Natural vertebrate hosts deﬁned arboviruses as:

include birds and rodents. Infection in the vertebrate is usually inapparent but can, under certain circumstances, cause disease and death. Transmission may

. . . viruses which are maintained in nature also occur to domestic animals and humans. Here principally, or to an important extent, through again the spectrum of disease varies from a clinically biological transmission between susceptible ver- inapparent infection to a severe disease and even tebrate hosts by haematophagous arthropods:

death.

they multiply and produce viraemia in the vertebrates, multiply in the tissues of arthropods, and are passed on to new vertebrates by the bites of arthropods after a period of extrinsic

MORPHOLOGY incubation.

The alphaviruses are essentially spherical, 60–70 nm in diameter (Figure 16.2) and sensitive to ether and

The deﬁnition was therefore based on biological detergent. The viron consists of three components: an criteria. The arboviruses were initially divided into

outer glycoprotein shell, a lipid bilayer and an RNA- group A (later known as alphaviruses) and group B

containing core or nucleocapsid (Peters and Dalrym- (later known as ﬂaviviruses), but this was followed by

ple, 1990). The lipid bilayer is derived from the host- the recognition of further antigenically related groups

cell plasma membrane. The viral-encoded glycopro- of arboviruses. Alphaviruses and ﬂaviviruses were later

teins, designated E1 and E2, form the outer surface of included as two separate genera in the family Togavir-

the virus and interact with cellular receptors and host- idae (Weaver et al., 2000). More recently, however,

derived antibodies. The glycoproteins are arranged in ﬂaviviruses have been classiﬁed as a separate family,

an icosahedral surface lattice (Harrison, 1986). Com- the Flaviviridae.

plete sequence information available suggests that the All medically important vector-borne togaviruses

viral genome is 11–12 kb in size and encodes four non- are members of the genus Alphavirus. Alphaviruses are

structural proteins (NSP1, NSP2, NSP3 and NSP4) at transmitted to their vertebrate hosts by arthropods

the 5’ end and the ﬁve structural proteins (C, E3, E2, (Figure 16.1) and have deﬁned geographic distribu-

6K and E1) at the 3’ end. The nsPs are translated from tions. The Eastern equine encephalitis (EEE) and

genomic RNA, and the structural proteins from Venezuelan equine encephalitis (VEE) lineages are

subgenomic RNA. Electron cryomicroscopy and closely related and restricted to the New World, while

image reconstruction of Sindbis, Semliki Forest, Ross the Sindbis-like (with the exception of Aura), Semliki

River and Aura viruses show that the envelope

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

Figure 16.1 Schematic transmission and maintenance cycles

glycoproteins form a T=4 icosahedral lattice (Cheng et al ., 1995; Mancini et al., 2000). The capsid proteins form a T=4 icosahedral nucleocapsid arranged in distinct pentameric and hexameric capsomeres on the exterior of the nucleocapsid.

BIOCHEMICAL AND BIOPHYSICAL PROPERTIES The nucleic acid of the alphaviruses consists of a

single-stranded (ss) positive-sense RNA, almost 12 000 nucleotides in length, which is capped at the 5’ end and polyadenylated at the 3’ end (Figure 16.3). The naked genome is infectious (Strauss and Strauss, 1986). The sedimentation coeﬃcient has been reported to be 42–49. The genome is divided into two regions; the 5’ two-thirds of the viral genome codes for the non- structural proteins and the 3’ third encodes the structural proteins (Strauss et al., 1984). Both the structural and non-structural proteins are translated as polyprotein precursors. The non-structural polyprotein precursor is cleaved into four non-structural proteins, NSP1, NSP2, NSP3 and NSP4, which function as the replicase–transcriptase of the virus (Strauss and Strauss, 1986). The structural polyprotein

Figure 16.2 Electron micrograph of a preparation of precursor is cleaved to form three major polypeptides. Sindbis virus (3% potassium phosphotungstate, pH 7.0; ﬁnal magniﬁcation 6200 000). Micrograph courtesy of Professor The capsid protein C (30–40 kDa) and the envelope

C. R. Madeley, The Royal Victoria Inﬁrmary, Newcastle glycoproteins E1 and E2 (45–59 kDa) are found in

upon Tyne, UK

ALPHAVIRUSES

Figure 16.3 Organisation of the alpha virus genome. The 5’ two-thirds of the viral genome encode the non-structural proteins, NSP1, NSP2, NSP3 and NSP4. The 3’ third encodes the structural proteins. The capsid and envelope glycoproteins (E1 and E2) are present in mature alphavirus particles. A third glycoprotein, E3, has been demonstrated in puriﬁed SFV. The 6 K polypeptide is not known to be a structural protein

mature alphavirus particles. In addition, a small neutralisation tests in cell culture (Calisher et al., glycoprotein, termed E3, has been demonstrated in

1980). The E1 glycoprotein of Sindbis virus contains Semliki Forest virus (Garoﬀ et al., 1974). A further

the antigen responsible for haemagglutination (Chanas small 6 K polypeptide is also encoded, but has not been

et al ., 1982). It is assumed that this is also the case for demonstrated in any alphavirus particles.

other members of the genus Alphavirus. The E2 glycoprotein induces virus-speciﬁc neutralising antibody. On the basis of this, alphaviruses have been

REPLICATION divided into six antigenic complexes: Western equine encephalitis (WEE), Venezuelan equine encephalitis

Viral attachment to a host cell occurs via the (VEE), Eastern equine encephalitis (EEE), Semliki glycoprotein spikes on the viral surface. The envelope

Forest (SF), Middelburg and Ndumu viruses (Peters glycoproteins have receptors that are essential to

and Dalrymple, 1990). A further virus, Barmah Forest initiate infection. Alphaviruses are able to infect both

virus, has been shown to be biochemically typical of vertebrate and arthropod hosts and enter the host cell

the genus, but is not related serologically to members by endocytosis or direct fusion with the plasma

of the six antigenic complexes (Table 16.1). Each membrane. After endocytosis the capsid is thought to

complex consists of either a single virus species having

be released by low-pH catalysed membrane fusion, no known close relatives, e.g. EEE, VEE, Middelburg initiated by the E1 glycoprotein (Wang et al., 1992).

and Ndumu viruses, or several species, subtypes and Once released, the positive-sense ssRNA probably

varieties that are more closely related to each other serves as an mRNA. A virus-encoded transcriptase

than the other members of the genus, e.g. Semliki transcribes the positive-sense RNA into minus-

Forest virus and WEE virus. Often only plaque stranded RNA, which then forms a template for the

reduction or kinetic HI tests can distinguish these synthesis of the positive-stranded progeny RNA.

variants. Kinetic HI tests have also been used to Nucleocapsids are assembled in the cytoplasm and

diﬀerentiate between geographical variants of EEE maturation occurs by budding through host cell

virus (Casals, 1964). Studies utilising monoclonal membranes.

antibodies have been able to deﬁne the alphavirus antigenic cross-reactivities more precisely and map the antigenic determinants.

A recent study sequenced one strain of each alphavirus species and this allowed for the classiﬁca- The alphaviruses have a group-reactive nucleoprotein.

ANTIGENIC AND GENOTYPIC PROPERTIES

tion of alphaviruses into six genotypes (as compared Complex and type-speciﬁc reactivity is determined by

with seven antigenic complexes): Sindbis, Ndumu, the envelope glycoproteins. This antigenic comparison

VEE, WEE, Barmah Forest and Semliki Forest of alphaviruses has been conducted using haemagglu-

viruses. The authors included EEE and WEE in the tination-inhibition (HI) and complement ﬁxation tests

same genotypic complex; this homology is to be (CF), as well as cross-protection tests in mice and

expected, as WEE is thought to be a recombinant

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

Table 16.1 Antigenic classiﬁcation of alphaviruses, global distribution, primary vertebrate hosts, primary and transmission vectors

Primary vector Barmah Forest

Antigenic complex

Virus

Geographical distribution

Vertebrate host

Cx. annulirostris Eastern equine encephalitis

Barmah Forest Australia

Birds

Enzootic—Culex sp. (EEE)

EEE

North and South America

Birds

Bridge vector—Aedes sp. Middelburg

Middelburg

South, West and Central

America

Semliki Forest

Africa and Asia

Ae. abnormalis Ae. africanus

Chikungunya

Africa and Asia

Primates

Ae. africanus Ae. aegypti Cx. tritaeniorhynchus

Getah

Asia and Australasia

Mammals

Ae. aegypti

Mayaro

South America

Western equine encephalitis

Birds, mammals Cx. tarsalis (WEE)

South America

Ae. serratus

Fort Morgan

North America

Birds

Cs. melanura Venezuelan equine encephalitis VEE

Highlands J

North America

Birds

Enzootic cycle—Culex sp. (VEE)

The Americas

Mammals

Epizootic cycle—Aedes sp.

virus which derives its non-structural genes from an humans. Some alphaviruses are also responsible for EEE virus ancestor (Pfeﬀer et al., 1997).

disease in animals, particularly Equidae (horses and related animals).

SPECTRUM OF DISEASE CAUSED BY ALPHAVIRUSES

DIAGNOSIS OF ALPHAVIRUS INFECTIONS Alphaviruses cause a wide range of disease in animals

The clinical features in a patient living or having and humans, ranging from a clinically inapparent

recently travelled to an appropriate geographical infection to a severe disease that may result in death.

region are the essential early components of establish- The main target organs are muscle, brain, reticuloen-

ing a diﬀerential diagnosis (Table 16.3). However, dothelial system and the joints. This group of viruses

diﬀerentiation from other viral infections may be may be clinically divided into those that are associated

diﬃcult, e.g., it is sometimes clinically diﬃcult to with fever, rash and polyarthritis found in the Old

diﬀerentiate infection with Ross River virus from World and those that cause encephalitis, primarily

rickettsial, rubella, parvovirus B19 or enterovirus found in the New World (Table 16.2). Middelburg and

infections. Laboratory conﬁrmation may therefore be Ndumu viruses are not known to cause disease in

required to identify the causative agent. Table 16.2 Range of human illness caused by alphaviruses

Clinical features

Virus

Fever, rash

Sindbis, Ockelbo

Fever, rash, polyarthritis

Ross River, Barmah Forest

Fever, rash, myalgia, arthralgia Chikungunya, o’nyong-nyong, Mayaro, Sindbis, Ockelbo, Ross River, Barmah Forest, Igo Ora Fever, encephalitis

Eastern equine encephalitis, Western equine encephalitis, Venezuelan equine encephalitis,

Everglades, Semliki Forest

513 Table 16.3 Generalised alphavirus case deﬁnition

ALPHAVIRUSES

Clinical description An illness with acute onset, with fever 538 8C, including variations of the following:

Rash Arthralgia Polyarthritis Myalgia, headache, nausea, vomiting Malaise and weakness Disorientation and drowsiness

Laboratory criteria for diagnosis Isolation of virus from blood, CSF or synovial joint ﬂuids in one or more areas

Positive IgM serology or seroconversion (IgG) in paired serum Demonstration of virus antigen in autopsy tissue by immunocytochemistry or in serum by

ELISA Positive PCR from serum, synovial joint ﬂuid, CSF or autopsy tissue

Case classiﬁcation Suspected

A case that meets the clinical case description

Probable A case compatible with the clinical description with one or more of the following: Supportive serology (comparable IgG serology or positive IgM antibody test in acute or

convalescent phase serum specimen) Occurrence at same location and time as other conﬁrmed alphavirus cases

Confirmed A suspected case that is laboratory-confirmed (National Reference Laboratory), that includes seroconversion and/or virus isolation or is PCR-positive or epidemiologically linked to a confirmed case or outbreak

Laboratory diagnosis includes viral culture, the infections. Class-speciﬁc IgM assays have been devel- detection of a speciﬁc antibody response and, more

oped to diagnose an acute or recent infection with a recently, the use of molecular techniques to detect the

particular alphavirus. However, care must be taken in viral genome. In general, virus isolation is often only

the interpretation of the result, as cross-reactions may successful when acute-phase antibody-negative serum

occur with other members of the same alphavirus samples are used, particularly when the serum sample

antigenic complex (Table 16.1) (Calisher et al., 1986). is taken in the ﬁrst 48 h of illness. Thereafter, the

A serological diagnosis of a recent alphavirus infection amount of virus in serum reduces rapidly and isolation

is demonstrated by a rise in specific IgM or IgG becomes increasingly difficult. Virus identification is

antibodies, when acute and convalescent serum sam- facilitated by the use of murine monoclonal alpha-

ples are tested in parallel. This is usually done using virus-speciﬁc antibodies. Virus may be isolated by

ELISA, IFA or neutralisation tests. intracerebral inoculation of suckling mice as well as in

a variety of cell culture systems, including monkey kidney (Vero) and mosquito (C6/36) cell lines. More recently methods have been developed to diagnose

MANAGEMENT AND PREVENTION alphavirus infections using genomic detection methods. As these are RNA viruses, any polymerase chain

Management of alphavirus infections is usually sup- reaction (PCR)-based assay must ﬁrst transcribe the

portive and directed at symptomatic relief, e.g. the RNA into DNA. A reverse transcription-polymerase

symptomatic relief of joint symptoms with non- chain reaction (RT-PCR) has been developed for the

steroidal antiinﬂammatory agents and aspirin. Preven- genus-speciﬁc detection of alphaviruses. This utilises

tion of alphavirus infections has focused on vector degenerate primers localised within a conserved region

control and the protection of the individual against of the non-structural protein 1 and has become a

mosquito bites. A detailed knowledge of the vector and routine sensitive and rapid alternative to virus isola-

vertebrate host range is therefore essential to allow for tion in the diagnosis of an acute infection (Pfeﬀer et al.,

a coordinated strategy to prevent and control alpha- 1997).

virus infections. Vaccines are also available against Detection of an appropriate speciﬁc antibody

some of the individual alphaviruses infections, but are response is frequently used to diagnose alphavirus

not generally available (see below).

ALPHAVIRUSES ASSOCIATED WITH FEVERS AND POLYARTHRITIS

Sindbis Virus Sindbis virus is considered the prototype of the

alphaviruses. It was originally isolated from Culex mosquitoes collected in the Egyptian village of Sindbis (Taylor et al., 1955). In Sweden, symptomatic disease resulting from infection with a Sindbis-like agent has been termed Ockelbo disease. A Sindbis-related virus has also been described as the cause of Pogosta disease in Finland and as Karelian fever in the Karelian region of the former USSR. Babanki virus is a Sindbis-like agent that occurs in West and Central Africa; the clinical signiﬁcance of the infection with this virus remains to be deﬁned (Peters and Dalrymple, 1990).

Epidemiology and Host Range Sindbis virus occurs in many parts of the world,

including Europe, Asia, Africa and Australia. The virus is known to infect humans, domestic animals and birds, with birds forming the principal reservoir. Humans are not considered to be essential to the survival of the virus in nature. Sindbis virus is transmitted among birds by Culex mosquitoes. Infec- tion in birds does not appear to result in disease. Where humans and birds exist in close proximity, e.g. in the Nile Valley, transmission to humans may occur (Peters and Dalrymple, 1990). Studies from South Africa have shown that the virus is distributed widely throughout the country. Transmission to the avian population, and probably to humans, occurs annually. Extensive human disease only occurs, however, during years of abundant rainfall and ﬂooding (McIntosh et al ., 1964; Jupp et al., 1986). In Europe, disease caused by Sindbis virus occurs after excursions into forested areas, e.g. by lumberjacks or berry pickers. Here the virus has been isolated from Culiseta, Aedes and Culex mosquitoes.

Clinical Disease Sindbis virus is among the least virulent of the

alphaviruses. Serological surveys suggest that infection with Sindbis virus is relatively common, but clinically apparent disease is unusual. When symptomatic, the spectrum of disease in humans varies from a mild illness to one consisting of a rash and arthralgia. The illness is usually characterised by a sudden onset of the

rash, but occasionally prodromal symptoms may be present. The rash is usually macular or papular but may become vesicular or haemorrhagic. Malaise, fatigue and headache are frequently present (McIntosh et al ., 1964; Jupp et al., 1986). The initial joint involvement is usually migratory, followed by a gradual resolution of symptoms.

Ockelbo disease was ﬁrst described in the central part of Sweden in the 1960s in clusters of patients with fever, arthralgia and a rash (Niklasson and Vene, 1996). Some patients with Ockelbo disease have reported joint symptoms persisting for more than a year and recurrent joint problems were documented in one-third of a group of Swedish patients interviewed 2 or more years post-infection (Niklasson et al., 1988). By contrast, reports from South Africa have emphasised the joint involvement less and have suggested that the involvement is of tendon and periarticular tissue, rather than a true arthritis (McIntosh et al., 1964).

Pathogenesis It is not clear whether the skin lesions are the result of

a direct viral cytopathic eﬀect or are immunopathological. Evidence to support the former hypothesis comes from Malherbe and Strickland-Cholmley (1963), who demonstrated the isolation of Sindbis virus from a vesicle in the absence of a serum viraemia. This was associated with a subsequent rise in speciﬁc antibody. However, other workers have failed to isolate Sindbis virus from skin lesions (McIntosh et al., 1964). The pathogenesis of the joint complications, and whether they are articular or periarticular, is also unclear.

Diagnosis Isolation of Sindbis virus from human sera has been

reported, but this is frequently not successful, even using sera taken early on in the illness. This is because the viraemia associated with Sindbis virus infection is often of low level and transient. In addition, medical attention is frequently sought relatively late in the course of the illness. As mentioned above, Sindbis virus has also been isolated from a skin lesion. When inoculated into 3–5-day-old mice, either intracerebrally or intraperitoneally, Sindbis virus causes a fatal infection, which is preceded by a short period of paralysis. The virus has also been isolated in vitro in ﬁbroblastic cell lines. Antibodies appear 7–10 days after the onset of illness and may be detected by HI, ELISA and immunoﬂuorescence (IF) (McIntosh et al.,

514

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

515 1964; Doherty et al., 1969; Espmark and Niklasson,

ALPHAVIRUSES

freshwater habitats, are also considered important 1984). Although the detection of Sindbis-speciﬁc IgM

vectors (Peters and Dalrymple, 1990). Besides humans, may be of use in diagnosis of an acute infection, this

other possible vertebrate hosts include marsupials, class of antibody may remain detectable 3–4 years after

domestic animals and rodents (Doherty, 1977). the initial infection. A positive IgM result must

In 1979 an explosive epidemic of RRV infection therefore be interpreted together with the available

occurred in Fiji (Aaskov et al., 1981). The virus spread clinical information.

to American Samoa and then to the Wallis and Futuna Islands and the Cook Islands. The mosquito vector in this region is unknown, although the virus has been

Ross River Virus isolated from Aedes polynesiensis, a mosquito that is found in many parts of the Eastern Paciﬁc (Rosen et

Infection with Ross River virus (RRV), also known as al ., 1981). Evidence suggests that humans are the most epidemic polyarthritis, is a major public health

important vertebrate hosts in this region. A study from problem in Australia. It is the most common arboviral

the Cook Islands showed that the incubation period infection of humans in Australia. In the late 1970s and

could be as short as 3 days, and the explosive nature of early 1980s, RRV also became endemic in the Paciﬁc

this epidemic led to speculation that mechanical Islands (Table 16.4). In recent years it has also been

transmission of RRV may occur. This theory has increasingly identiﬁed in travellers returning to Europe.

been supported by laboratory evidence of mechanical transmission of RRV from viraemic donors to uninfected recipient suckling mice. In addition, it has also

Epidemiology and Host Range been suggested that transovarial transmission in mosquitoes is a potential survival mechanism for this

Until 1979 the distribution of RRV infections was

virus (Kay, 1982).

thought to be limited to Australia, Papua New Guinea The number of annual reported cases of RRV and the Solomon Islands, all in the western half of the

disease has increased during the past decade to 4000– Paciﬁc basin. In Australia both epidemic and sporadic

6000. However, these ﬁgures represent a combination transmission of RRV occurs. Epidemics are character-

of improved laboratory diagnostic reagents, greater istically preceded by heavy rainfall and are located in

awareness by clinicians, housing developments on salt river valleys and irrigated lands in coastal areas.

marshes and migration northwards to warmer climates However, other factors, such as temperature, are

in retirement (Mackenzie et al., 2001). important—a sudden period of cold can delay the build-up of the vector and so abort an epidemic (Hawkes et al., 1985). The virus was ﬁrst isolated

Clinical Disease

from Aedes vigilax, which is accepted as being the major vector in the coastal regions of Australia. Other

Infection with RRV does not usually result in disease in mosquito species, including the salt marsh arthropod

humans and it has been estimated that 50 subclinical Aedes comptorhynchus , found in northern and southern

infections occur for each clinically recognised case coastal regions, and Culex annulirostris, which breeds in

(Table 16.2). The disease is not fatal. Adults are more

Table 16.4 Geographic distribution of the Semliki Forest virus complex Virus species

Other features Semliki Forest

Subtype

Main geographic distribution

Epidemic in Central African Republic Chikungunya

Africa

Chikungunya

Epidemics in E. Africa, Asia O’nyong-nyong

Africa, S.E. Asia, The Philippines

Epidemics in E. Africa, Zimbabwe Getah

West and East Africa, Zimbabwe

Getah

Malaysia, Japan, Australia, Cambodia, The

Ross River

Australasia, South Paciﬁc

One epizootic in Japan Mayaro

Sagiyama

Japan, Okinawa

Mayaro

Trinidad, Brazil

Una

Brazil, Colombia, Panama, Surinam, Trinidad,

French Guyana

likely to be symptomatic than children. The onset of clinical symptoms is characterised by joint pains (epidemic polyarthritis) accompanied by a rash and fever. In a recent postal survey of general practitioners in south Australia the complaint of ‘pain in the joints’ was the most important symptom for suspecting an infection with RRV, with joint eﬀusion, rash and pyrexia being important signs (Stocks et al., 1997; Flexman et al., 1998). The rash may occur simultaneously with the joint pains or may follow 1–2 days later. Although the rash is usually macular, maculopapular or papular, vesiculation and petechiae have been described. The rash and joint pains are commonly accompanied by some degree of constitutional upset, including myalgia, headache, anorexia and nausea. The joint involvement is usually asymmetrical and migratory and most often aﬀects the small joints of the hands and feet, together with the knees. In addition, there may also be periarticular swelling and a tenosynovitis (Clarke et al., 1973; Hawkes et al ., 1985). The arthralgia and arthritis tend to run a relapsing course but with an overall gradual improvement. The symptoms of epidemic polyarthritis may last for 30–40 weeks, with some patients having symptoms for more than a year. While joint pain, rash and fever are the most common clinical features, a case of glomerulonephritis has also been reported in the acute phase of RRV disease (Fraser et al., 1988). This report described

a patient presenting with haematuria and proteinuria coincident with an acute RRV infection; the frequency of this complication has yet to be deﬁned.

Pathogenesis The pathogenesis of the disease is not completely

understood. While virus has been isolated from the blood, it has not been isolated from either the skin or joints. However, RRV antigen has been detected by immunofluorescence at both of these sites. Histological examination of the skin lesions shows a mononuclear cell infiltration (Fraser et al., 1983). It has also been shown that RRV is capable of infecting human synovial cells (Cunningham and Fraser, 1985). Serum complement is normal and circulating immune complexes do not usually exceed normal levels. It has therefore been suggested that RRV plays a direct role at these sites of inflammation.

Diagnosis

A key factor in diagnosis is knowledge of travel history. A diﬀerential diagnosis would also include

rubella, other alphavirus-induced arthritis, dengue, rheumatoid arthritis and other chronic rheumatic diseases (Fraser, 1986).

Isolation of RRV is only successful using acute- phase antibody-negative serum samples. Both mosquitoes and mosquito cell lines have been used. The current method of choice for RRV isolation is the inoculation of the mosquito cell line, C6/36. As this virus does not display a cytopathic eﬀect in C6/36 cells, viral antigen is detected using IF (Rosen et al., 1981). RRV may also be isolated in Vero (E6) cells. More recently, methods have been developed to detect RRV RNA using RT-PCR. This methodology has potential application to virus surveillance programmes in mosquito populations (Sellner et al., 1994).

The most sensitive and established serological assay involves the use of an IgM capture EIA. However, there is a complication in that the assay can remain positive for up to 2 years after the acute infection (Carter et al., 1985). It should be noted that IgM antibody cross-reactions with Mayaro, chikungunya and Semliki Forest viruses occurs with sera from patients with RRV infections (Table 16.4). However, ratios of homologous to heterologous IgM titres are usually high and so, although this cross-reaction somewhat limits the diagnostic usefulness of this test, it can provide a reasonably speciﬁc and rapid diagnosis in an epidemic situation (Calisher et al., 1986). The presence of RRV-speciﬁc IgA or low-avidity IgG can assist the diagnosis of a recent infection (Carter et al., 1987). A more precise diagnosis can be made using neutralisation tests (Kanamitsu et al., 1979).

Vaccine An inactivated RRV vaccine is being developed.

Preliminary studies have shown that antibodies to this candidate vaccine neutralised all strains of RRV tested, but to diﬀerent degrees (Aaskov et al., 1997). Further studies are awaited with interest as, while RRV does not usually cause a severe disease, it may result in considerable morbidity with an associated loss of productivity.

Barmah Forest Virus

This alphavirus has been recently reported to cause both clinical and subclinical infections in Australia. Clinical disease resulting from BFV infection was ﬁrst reported in 1986. The ﬁrst epidemic of human disease occurred in 1992 in the Northern Territory (Mackenzie

516

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

et al ., 1994). To date, Australia is the only country in which this virus has been detected (Hills, 1996).

Barmah Forest virus (BFV) is the sole member of the seventh alphavirus serocomplex (Table 16.4). The nucleotide sequence of the BFV genome has recently been described and, from amino acid sequence comparisons with sequenced alphaviruses, BFV has been shown to be most closely related to RRV and Semliki Forest virus (Lee et al., 1997). In recent years there has been an apparent increase in incidence of polyarthritis caused by BFV. This prompted a study on the molecular epidemiology of this alphavirus, which showed a high degree of sequence homology between BFV isolates with no evidence of geographical or temporal divergence (Poidinger et al., 1997).

Epidemiology and Host Range Barmah Forest virus is an alphavirus that is enzootic in

Australia and has been isolated from the two most common vectors of RRV, Culex annulirostris and Aedes vigilax . It has been shown to circulate among mosquitoes and terrestrial animals, most noticeably marsupial species. Occupational and recreational exposure to these vectors is therefore an important risk factor for infection with this virus. A higher incidence of antibodies to BFV in RRV antibody- positive blood donors compared with RRV antibody- negative blood donors suggests a common ecology. Further information is required on the duration of viraemia in humans to determine whether the other vertebrate hosts are of importance in viral maintenance. Currently, human disease caused by BFV has not been recognised in southern Australia or Tasmania.

Clinical Disease Human infections have been recognised since 1986 and

its incidence has increased through greater clinical awareness and improved laboratory diagnostic methods. Currently, approximately 500 cases are recorded each year (Lindsey et al., 1995). As mentioned above, both clinical and subclinical infections occur. The most common symptoms noted in a study from Queensland were arthritis, arthralgia, myalgia and fever, which were present in approximately 75% of patients (Table 16.2). In addition, half the patients in this study had a rash (Phillips et al., 1990). A more recent study from New South Wales reported lethargy (89%), joint pain (82%) and rash (68%) as the most common symptoms. In the latter study, just over half of the

patients reported an illness lasting for more than

6 months. These authors also reported a possible association between the presence of a rash and an improved prognosis (Beard et al., 1997). In general, the symptoms are similar to those associated with RRV infection; laboratory conﬁrmation is therefore necessary to achieve a precise diagnosis. The wider availability of serological testing for BFV infections has also enabled their more unusual features to be described. Recently the ﬁrst case of glomerulonephritis after BFV infection was diagnosed. The authors suggest that BFV infection should be considered as a possible aetiological agent for glomerulonephritis (Katz et al., 1997).

Diagnosis Clinical diﬀerentiation from RRV infection in patients

living in endemic areas is difficult. Laboratory confirmation is therefore necessary for a precise diagnosis to be made. BFV has been isolated from a patient in the mosquito cell line, C6/36. The serological response to infection with this virus in humans remains to be defined precisely. However, in contrast to RRV infections, where the antibody response may be delayed for 7 days, a recent study demonstrated the presence of both HI and neutralising antibody titres in two patients with BFV infection at the onset of symptoms (Phillips et al., 1990). This could be due to either a different serological response to these two viruses or, alternatively, BFV-associated disease may have a less clearly defined onset than that caused by RRV infections.

Chikungunya Virus

This virus was ﬁrst isolated from patients and mosquitoes in the Newala district of Tanzania in 1952–1953. The name is derived from a local term for the illness and means ‘that which bends up’, referring to the crippling arthralgia and arthritis associated with infection with this virus. Chikungunya virus is found in the savannahs and forests of tropical Africa, as well as in many parts of Asia, including Thailand, Cambodia, Vietnam, Burma (now Myanmar), Sri Lanka and India (Table 16.4). The chikungunya strains from Africa and Asia have been shown to be closely related, using a panel of monoclonal antibodies prepared against strains from Africa and Asia (Blackburn et al., 1995).

ALPHAVIRUSES

517

Epidemiology and Host Range The epidemiology of chikungunya virus infections

diﬀers in Africa and Asia. In Africa this virus is transmitted in savannahs and forests by Aedes mosquitoes. The most important vertebrate host in the cycle of infection is the non-human primate, e.g. baboons and Cercopithecus monkeys. Bushbabies and certain species of bats may also be infected in nature, but their role in viral maintenance is likely to be of secondary importance. Humans may be infected in African villages and rural areas, particularly where Aedes aegypti is present in large numbers. In contrast to the situation in Africa, transmission in Asia is primarily from human to human by the vector Aedes aegypti . It has been suggested that infection with this virus may be common in certain parts of Africa, e.g. 47% of sera collected from 132 adults living in the Karamoja district of Uganda had antibodies to chikungunya virus detectable by HI (Rodhain et al. 1989).

Although the most important vector for chikungunya virus transmission to humans is the Aedes mosquito, transmission has also been described by Mansonia africana . The active replication of alphaviruses in the mosquito is essential for perpetuation in nature, but the explosive nature of the chikungunya epidemics has led to speculation that mechanical transmission of the virus may also occur from a viraemic host by an uninfected mosquito (Peters and Dalrymple, 1990).

Clinical Disease The incubation period varies but is usually 2–4 days,

with a range of 1–12 days. Chikungunya is an acute infection of abrupt onset of fever, headache and severe joint pains without prodromal symptoms. The joint pains are the dominant complaint and aﬀect mainly the small joints of the hands, wrist and feet. This may

be associated with an erythematous flush over the face and upper chest in approximately 80% of patients. A maculopapular rash, together with a generalised lymphadenopathy, appears 3–4 days later. Although the arthralgia may resolve within a few weeks, pain, swelling and morning stiffness may continue for months and even years after infection. Petechiae and bleeding from the gums does occur, but there are no significant haemorrhagic manifestations.

In contrast to the clinical presentation in adults, a study from Bangkok concentrating on a paediatric

outpatient department showed that the most frequent presenting symptom of chikungunya virus infection in children was vomiting, which was present in 35% of patients. In addition, 18% had abdominal pain or anorexia. None of the patients in this study were noted to have joint symptoms; arthritis and arthralgia therefore appear to be less prominent features in children. On clinical examination of this paediatric population, the most frequent sign was a pharyngitis, which was present in 71% of patients, followed by facial ﬂushing (24% of patients). None of these children had a rash. This study illustrates very clearly that the clinical presentation of chikungunya infections in adults and children diﬀer (Halstead et al., 1969). In Asia, several virus isolations have been made from severely ill children diagnosed as having haemorrhagic fever, similar to dengue haemorrhagic fever (DHF).

Haemorrhagic forms of chikungunya virus infection have been described in India and southeast Asia, but these are seldom as severe as dengue haemorrhagic fever. In a hospital-based study in Bangkok about 8% of children with suspected haemorrhagic fever had chikungunya virus infection (Nimmannitya et al., 1969).

Pathogenesis Chikungunya virus may be isolated from the blood

early in the course of disease. As the disappearance of the viraemia correlates with the appearance of HI and neutralising antibodies, it would appear that recovery from infection correlates with the development of viral-speciﬁc antibody rather than cell-mediated immunity. This is further substantiated by the eﬀect of serum antibody in mouse protection tests (Carey et al., 1969). The haemostatic abnormality seen in both children and adult patients is possibly the result of an acquired platelet defect, as it has been shown that the association of chikungunya virus with human platelets in vitro promotes platelet clumping (Chernesky and Larke, 1977).

Diagnosis The clinical features described above in a patient who

has recently returned from, or is living in, sub-Saharan Africa or Asia enables a presumptive clinical diagnosis to be made. In addition, a minority of patients will have a leukopenia with a relative lymphocytosis. In most, however, the white cell count will be normal. The

518

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

519 platelet count may be decreased, but this is usually not

ALPHAVIRUSES

Epidemiology and Host Range signiﬁcant.

Chikungunya virus can be isolated by the intracere- The initial epidemic began in 1959 in north-western bral inoculation of suckling mice or by viral culture in

Uganda and spread from there to Kenya, Tanzania, either mosquito or mammalian cell culture systems

Mozambique, Malawi and Zaire (now Democratic (Peters and Dalrymple, 1990). Viraemia will be present

Republic of Congo). It involved some 2 million people. in most patients during the ﬁrst 48 h of disease and

Within an infected area spread was rapid, with an may be detected in sera as late as day 4 post-onset.

extremely high attack rate. In an aﬀected village a high Thereafter the amount of virus in serum drops rapidly

proportion of the population, regardless of age or sex, and recovery becomes increasingly diﬃcult. Both HI

were incapacitated. This disease was also apparently and neutralising antibodies appear on day 5–7 of the

unknown to the tribes who were aﬀected. The origin of illness. The appearance of these antibodies is asso-

o’nyong-nyong virus is not clear. It might have existed ciated with a decrease in viraemia (Carey et al., 1969).

unrecognised prior to the initial epidemic described HI antibodies rise rapidly and peak in the second

above or, conversely, the epidemic may well have week. Virus-speciﬁc IgM antibodies are readily

resulted from a mutant or recombinant virus. The detected by capture ELISA in patients recovering

vectors of this virus are Anopheles funestus and from infection and they persist in excess of 6 months.

Anopheles gambiae . It is not known whether a non- All patients will be serologically positive by day 5–7

human vertebrate host exists. After this initial epi- post-onset of infection. However, cross-reaction occurs

demic very little further information on this virus was with other members of the same alphavirus antigenic

obtained until 1978 when it was isolated from complex, so the results obtained must be interpreted

Anopheles funestus mosquitoes in the Kao Plain in with caution (Table 16.3) (Calisher et al., 1986). The

Kenya (Johnson et al., 1981). The re-emergence of use of a speciﬁc IgM assay may be especially relevant

epidemic o’nyong-nyong fever in south-western in an epidemic situation. This was illustrated in an

Uganda after an absence of 35 years has recently outbreak in Yangon in Burma (now Myanmar).

been described (Rwaguma et al., 1997). Utilisation of a simple indirect ELISA was helpful in achieving an acute-phase diagnosis and results showed

a strong concordance with HI (90% agreement), although ELISA was able to identify twice as many

Clinical Disease

patients at initial hospital admission (Thein et al., 1992). The diﬀerential diagnosis within the laboratory

The clinical features are similar to those of chikungu- should include Ross River, dengue, Sindbis and West

nya virus infections, with a sudden onset of a 5 day Nile virus infections.

fever often accompanied by a rigor. Characteristic features of the disease include joint pains followed by the development of a rash. Joint pain primarily aﬀects the knees, although ankles, elbows or wrists also can

Vaccine

be involved. The morbilliform macupapular rash erupts on the

A formalin-inactivated chikungunya virus vaccine face, extending to the trunk and extremities, 4–7 days prepared in monkey kidney tissue culture has been

post-onset of infection and is similar to that of described. Despite being apparently safe and immuno-

chikungunya virus. Some fatalities have been genic, its use has been limited to initial safety studies

described, but morbidity is substantial (Kiwanuka et and laboratory workers (Harrison et al., 1971). More

al ., 1999).

recently, a live-attenuated vaccine has been produced.

Diagnosis

O’Nyong-Nyong Virus Virus may be isolated in 1 day-old mice from serum ‘O’nyong-nyong’ literally means ‘weakening of the

samples taken during the acute stage of the infection. joints’. It was ﬁrst described as an epidemic viral

However, mouse passage or subinoculation into chick disease in East Africa (Haddow et al., 1960). Anti-

ﬁbroblasts may be necessary. The mice demonstrate genically, o’nyong-nyong virus is a subtype of

alopecia and runting. The virus may then be further chikungunya virus (Table 16.1).

identiﬁed using plaque inhibition, cross-neutralisation identiﬁed using plaque inhibition, cross-neutralisation

Igbo Ora Virus Igbo Ora virus is closely related to both chikungunya

and o’nyong-nyong viruses. Human disease associated with infection with this virus is characterised by fever, arthritis and the development of a rash (‘the disease that breaks your wings’). The virus was isolated from human sera from patients in Africa (Nigeria, central African Republic) in the latter part of the 1960s (Moore et al., 1975) and was responsible for an epidemic that involved four villages in the Ivory Coast in 1984 (Peters and Dalrymple, 1990).

Mayaro Virus Although infection with Mayaro virus was ﬁrst

recognised in Trinidad in 1954, it was an epidemic in Brazil in 1978 that permitted a detailed evaluation of human infections (LeDuc et al., 1981; Pinheiro et al., 1981).

Epidemiology and Host Range During the extensive epidemic described by LeDuc and

colleagues (1981), 20% of the entire population of approximately 4000 persons in the rural village of Belterra in Brazil were infected. The numbers infected were higher amongst those who lived near a plantation forest. The epidemic began with the onset of the wet season and ended with the onset of the dry season. This correlated with the abundance of the mosquito vector, Haemagogus janthinomys. In addition to humans, marmosets and other primates are susceptible to infection (Hoch et al., 1981). It has therefore been suggested that non-human primates play an important role in the maintenance of this virus, as experimental infection of marmosets with Mayaro virus showed that

a signiﬁcant viraemia develops. The role of other vertebrates, e.g. birds, remains to be deﬁned.

Clinical Disease

A detailed evaluation of the clinical features of infection with this virus in humans is available from studies done during the 1978 epidemic in Brazil. Fever, arthralgia and rash were the most frequently encountered clinical manifestations. Other symptoms included headache, myalgia and chills (Pinheiro et al., 1981).

Diagnosis Leukopenia, sometimes accompanied by a modest

lymphocytosis, is a frequent ﬁnding. A mild thrombocytopenia may also be present. Mayaro virus may be isolated from acute-phase serum samples in an in vitro cell culture system using Vero cells. The virus may then

be identified using HI or plaque reduction neutralisation tests. In addition, Mayaro virus is also pathogenic for infant mice on intracerebral inoculation. In the Belterra epidemic, virus was isolated from the serum in 97% of the patients bled in the first 24 h after the onset of symptoms. Thereafter, viral recovery rate decreased to only 7% on day 4. Serological evidence of a Mayaro virus infection can be achieved by demonstrating a seroconversion using HI in paired serum samples taken from a patient during the acute and convalescent phase of the illness (Pinheiro et al., 1981). In addition, the use of a Mayaro virus-specific IgM assay has been described. Cross-reactivity of IgM antibody occurs with alphavirus infections by viruses belonging to the same antigenic complex (Table 16.1). However, the ratios of the homologous to heterologous IgM titres are high, allowing a reasonably specific and rapid diagnosis to be made. The IgM assay is therefore a useful diagnostic tool in an epidemic situation (Calisher et al., 1986).

ALPHAVIRUSES ASSOCIATED WITH ENCEPHALITIS

The genus Alphavirus contains four viruses that produce encephalitis in humans; Eastern equine encephalitis (EEE) virus, Western equine encephalitis (WEE) virus, Venezuelan equine encephalitis (VEE) virus and Everglades virus. The EEE, WEE and VEE viruses were ﬁrst isolated from the brains of dead horses in the 1930s. The severity of disease associated with infection with this group of viruses diﬀers: EEE virus is the most neurovirulent, while infection with VEE virus is associated with a febrile illness, with

520

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

encephalitis occurring infrequently. Focal epidemics of EEE virus have periodically occurred in eastern USA; WEE is endemic in western USA and Canada; and VEE virus in Central and South America and occasionally in North America. The Everglades virus is related to EEE but is restricted to the state of Florida, USA, and only ﬁve cases of encephalitis associated with it have been reported, despite a high seroprevalence (27%) in southern Florida.

Eastern Equine Encephalitis Virus Eastern equine encephalitis virus is an important cause

of human disease in the eastern states and some inland mid-western locations of the USA. Its distribution extends through Central America to Trinidad, Brazil, Guyana and as far south as Argentina (Weaver et al., 1994). This virus is among the most virulent of the alphaviruses. It has been reported that there are two distinct antigenic variants (North American and South American), the former being genetically quite stable (Roehrig et al., 1990). It causes severe disease in humans, certain species of birds such as the pheasant, as well as horses, all of whom have high fatality rates after infection.

Epidemiology and Host Range The physical, biological and ecological factors asso-

ciated with epizootic transmission are complex, but the abundance of EEE virus circulating in the enzootic cycle and the various characteristics of the epizootic vectors are important determinants of risk. In endemic areas birds are the most important vertebrate hosts and the natural cycle is between birds and the ornithophilic mosquito, Culiseta melanura. Birds vary in their susceptibility, with some, e.g. the pheasant, developing severe disease, while other avian species suﬀer no appreciable morbidity or mortality (Peters and Dalrymple, 1990). Culiseta melanura is thought to

be the main endemic vector (Howard and Wallis, 1974). However, because of its ornithophilic nature, this mosquito is unlikely to play a major role in the transmission of EEE virus to horses or humans. However, the virus escapes from enzootic foci located in swamp areas through bridge vectors such as Aedes sollicitans and Coquillettidia perturbans, which are thought to be important in the transmission to these vertebrates. These species also feed on both mammals and birds and can transmit the virus to humans, horses and other hosts. Both humans and horses are ‘dead-

end’ hosts for EEE virus infections; infected birds are therefore necessary for the epidemic spread of these species. In South and Central America the enzootic cycle is maintained in most forests where Culex melanoconion spp. are considered the primary vectors (Scott and Weaver, 1989). Disease in humans often follows an epizootic course in horses. Fewer than 10 cases of EEE are reported annually in the USA. Even during a major equine epizootic episode, EEE virus- associated disease in humans is rare. In addition to being limited to certain geographical regions, the prevailing climatic conditions, in the form of an unusually hot, wet summer, also play an important role, for this allows for an abundance of the vector Culiseta melanura . This, together with the presence of a susceptible bird population and an additional vector that can transmit virus from viraemic birds to susceptible horses and humans, are important predisposing factors for the occurrence of human and equine cases. Although the transmission cycles have not been well established, additional reservoirs of infection have been reported in forest-dwelling rodents, bats and marsupials (Ubico and McLean, 1995), reptiles and amphibians (Morris, 1988).

Clinical Disease EEE virus infections may produce a severe, often fatal

encephalitis in humans (Deresiewicz et al., 1997; Farder et al., 1940). The ratio of inapparent to apparent infections is approximately 23:1 in adults and 8:1 in children under the age of 4 (Goldﬁeld et al., 1968). The encephalitis associated with EEE virus infection tends to be fulminant; a recent study that reviewed all cases of EEE in the USA during 1988– 1994 showed a mortality of 36%, with 35% of patients being left moderately or severely disabled. The remaining patients were left mildly disabled, with only one patient recovering fully. The most common clinical features were fever (83%), headache (75%), nausea and vomiting (61%) and malaise and weakness (58%). The incubation period for EEE virus is 4–

10 days. However, the onset of neurological symptoms was associated with a rapid deterioration of the patient; 89% (32/36 patients) were or became stuporous or comatose, with about one-quarter having seizures. Interestingly, abdominal pain was present in 22% of cases, with two patients presenting with acute abdominal pain in the prodromal period (Deresiewicz et al ., 1997). Although it has been suggested that an age of 40 and a long prodromal course correlates with

a good functional recovery (Przelomski et al., 1988),

ALPHAVIRUSES

521

others have found that these parameters do not significantly predict outcome (Deresiewicz et al., 1997). Deresiewicz et al. (1997) did, however, demonstrate that high initial cerebrospinal fluid white cell counts and the development of severe hyponatraemia were poor prognostic signs, possibly because both could be markers of intense cerebral inflammation.

Pathogenesis and Pathology Post mortem histopathological studies have demon-

strated necrotic foci together with arteriolar and venular inflammation and perivascular cuffing. An inflammatory meningeal infiltrate may be present. Although the distribution of the focal lesions may vary, involvement of the basal ganglia and thalami appears to be prominent (Deresiewicz et al., 1997). Viral particles have been visualised in the oligodendroglial cells by electron microscopy (Bastian et al., 1957). The neurological damage is probably due to a combination of a direct viral cytopathic effect, inflam-

matory damage and a vasculitis (Peters and

Dalrymple, 1990).

Diagnosis

A diagnostic clue may be obtained from the prevailing climatic conditions (a hot wet summer), together with associated illness in the horse and pheasant populations. However, the symptoms are non-specific and confirmation needs to be obtained serologically, or by demonstration of virus in cerebrospinal fluid, or both. Neuroradiological imaging may be helpful, with focal lesions visible in the basal ganglia, thalami and brainstem (Deresiewicz et al., 1997).

Laboratory diagnosis is based on molecular detection of viral antigen, virus isolation or antibody detection. Samples for isolation can be CSF, blood or CNS tissue by inoculation into newborn mice or tissue culture using Vero cells or MRC-5 (Sotomayor and Josephson, 1999). As with all alphavirus infections, an acute serum sample should be taken as soon as possible after the onset of illness. EEE virus has been isolated from a laboratory worker on the second day of illness (Clarke, 1961). In this case, four of 25

1 day-old mice died after intracerebral inoculation with

a serum sample taken from this patient. Further identiﬁcation was done serologically. In addition, virus has also been isolated from post mortem brain tissue taken from a fatal case of EEE. More recently,

an RT-PCR-based assay has been developed to detect EEE virus RNA (Voskin et al., 1993).

Serology is an important diagnostic tool. Paired serum samples may be tested in parallel using ELISA (Scott and Weaver, 1989) or neutralisation tests. A class-speciﬁc IgM assay has also been described as a rapid and speciﬁc diagnostic tool. As EEE virus is the only species of virus belonging to the EEE complex (Table 16.1), cross-reaction with heterologous viruses belonging to the same antigenic complex does not pose

a diagnostic problem (Calisher et al., 1986).

Prevention and Control Protection against mosquito bites and control of the

vector is important. An effective equine vaccine is licensed in the USA and is recommended for livestock in areas where EEE virus transmission is known to occur. Specific control measures to prevent human disease include a formalin-inactivated vaccine derived from a North American strain of EEEV (PE-6) is available for horses but not for general human use. The vaccine has been shown not to induce significant levels of neutralisation or anti-E@ antibody responses to South American strains of EEE virus (Strizki and Repik, 1995). However, as human disease is rare, even during an equine epizootic episode, this is only available to those considered to be at risk of exposure. Prompt notification of suspected cases of arboviral encephalitis to the local public health authority is also essential.

Western Equine Encephalitis Virus

The WEE complex in the New World contains three additional viruses in addition to WEE, named High- lands J (HJ), Fort Morgan (FM) and Aura. Only WEE is recognised as causing human disease (Calisher, 1994). Viruses in the WEE complex found in the Old World include Sindbis, Whataroa and Kyzlagach. Western equine encephalitis virus causes more human infections than the EEE virus, but the illness is less severe and mortality seldom exceeds 10%. Its distribution covers the Paciﬁc coast of the USA, but also includes the great plains of the USA and Canada and extends into Central America and the northern parts of South America (Chamberlain, 1987; Reeves, 1987; Iversen, 1994). The WEE antigenic complex consists of six species (Table 16.5). It has been suggested that WEE virus arose through recombination between an

522

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

EEE virus-like virus and a Sindbis-like virus (Hahn et al ., 1988).

Epidemiology and Host Range WEE virus is transmitted in the western USA by the

mosquito Culex tarsalis. Birds are the most important vertebrate host. Endemic transmission results in a few human cases. In addition, major equine epidemics may result in a signiﬁcant number of human cases. As with other arboviruses, climatic factors exert a major inﬂuence on the epidemiology and distribution of WEE virus.

In the eastern USA WEE virus is replaced by Highlands J virus, whose primary vector is Culiseta melanura , which is also the primary vector of EEE virus. The strictly ornithophilic nature of this vector explains the absence of signiﬁcant disease outbreaks in the eastern USA. WEE virus and Highlands J viruses are closely related, both serologically and at a molecular level.

Clinical Disease Most WEE infections of adults are asymptomatic,

patients presenting with a sudden onset of fever, headache, nausea, vomiting, anorexia and malaise. This progresses in the most serious cases to restlessness, tremor, irritability, photophobia and altered mental status. Paralysis is not uncommon. The estimated case:infection ratio in adults is approximately 1:1000. This decreases to approximately 1:60 in children and 1:1 in babies (Peters and Dalrymple, 1990). Most cases of encephalitis occur in children, frequently under the age of 4 years, in whom the onset is marked by convulsions. This may

result in permanent neurological damage. Drowsi- ness, headache and mental confusion, sometimes leading to coma, may be seen in adults. In utero infection followed by an acute encephalitis has been documented (Shineﬁeld and Townsend, 1953). Recovery from the acute illness may be slow and symptoms such as fatigability, irritability and headache may persist for up to 2 years (Earnest et al., 1971).

Pathogenesis and Pathology WEE virus is less neurovirulent than EEE virus in both

humans and horses. Post mortem examination of the brain of fatal cases shows a perivascular mononuclear and polymorphonuclear inﬁltrate, together with par- enchymal necrosis.

Diagnosis During the acute phase of the illness, viral isolation

may be attempted from blood, throat swabs or cerebrospinal ﬂuid samples, using suckling mice, but this is frequently not successful. Virus may usually be isolated from brain biopsy material or post mortem brain tissue taken early on in the illness. However, serology, together with the appropriate clinical manifestations in a patient living in, or who has recently travelled to, an endemic area, forms the most important method of diagnosing of disease associated with infection with WEE virus. Classical serological techniques, e.g. HI, ELISA or neutralisation, can be used to demonstrate a rise in antibody titre between acute and convalescent serum samples. In addition, the detection of WEE virus-speciﬁc IgM can provide a rapid acute-phase diagnosis. However, cross-reaction

ALPHAVIRUSES

523 Table 16.5 Geographical distribution of the western equine encephalitis virus complex

Virus species Subtype

Main geographic distribution

Other features Western equine

encephalitis (WEE)

N. America, Mexico, Guyana, Brazil, Argentina

Epizootics: N. America only

Aura

Brazil, Argentina

Fort Morgan

Western USA

Highland J

Florida, Eastern USA

Sindbis Sindbis

Africa, E. Mediterranean, Borneo, The Philippines, Australia, Sicily

Babinki

West and Central Africa

Whataroa

New Zealand Kyzylagach Azerbaijan Ockelbo

Scandinavia, former USSR

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

may occur with other viruses within the complex virus, is endemic in Florida and another variant, (Table 16.1). Knowledge of the geographical distribu-

Mucambo virus, is found in Brazil, Trinidad and tion of the viruses within the complex allows

Surinam (Table 16.6).

interpretation of a positive IgM result, together with the appropriate patient details (Calisher et al., 1986). Antigen detection using RT-PCR with patient serum

Epidemiology and Host Range or CSF is well established (Pfeﬀer et al., 1997).

Both epizootic and enzootic strains of VEE virus occur. Serotypes IAB and IC are equine virulent, while ID, IE

Prevention and Control and II, III and IV are equine avirulent (Table 16.6) (Weaver et al., 1996). The principal vertebrate host for

Vector control is an important aspect of the control of enzootic transmission is the rodent, although birds and all mosquito-borne encephalitides. Outbreaks in

other species may play a less important role. Horses humans and horses are associated with unusually high

may be infected by several subtypes or variants of VEE densities of mosquitoes. There is therefore a correlation

(Table 16.1) but they do not play an important role in between Culex tarsalis density and human cases of

enzootic transmission, which occurs primarily between WEE virus infection. Besides vector control, changing

small mammals and the Culex mosquitoes of the behaviour patterns, e.g. air-conditioning houses and

subgenus Melanoconion (Cupp et al., 1986). In con- promoting indoor activities, may also protect from

trast, during equine epizootics horses do play an vector-borne diseases and are complementary to

important role in viral spread. It has been suggested mosquito control programmes (Gahlinger et al.,

that epizootic strains of the VEE are maintained in 1986). An inactivated vaccine is available to those at

other mammalian hosts and birds until the conditions risk. An inactivated vaccine is also available for horses.

are favourable for the development of an equine epizootic. A number of mosquito species have been implicated as likely vectors during epizootics. Human

Venezuelan Equine Encephalitis Virus disease usually follows equine disease, but humans are not thought to be important in the maintenance of the

As the name suggests, Venezuelan equine encephalitis epidemic, due to low-titre viraemia. virus was ﬁrst isolated in Venezuela, where it causes

The major outbreak of VEE that occurred in important epizootics in horses, but also infects

Venezuela and Colombia in 1995 was remarkably humans. The geographical distribution includes Cen-

similar to an outbreak occurring in 1962–1964. Both tral and South America. VEE virus caused epidemics/

outbreaks followed unusually heavy rains, which led to epizootics among people and horses in Latin America

an increase in the mosquito vector populations, and from the 1920s to the 1970s. It reached the USA in

both occurred in the same geographic region. Phylo- 1971, but no further activity was reported until 1992–

genetic analysis showed that isolates from the 1995 1993, when a small outbreak in Venezuela conﬁrmed

epidemic of VEE were closely related to the serotype the re-emergence of VEE from its epizootic habitat to

IC viruses isolated during the 1962–1964 epidemic. As humans. This was followed by a major outbreak

a similar virus was identiﬁed in mosquitoes in occurring in 1995, involving an estimated 75 000–

Venezuela in 1983, the authors raise the possibility of 100 000 people (Rico-Hesse et al., 1995; Weaver et al.,

a serotype IC enzootic transmission cycle in northern 1996). A variant of the virus, known as Everglades

Venezuela (Weaver et al., 1996). Table 16.6 Geographical distribution of the Venezuelan equine encephalitis virus complex

Virus species

Main geographic distribution Venezuelan equine encephalitis (VEE)

Subtype

Variant

I 1 A–B, C, D, E, F

Venezuela, Colombia, South and Central America

II Everglades

Florida, USA

IIIA

Mucambo

Brazil, Trinidad, Surinam

IIIB

Tonte

South America

Bijou Bridge

North America

IV Pixuna

Brazil

V Cabassou

South America

VI AG80-663

Africa

525 Clinical Disease

ALPHAVIRUSES

species, there is a clear diﬀerence in the pathogenicity of the epizootic and the enzootic strains of VEE.

In horses, enzootic strains of VEE virus usually Epizootic strains are generally more virulent, with produce a fever and mild leukopenia. In contrast to

infection in horses resulting in viral replication in the this, horses infected with epizootic strains exhibit high

lymphoid tissue and bone marrow. This is associated fevers, severe leukopenia and signs of encephalitis.

with lymphoid necrosis and a lymphopenia and is Infections in humans result in a range of symptoms.

accompanied by a high-titre viraemia. Spread to the Natural human epidemics in Colombia in 1952 and

central nervous system probably occurs in the blood- 1995 and in Venezuela and Panama during 1962–1964,

stream, resulting in a fatal encephalitis in horses, as resulted in over 100 000 cases with 500 deaths,

well as rodents and some primates (Peters and demonstrating the highly infectious nature and severity

Dalrymple, 1990).

of the VEE virus. These may be subclinical (usually caused by enzootic strains) or result in signiﬁcant disease caused by

Diagnosis

epizootic strains of VEE (Table 16.3). Symptoms and signs in patients seeking medical care include fever,

The diagnosis of VEE-associated disease should be chills, severe headache, myalgia, vomiting and diar-

suspected in patients presenting with a ‘ﬂu-like’ illness rhoea. Most patients will have an acute and self-

in an appropriate geographical region when there is a limiting febrile illness; however, convulsions, disorien-

concurrent equine epizootic. Material obtained from tation and drowsiness may also be present (Weaver et

sick horses conﬁrming an epizootic of VEE may al ., 1996). The proportion of cases that develop

therefore be an important indicator of human disease. neurological sequelae appears to vary from outbreak

Isolation of virus may be attempted from acute-phase to outbreak. A review of the medical records of

serum and throat swabs, as well as from brain tissue patients with convulsions in the 1995 epidemic showed

obtained from aborted and stillborn fetuses (Weaver et that, although the age varied greatly (2 months–

al ., 1996). Culture of VEE virus has been achieved by

48 years), most were children; however, only approxi- inoculation into Vero or mosquito cells or suckling mately 20% of these were under 3 years of age. Of

mice (Dietz et al., 1979). Intracerebral inoculation of interest is that about half of these patients had their

suckling mice may result in death within 72 h. Isolates ﬁrst seizure on days 6–10 after the onset of their illness

identiﬁed in cell culture may be characterised further and when their temperature had returned to normal

using VEE serotype-speciﬁc polyclonal sera. Alterna- (Weaver et al., 1996). A further prospective study,

tively, detection and genetic characterisation may be which included a total of 13 patients, showed that six

done using RT-PCR with sequencing of the products patients had a severe incapacitating febrile illness

generated from the E3 and E4 genes (Weaver et al., lasting 3–5 days, two others had a ‘ﬂu-like’ illness,

1996; Brightwell et al., 1998). and the remaining ﬁve (38%) were asymptomatic

A number of serological assays (IFA, ELISA, HI) (Martin et al., 1972). The overall mortality is thought

have been described. Acute and convalescent serum to be less than 1%. Fetal loss may occur in pregnant

samples may be tested in parallel by HI. This may women with VEE.

provide diagnostically useful information. More Transmission can occur by the respiratory route as

recently attention has been focused on the use of an well as by mosquitoes. Accidental laboratory aerosol

IgG ELISA based on an antigen prepared from the infection with epizootic strains of VEE has been

attenuated VEE vaccine strain of virus. In experimen- reported to cause a febrile illness with abrupt onset

tally infected guinea-pigs, VEE-speciﬁc IgG could be of chills, headache, myalgia, vomiting and pharyngitis

detected at 6 days post-inoculation, compared with 2–5 days after exposure, without evidence of encepha-

10 days for HI antibodies. However RRV, EEE and litis (Erenkranz and Ventura, 1974).

WEE virus-speciﬁc IgG exhibit a weak cross-reaction on the IgG ELISA, so results must be interpreted with caution. The appearance of IgG antibodies detectable by ELISA coincides with the development of antibodies

Pathogenesis and Pathology detectable by plaque reduction neutralisation assay (PRN). While less sensitive than the IgG ELISA, PRN

The disease caused by VEE virus in humans is is more speciﬁc and therefore eliminates some of the relatively mild compared with that caused by either

problems of cross-reaction discussed above. It may WEE or EEE viruses. In horses, as well as other

therefore be used as a conﬁrmatory test. VEE-speciﬁc

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

IgM antibody has been detected as early as 4 days post- rash and limb oedema). Although Getah virus has inoculation in experimentally infected guinea-pigs (at a

not been implicated in human disease, serological time at which infectious virus was present in the serum).

studies have demonstrated antibodies to Getah In summary, suspected cases of VEE virus infection

virus in sera taken from individuals from the may be investigated serologically using a VEE-speciﬁc

Paciﬁc Basin, Japan and Hanna Island in China IgM and IgG ELISA, together with the more speciﬁc

(Johnson and Peters, 1996). PRN as a conﬁrmatory test (Coates et al., 1992).

. Una virus was ﬁrst isolated from Psorophora ferox mosquitoes originating from the Amazonian region of Brazil in 1959. The virus has also been isolated

Prevention and Control in Trinidad and is considered to be part of the SFV complex of viruses. As yet, this virus has not been

Personal protection against mosquito bites, together recognised as causing human disease. with vector control, as for other alphavirus infections, is important. Control of an equine epizootic by immunising horses is also important in the prevention

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17 Flaviviruses

Barry D. Schoub and Nigel K. Blackburn

National Institute for Communicable Diseases, Sandringham, South Africa

INTRODUCTION by alternate infection of a variety of mammals or birds, or occasionally other vertebrate hosts, and their

The Flaviviridae are a family of viruses comprising over respective mosquito or tick vectors. Man is infected

70 members and are responsible for a major portion of as an accidental event when he intrudes into this disease and death in man and animals. The family is

natural ecosystem. In these situations he is a dead-end subdivided into three genera—Flavivirus, Pestivirus

host of no biological signiﬁcance to the life-cycle of the and Hepatitis C virus.

virus. The non-arboviruses that are members of the genus Flavivirus may be found in either arthropods or in vertebrates, but not in both, and are of little medical

Flavivirus

importance.

Whether ﬂaviviruses have a narrow or wide host The genus Flavivirus was formerly termed ‘group B

range, their distribution is dependent on the ecology of arboviruses’ and prior to 1984 was placed together with

their speciﬁc vertebrate and invertebrate hosts. Some the ‘group A arboviruses’ within the family Togaviridae.

ﬂaviviruses have very restricted geographic distribu- However, as knowledge of the ﬂaviviruses increased, it

tions, such as Kyasanur Forest virus, found to date only became apparent that their features and properties, e.g.

in Karnataka (previously Mysore) State in India, their replication strategies, structure and biochemistry,

Omsk haemorrhagic fever virus , found in the Omsk were suﬃciently distinctive that they would need to be

district of western Siberia, or Rocio virus in the placed in a separate family, the Flaviviridae, with the

Santista lowlands and the Ribeira valley of Sao two additional genera, Pestivirus and Hepatitis C virus,

Paulo state in south-east Brazil. By contrast, ﬂavi- being added subsequently. More recently, GBV-C, also

viruses such as West Nile virus are distributed widely referred to as hepatitis G virus, has been tentatively

through Africa, Asia, large parts of Europe and, more added to the family Flaviviridae, although its taxonomic

recently, the USA and Canada. Japanese encephalitis designation remains to be deﬁned.

virus is found in at least 16 countries in a wide belt The majority of the genus Flavivirus are arboviruses,

stretching from eastern to south-eastern to southern which are transmitted between a vertebrate host and

Asia, aﬀecting a combined population in excess of two an invertebrate (mosquito or tick) host. Replication

billion. Dengue virus has moved over the past 20 years and ampliﬁcation of virus occurs in both the vertebrate

from south-east Asia into the Paciﬁc, the Americas, and invertebrate hosts. With some of the ﬂaviviruses

Africa and Australia, progressively establishing itself man may be the major vertebrate host, e.g. in the

in local populations of the widespread man-biting viruses causing dengue and urban yellow fever, and the

mosquito Aedes aegypti.

virus is maintained by being alternately transmitted The clinical manifestations of the majority of from man to mosquito to man. In most of the

flavivirus infections are relatively non-specific. In flavivirus infections, the virus is maintained in nature

endemic areas, the diagnosis of infection is often

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

made on clinical suspicion, especially if the physician is characteristics of the virus have been determined and alerted by epidemic activity in the region. Occasionally

have been shown to be characteristic of the family outbreaks of ﬂavivirus infection may follow climatic

Flaviviridae . Hepatitis C virus is not an arbovirus and is events, e.g. heavy rains after a period of drought,

not known to infect any non-human host in nature. which result in the formation of numerous stagnant

Experimentally, the only animal able to be infected is pools, facilitating the breeding of mosquito vectors

the chimpanzee. Infection is transmitted by blood and also attracting bird life. Outbreaks may follow

transfusion, penetrating injuries with blood-contami- uncontrolled urbanisation and the breakdown of

nated needles and instruments, sexually, and from vector control measures, as has occurred in recent

mother to child during birth. Since the implementation yellow fever outbreaks in West Africa. In some

of widespread screening for hepatitis B virus in blood, situations human cases may follow an extensive

hepatitis C virus has become the major cause of post- zoonosis in the vertebrate host, especially where birds

transfusion hepatitis. Current screening programmes or livestock are involved. Surveillance and epidemio-

for hepatitis C have, in turn, reduced signiﬁcantly the logical monitoring, which involve, amongst other

incidence of post-transfusion hepatitis due to this things, the continual and sustained sampling of

virus.

arthropod vectors and vertebrate hosts for arbovirus The most recent member of the family Flaviviridae is activity, is of critical importance in the prevention and

GBV-C virus. The virus was ﬁrst detected in 1995 in management of outbreaks of ﬂavivirus infections.

laboratory tamarins experimentally inoculated with Recently calls have been made for mobile units to be

blood from a surgeon with clinical hepatitis. Three established and sent into the ﬁeld to conduct clinical

distinct agents were deﬁned and called GBV-A, GBV-B diagnosis and therapeutic research, as well as for

and GBV-C, although it appeared that only GBV-C epidemiological surveillance in endemic areas.

was a human virus (GBV-A and GBV-B were The diagnosis of infection due to ﬂaviviruses

probably tamarin agents). Hepatitis G virus (HGV) becomes particularly diﬃcult in individuals who have

was described a year later by a diﬀerent laboratory, but travelled from an endemic area and present themselves

subsequent analysis of HGV and GBV-C has suggested for medical management many thousands of miles

that they are identical viruses. Both are, however, away from their source of infection. In the modern era

distinct from HCV, having only 29% amino acid of jet travel, a history of travel and also a knowledge of

homology with it. The pathological role, if any, of the prevalent infections in diﬀerent parts of the world,

GBV-C or HGV has not yet been established. are now an important component of infectious diseases

Hepatitis C and GBV-C viruses are dealt with fully medicine. In addition, travellers should be educated to

in Chapters 2 and 3.

alert their attending physician should they become ill on returning home.

PROPERTIES OF THE VIRUS Pestivirus The type species of the genus Flavivirus, yellow fever The second genus in the family Flaviviridae, Pestivirus,

virus, has been the most extensively studied member of is also referred to as the mucosal disease virus group.

the family.

The viruses of this genus are the causes of important veterinary diseases worldwide, such as bovine viral diarrhoea, border disease of sheep and hot cholera. They are not known to be spread by arthropod

Morphology and Morphogenesis vectors, transmission taking place by direct contact and also via faeces, urine and nasal secretions, as well

The virus particles are spherical, with a diameter of as vertically. The pestiviruses are not able to infect

some 40–50 nm. The envelope is tightly applied to a humans, therefore will not be discussed further.

25–30 nm spherical core and surface peplomers are often visible. Replication of virus takes place in the cytoplasm of the cell in association with the rough and

Hepatitis C Virus smooth endoplasmic reticulum. Accumulations of viral particles are seen within the lamellae and vesicles and

The third and newest genus of the family Flaviviridae, replication is characteristically associated with the is Hepatitis C virus. The biochemical and biophysical

proliferation of intracellular membranes.

533 Biophysical and Biochemical Properties

FLAVIVIRUSES

The lipid content of the ﬂavivirus envelope, which comprises some 17% of the viral weight, is derived

The S 20 W of ﬂaviviruses is 70–210, that of pestiviruses from the host cell membrane. The E and M proteins is 140 and that of hepatitis C virus is 5150. The

are inserted into the envelope. The glycoprotein E is buoyant density of the ﬂaviviruses in CsCl is 1.22–

rich in mannose and complex glycans.

1.24 g/cm 3 and 1.15–1.20 g/cm 3 in sucrose.

ANTIGENIC PROPERTIES Flaviviruses

The antigenic properties of the genus Flavivirus are deﬁned by serological tests, such as the highly cross-

The nucleic acid of ﬂaviviruses consists of a single reactive pH-dependent haemagglutination-inhibition molecule of positive-sense ssRNA. A single open

(HI) test, the less cross-reactive complement ﬁxation reading frame (ORF) on the genomic RNA is

(CF) test, and the much more speciﬁc neutralisation translated directly into a polyprotein, which is further

(NT) or plaque reduction neutralisation (PRNT) test. processed into the three structural proteins. In order

Other techniques mainly used for diagnostic serology from the N terminal, these are the internal RNA

are IgG and IgM detection by enzyme-linked immuno- associated C protein and then the two envelope

sorbent assay (ELISA), and immunoﬂuorescent (IF) proteins, pre-M and E. The pre-M protein is a

assays, for antigen detection the polymerase chain glycosylated precursor protein which is cleaved during

reaction (PCR) or related molecular techniques are or shortly after release from the cell into the non-

used. The antigenic features of the virus are char- glycosylated M membrane protein with no disulphide

acterised by reactivity with antigenic domains and bridges (molecular weight 7–9 kDa). The E membrane

epitopes on the membrane E protein of the virus. protein is usually glycosylated and is considerably

The genus Flavivirus is subdivided further into larger, with a molecular weight of 51–59 kDa and

subgroups on the basis of cross-neutralisation tests, possessing six disulphide bridges formed by 12 con-

using a polyclonal hyperimmune mouse ascitic ﬂuid served cysteine residues. The core protein C is rich in

prepared against each member virus. The members of arginine and lysine, with a molecular weight of 14–

each subgroup give signiﬁcant cross-neutralisation

16 kDa. results against each other, whereas the ‘unassigned’ Following the translation of the three structural

subgroup contains a miscellany of ﬂaviviruses which proteins, seven non-structural proteins are produced—

will display only limited cross-reactivity with each the glycoproteins NS1, NS2A, NS2B, NS3, NS4A,

other or with at least one other virus in any of the NS4B and NS5. Two of these proteins, NS3 and NS5,

established subgroups. The antigenic classiﬁcation of are probably components of the RNA replicase.

members of the genus Flavivirus of medical importance The gene order is thus 5’-C–pre-M–E–NS1–NS2A–

together with their respective vectors, disease manifes- NS2B–NS3–NS4A–NS4B–NS5-3’ (Figure 17.1).

tations, geographic distribution, vaccine availability

Figure 17.1 Schematic representation of the organisation of the gene of ﬂavivirus

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

and frequency of reported cases, is given in Table 17.1. monkeys. The Asibi strain was later to be the parent The ﬂaviviruses which either cause no human disease

strain of the 17D vaccine, developed by Nobel laureate or have only rarely been reported in association with

Max Theiler at the Rockefeller Foundation in New illness will not be dealt with further here.

York in 1937. The 17D vaccine strain was developed by serial passage through mouse embryo culture to chick embryo culture and then through minced chick

YELLOW FEVER embryo devoid of nervous tissue. The ﬁnal vaccine virus, propagated through fertilised chicken eggs, is remarkably attenuated yet immunogenic, and the

The yellow fever virus is the type species of the genus vaccine has proved to be very safe and eﬀective, Flavivirus and also the source of its name—ﬂavus is probably producing lifelong immunity after a single Latin for yellow. Yellow fever has been one of the

classical diseases of antiquity, instilling dread and

injection.

terror in the Americas, Europe and Africa from the seventeenth to the twentieth century. The disease has been romanticised in the classic works of Samuel Taylor Coleridge’s Rime of the Ancient Mariner and

Epidemiology Wagner’s opera The Flying Dutchman. At the turn of

the century, the disease almost put paid to the Yellow fever occurs today in the tropics on both sides construction of the Panama Canal, and in West Africa

of the Atlantic. The endemic zone stretches between yellow fever more than any other disease was

the latitudes of 108N and 408S in the Americas and responsible for the appellation ‘the white man’s

168N to 108S in Africa.

grave’. As late as 1988, an estimated 44 000 cases In the Americas, eﬀective control of Aedes aegypti with 25 000 deaths were estimated to have occurred in

has resulted in the virtual disappearance of urban Nigeria in the 3 years 1986–1988.

yellow fever from the western hemisphere. The last such epidemic of urban yellow fever occurred in Trinidad in 1954. However, jungle fever remains endemic in Bolivia, Brazil, Colombia, Ecuador, Peru,

History Panama, Venezuela and the Guyanas. The annual incidence by notiﬁcation is 100 cases, although this is

The disease entity of yellow fever was ﬁrst described in probably a substantially underestimated number. 1667 in Barbados, and in the following two centuries

Remarkably, the infection has not extended to the devastating epidemics raged on the continents of

heavy concentrations of Aedes aegypti which have built America and Africa and also, to some extent, in

up in urban centres.

Europe. The panic and chaos was matched by the In Africa, recent epidemics of yellow fever have been extravagance of the speculations as to the cause. The

far more extensive than those in the Americas. The confusion was further aggravated by the diﬃculty in

features of jungle yellow fever diﬀer in East and West distinguishing it from the other tropical plagues of

Africa. In East Africa, endemic yellow fever activity is malaria and dengue. In 1848, Nott proposed that

relatively quiet with few notiﬁed cases. However, two yellow fever was transmitted by the bite of a mosquito,

vast epidemics took place in 1940 in the Nuba

a suggestion supported by Beauperthuy in 1854 and mountains of Sudan, causing 40 000 infections with Carlos Finlay in 1881. In 1900 the US Army Yellow

over 15 000 clinical cases and 1500 deaths. The second Fever Commission, under Major Walter Reed, demon-

epidemic in 1960–1962, the largest epidemic of yellow strated in historical experiments on human volunteers

fever ever recorded, took place in south-west Ethiopia, that the infection was indeed transmitted by mosqui-

causing 30 000 deaths in 100 000 clinical cases in a rural toes. The following year, Walter Reed demonstrated

population of some 1 million. In contrast to this, in that yellow fever was due to a ﬁlterable agent, the ﬁrst

West Africa, frequent outbreaks have occurred, human disease shown to be due to a ﬁlterable agent.

usually on a considerably smaller scale than the two The discovery of the aetiological agent of yellow fever,

East African epidemics, with the exception of the however, had to wait a further 26 years when workers

Nigerian epidemic of 1986–1988, when an estimated of the Rockefeller Foundation’s West Africa Yellow

44 000 cases and 25 000 deaths may have occurred Fever Commission demonstrated the transmission of

(WHO, 1991). Over the last two decades signiﬁcant infection from a Ghanaian patient called Asibi to a

epidemics have been reported from several West rhesus monkey, and the subsequent passaging between

African countries, both preceding the Nigerian

535 Table 17.1 Antigenic classiﬁcation of ﬂaviviruses of medical importance and their clinical and epidemiological features in man

FLAVIVIRUSES

Subgroup Virus

Vaccine Frequency of

manifestations

distribution

reported cases

Unassigned Yellow Fever

Fever, HF, jaundice,

Tropical Africa

Yes Numerous

etc.

and South America

No Uncommon

Brazil

Bussuquara*

No Rare Ilheus*

Fever, arthralgia

South America

No Rare Sepik*

Fever, encephalitis

South America

No Rare Spondweni*

Fever

New Guinea

No Rare Wesselsbron

Fever

Africa

Africa, Thailand No Uncommon Zika*

Fever

Africa, Malaysia No Rare Dengue

Fever, rash

Dengue types 1–4

Fever, rash, myalgia, Asia, Paciﬁc,

No Numerous

HF Americas, Africa

Japanese encephalitis Japanese

Yes Numerous (mosquito-borne

Encephalitis

East Asia,

encephalitis

Australasia

encephalitis) St Louis

North America, No Numerous encephalitis

Encephalitis

Jamaica, Haiti, South America

West Nile

Fever, rash,

Africa, Asia,

No Numerous

encephalitis

Europe, USA, Canada

Murray Valley

No Uncommon encephalitis

Encephalitis

Australia, New

Guinea

Kokobera*

No Rare Kunjin*

Arthritis

Australia

Fever, encephalitis

Australia,

No Rare

Borneo, Indonesia, Malaysia

No Rare Tick-borne encephalitis TBE—European

Usutu*

Fever, rash

Africa, Austria

Yes Numerous subtype (CEE)

Encephalitis

Central and

Western Europe

TBE—far eastern

Developmental Numerous subtype (RSSE) Omsk HF

Encephalitis

Asiatic Russia

Western Siberia CEE? Numerous Kyasanur forest

Fever, HF

Kanataka State, Yes Uncommon disease

Fever, encephalitis

India

No Uncommon Louping Ill*

Powassan

Encephalitis

North America

No Rare Negishi*

Encephalitis

No Rare Kumlinge*

Encephalitis

Japan, Russia

No Rare Rio Bravo

Encephalitis

Australasia

No Rare Dakar Bat*

Rio Bravo*

Fever

USA, Mexico

No Rare Uganda S

Fever

Africa

Uganda S*

Africa, ?Far

No Rare

East

No Rare *These viruses are of very little importance to clinical medicine and will not be discussed further.

**Numerous, 41000 cases reported, usually many thousands to millions; uncommon, 10–1000 cases reported; rare, 510 cases reported. M, mosquito; T, tick; HF, haemorrhagic fever; TBE, tick-borne encephalitis; CEE, Central European encephalitis; RSSE, Russian spring-summer encephalitis.

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

epidemic, in the Ivory Coast (1982) and Burkina Faso

3. The urban yellow fever cycle is maintained by Aedes (1983), as well as after it, Mali (1987), Angola (1988),

aegypti and was the dominant form of yellow fever Cameroon (1990) and Niger (1990). Yellow fever has

before extensive mosquito control eliminated the expanded into Gabon, Liberia and Kenya, which

disease from South American towns. Periodic reported their ﬁrst cases since 1950 between 1992 and

reinfestations with Aedes aegypti have been 1995 (WHO, 1996). Smaller, more recent outbreaks,

observed in many South American countries and

e.g. in Guinea in 2000–2001, highlighted problems in have revived fears of a resurgence of urban yellow the availability of yellow fever vaccine stocks for

fever. In West Africa, Aedes aegypti transmitted satisfactory outbreak response (Nathan et al., 2001).

yellow fever is still responsible for outbreaks in The transmission cycles of yellow fever and the

towns and rural villages.

ecological interrelationships of its vectors and reservoir hosts are complex. Essentially, three cycles of transmission are recognised:

Clinical Features

1. The enzootic forest cycle represents the predominant maintenance of infection in its major

The clinical presentation of yellow fever follows the vertebrate host, the monkey. In South America,

general pattern of arbovirus disease—a short incuba- Alouatta , Cebus and Ateles monkeys are the major

tion period of 3–6 days followed by an acute biphasic primate reservoir hosts and they are infected by tree

illness. It is the severity and extent of the second phase hole-breeding Haemagogus mosquitoes in the forest

of the acute illness which has imparted to this infection canopy. The monkey is, however, only a transient

its classical awesomeness.

host because of the short-lived viraemia. The major The initial phase of illness is characterised by a ampliﬁcation host is the mosquito, which remains

viraemia which renders the patient infectious to biting infected for life and is also able to pass infection on

mosquitoes and is also responsible for the acute transovarially. Occasionally a single or a few

constitutional symptoms. These symptoms last about human cases may occur when man ventures into

3 days and are generally those of a non-speciﬁc febrile the forests. The cycle in Africa is similar and

illness: headache, malaise, nausea, lassitude and wide- involves Cercopithecus and Colobus monkeys, with

spread muscle pain, especially in the back. The the Aedes africanus mosquito as the principal

diﬀerential diagnosis is wide and includes malaria, vector.

other arboviral infections including dengue, typhoid,

2. The jungle yellow fever cycle represents the most rickettsial infections, inﬂuenza, enterovirus infections, important epidemiological form of yellow fever

acute HIV, etc. With more intensive and careful with respect to human infection. Epizootic upsurges

physical examination of patients, signs and symptoms of yellow fever are frequent, both on the fringes of

more suggestive of yellow fever may be revealed, such the rain forests and in the surrounding riverine

as ﬂushing of the head and neck, conjunctival gallery forests. Human infection occurs when forest

injection, strawberry tongue and a relative bradycar- mosquitoes invade adjacent plantations, clearings

dia. Probably the majority of clinically manifest yellow and villages. Once infection has been introduced

fever infections are aborted at this phase of the illness, into the human host, man-to-man transmission

accounting for the underestimation of the true sustains the epidemics, resulting in the dramatic

numbers of cases by up to 500-fold (WHO, 1991). outbreaks reported in recent years. In South

In cases of severe yellow fever, the early acute illness America, Haemagogus mosquitoes and possibly

is followed by a brief period of remission before the other mosquito species may establish epidemics in

onset of the haemorrhagic, hepatic and renal disease. man. In East and Central Africa east of Cameroon,

The latter is heralded in by a return of fever, vomiting, Aedes simpsoni will readily bite monkeys and man.

abdominal pain, dehydration and prostration. The In the East African epidemics in Sudan and

onset of the haemorrhagic diathesis is usually marked Ethiopia, the anthropophilic Aedes aegypti was

by coﬀee ground haematemesis, the classic ‘black the most frequently responsible mosquito, as well as

vomit’, and bleeding from puncture sites where

a number of ‘wild’ mosquitoes. In West Africa and injections or drip needles have been inserted. This is Central Africa west of Cameroon, Aedes simpsoni

accompanied by jaundice, albuminuria and oliguria. does not bite man and Aedes furcifer is the major

Deepening jaundice, massive haematemesis or hae- vector. In the Nigerian epidemic the major vector

moptysis or intra-abdominal bleeding, progressive was Aedes africanus.

renal failure, hypotension and shock may occur, renal failure, hypotension and shock may occur,

Patients who survive generally recover completely, although a chronic phase of illness lasting weeks or sometimes even months may occur in some individuals. This is characterised by prolonged jaundice and disturbances of liver function as well as prolonged renal failure. Occasionally sudden death may occur in the chronic phase as the result of myocardial damage or cardiac arrythmias.

A number of host factors may affect the clinical expression and severity of yellow fever virus infection. Age has played a significant role in South American epidemics, the majority of infections occurring in young adults, especially between the ages of 20–25. Age distribution has not, however, been a significant characteristic of African epidemics. In some epidemics gender has played a significant role in the distribution of cases, e.g. in the 1972–1973 Brazil epidemic there was a marked predominance of males affected: a 9:1 ratio, whereas in others there was only a slight male preponderance (e.g. 53% males in the 1986 Nigerian epidemic). More controversial has been the purported association of race and susceptibility to yellow fever, especially in the classical literature, which frequently made reference to the mildness of the disease in the indigenous inhabitants of the African jungle. There is, however, no evidence confirming any difference in susceptibility between races.

There are no known viral factors aﬀecting either transmissibility or virulence. Although genomic heterogeneity has been demonstrated by ﬁngerprinting and sequencing studies and also some antigenic heterogeneity has been observed between isolates, there has been no demonstrable clinical or

epidemiological diﬀerences between American and African isolates.

Diagnosis

The development of reliable rapid tests for the urgent diagnosis of yellow fever has become a major public health priority in the management of the viral haemorrhagic fevers. The IgM antibody capture ELISA has become the test of choice for rapid serological diagnosis of yellow fever virus infection. Cross-reactions may occur but IgM antibody levels will normally be higher against yellow fever. Immuno- fluorescence tests using infected cells spotted onto microscope slides and then acetone-fixed can be used for detecting IgG and IgM in patients’ sera. In the IF test, however, the rheumatoid factor may be a much greater problem than in the IgM antibody capture ELISA, and pre-treatment to remove IgG prior to IgM testing is necessary. The classical techniques, such as haemagglutination-inhibition (HI), complement fixation (CF) and neutralisation tests (NT), still have a place in yellow fever surveillance and diagnosis but a four-fold or greater rise in titre would have to be demonstrated before a definitive diagnosis could be made. Specific diagnosis still may be difficult, particularly if the patient has had previous experience of flavivirus infection. In these instances higher titres may appear against heterologous viruses, in keeping with the doctrine of original antigenic sin.

Isolation of virus may be performed in specialised reference laboratories, either by intracerebral or intraperitoneal inoculation of suckling mice or by the intrathoracic inoculation of male Aedes aegypti or Toxorhynchites mosquitoes. These techniques are particularly sensitive and are essential for epidemiological monitoring and research, but unfortunately take up to 3 weeks to provide an answer. More rapid viral isolation techniques using mosquito cell lines, such as the lines from Aedes albopictus (C6-36) and Aedes pseudoscutellaris (MOS 61), which are sensitive to infection and, combined with the IF test using monoclonal antibodies, may give a diagnosis within 3–

4 days. It is recommended that early antibody is dissociated from viral antigen using dithiothreitol prior to isolation attempts in mosquito cell cultures. An antigen capture ELISA technique is available which is slightly less sensitive than virus isolation but is able to produce a speciﬁc result with the use of monoclonal antibodies in less than 24 h. A sensitive method for detecting and quantifying viral RNA has been

FLAVIVIRUSES

537

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

developed using real-time reverse transcription PCR Side-eﬀects to immunisation occur in less than 5% (RT-PCR).

of recipients and are generally mild, low-grade fever, Liver biopsy is contraindicated in acute yellow fever.

myalgia and headache. Occasionally, hypersensitivity However, liver tissue from post mortems may provide

reactions have been reported, especially in individuals useful histopathological information. The classical

allergic to egg protein. The most serious side-reaction, liver pathology is that of a coagulative mid-zonal

encephalitis, has been reported in 18 cases out of more necrosis. The inclusion bodies which have been held to

than 35 million doses of vaccine which have been

be pathognomonic of yellow fever are those in the administered to date. Only two cases have occurred in cytoplasm due to eosinophilic degeneration (Council-

children over the age of 7 months, although the only man bodies) and intranuclear eosinophilic inclusion

fatality was in a 3 year-old child. Regarding the latter bodies (Torres bodies). Many of these features are,

case, it was postulated recently that a change identiﬁed however, also found in fatal cases of viral haemor-

in amino acid position 303 of the isolate P-16065 as rhagic fever due to other viruses, and the

compared to the parent vaccine virus 17D-204 USA histopathology is now no longer regarded as being

may have aﬀected the virulence of the vaccine virus diagnostic of yellow fever.

(Jennings et al., 1994).

The contraindications to immunisation are those determined by age, pregnancy, history of egg allergy and immunosuppression (Centers for Disease Control,

Control 1990). The vaccine should not be given to children under the age of 9 months unless travel to an endemic

The worldwide control of yellow fever has been area cannot be avoided but should, at any rate, never achieved by immunisation and eﬀective vector control,

be given to infants less than 4 months of age. The eﬀect which have largely eliminated the urban yellow fever

of immunisation in pregnancy has not been deter- cycle due to Aedes aegypti. However, in recent times,

mined; however, being a live attenuated vaccine, it poverty, war and competing health priorities have led

should not routinely be given to pregnant women. to a reduction in immunisation and surveillance

However, if travel to an area with ongoing yellow fever eﬀorts, resulting in a resurgence of disease, especially

cannot be avoided, the danger of infection would far in the endemic zone of Africa. In addition, reinfesta-

outweigh the theoretical risk to a pregnant mother and tion of towns and villages adjacent to forests with

her fetus. The vaccine should also be avoided in Aedes aegypti , aggravated by the massive uncontrolled

persons with a history of egg allergy or in immuno- urbanisation and population migrations to the towns

suppressed individuals, e.g. due to HIV infection, of developing countries, also exacerbated particularly

malignancy or immunosuppressive treatment. How- by severe drought, has renewed the spectre of urban

ever, as in the case of pregnancy, the relative risks of yellow fever in Africa.

travel to an endemic area vs. the slight or even It is over 50 years since the development of the ﬁrst

theoretical risk of the vaccine, would need to be yellow fever vaccine by Theiler, and the original

evaluated on an individual basis. method for the production of chick embryo-passaged

Immunisation policies with regard to yellow fever 17D vaccine has undergone little modiﬁcation over the

immunisation involve three aspects of the control of years. The French neurotropic vaccine, developed by

infection:

passage in mouse brain, is now no longer used because of the prohibitive danger of encephalitic complica-

1. International travellers going to yellow fever tions. The vaccine is a live attenuated puriﬁed product

endemic areas require prophylactic immunisation produced by growing vaccine virus in chick embryos

as a condition of entry to these countries, or exiting and is supplied as a lyophilised preparation, which

from endemic countries. There were two reports in should be stored frozen or at least kept at temperatures

1996–1997 of travellers to the Amazon region of of not more than 58C. After reconstitution it should be

Brazil who died from yellow fever after returning used immediately and any remnants discarded within

to their respective countries. Neither had been an hour. A single dose of 0.5 ml given subcutaneously

vaccinated.

provides excellent, long-lasting immunity to 99% of

2. Within endemic zones, two kinds of immunisation vaccinees. Although international health regulations

policy have been practised. These are (a) the so- demand booster doses every 10 years, neutralising

called ‘ﬁre-ﬁghting’ policy, where there is a response antibodies have been shown to persist for over 30 years

to an outbreak, or alternately (b) proactive routine and immunity is probably lifelong.

immunisation to prevent outbreaks occurring.

539 (a) ‘Fire-ﬁghting’ mass immunisation is usually put

FLAVIVIRUSES

resources to implement what is already known, but into operation in the early phases of an out-

also the elucidation of the remaining enigmas regarding break or in advance of an imminent epidemic, if

the infection.

effective surveillance is able to predict it. Although the ‘fire-fighting’ strategy may be cheaper than routine immunisation, it can often only be implemented after a considerable num-

OTHER MEMBERS OF THE ‘UNASSIGNED’ ber of individuals have already been infected and

SUBGROUP OF FLAVIVIRUSES the protective eﬀect will be further delayed by

another 7 days for antibodies to develop. Rocio (b) A far more eﬀective way of controlling yellow

Rocio virus is an arboviral cause of encephalitis fever is proactive routine and sustained admin- localised to the Ribeira valley and Santista lowlands istration of vaccine. In Africa, 33 countries have in the southern coastal region of Sao Paulo state in been identiﬁed by the World Health Organiza- south-eastern Brazil. The virus was ﬁrst isolated in tion as endemic high-risk regions where yellow 1975 from the brain of a fatal case of encephalitis fever vaccine should be incorporated into the diagnosed during an unusual epidemic of encephalitis Expanded Programme on Immunization (EPI)

in 1975–1976. No further cases have occurred since schedule. However, only one of these countries,

1980, although two children from the Ribeira valley Gambia, has exceeded the target vaccine cover-

tested positive for IgM antibodies in 1989. A total of age of 80% (87% in 1994). In other countries

821 cases were diagnosed during 1975–1978, twice as coverage has ranged from 1% in Nigeria to many in males and usually aged 15–30 years. The virus 55% in Mauritania (WHO, 1996). Yellow fever has been isolated from pools of mosquitoes, such as vaccine should preferably be given together Psorophora ferox , and also from wild birds. The with measles vaccine at 9 months of age and can transmission cycle of the virus has not, however,

be administered simultaneously with measles

been established.

vaccine with no reduction in efficacy of either Clinical and epidemiological features of the disease vaccine. It can also be given simultaneously have been reviewed in 821 cases between 1975 and with other viral vaccines and also BCG, with no 1978 (Iversson, 1980). The disease commences with increase in reactivity or decrease in efficacy non-specific signs of pyrexia, headache and vomiting. (however, yellow fever and cholera vaccines This may then be followed by disturbances of should preferably be separated by an interval of consciousness and signs of encephalitis, including

3 weeks). localising signs. Death may follow a prolonged coma or there may be a sudden fulminant course. Serious

The current worldwide production of yellow fever neurological sequelae occur in some 20% of clinical vaccine is estimated to be of the order of 15 million cases and the overall case fatality is found to be 10%. doses/year. There are concerted eﬀorts to improve on The IgM capture ELISA has been used to diagnose techniques for the production of yellow fever vaccine, Rocio virus infection in children and is preferable to the

e.g. by the use of tissue culture techniques and also the HI test for identifying recent infection. development of recombinant technology with reference

to yellow fever immunisation. Vector control strategies such as aerial spraying, domiciliary spraying and the enforcement of public

Wesselsbron health regulations to reduce collections of stagnant water and other breeding sites, have had successes in

Wesselsbron virus was ﬁrst isolated from a lamb in the the past in eliminating Aedes aegypti and controlling

village of Wesselsbron in South Africa in 1955. Virus urban yellow fever. Present-day socioeconomic diﬃ-

has been isolated from 23 cases of infection, 11 of them culties have, however, hampered recent eﬀorts to again

resulting from laboratory infection or infection of control the reinfestation of villages and towns in

laboratory ﬁeld workers. The vertebrate host is chieﬂy Africa. Control of vectors responsible for the jungle

livestock, especially sheep, and isolations have been yellow fever cycle is impractical.

made from throughout sub-Saharan Africa, especially There still remain many gaps in our knowledge of

South Africa. It has also been isolated from pools of the epidemiology of the infection and the permanent

wild-caught Aedes mosquitoes, such as Aedes (neo) control of the disease will probably need not only

lineatopennis . The major vector in sheep is Aedes

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

caballus-juppi . Man is infected by mosquito bite or by regarded as a relatively benign illness, aﬀecting direct contact in handling carcasses or tissues of

predominantly colonial expatriots living in tropical animals that have died of the disease.

countries and, although responsible for severe and Human infection is characterised by a sudden onset

often incapacitating muscle pain, it was seldom lethal. of pyrexia, severe headache and retro-orbital pain

However, the gravity of the dengue pandemic was associated with photophobia and hyperaesthesia of the

really conﬁrmed by the recognition in the 1950s of the skin, with an evanescent skin rash frequently present.

severe complications of infection, viz. DHF/DSS Muscle and joint pains are also commonly seen. In

aﬀecting mainly children in the endemic areas. Epi- severe cases signs of encephalitis, such as blurred vision

demics of DHF/DSS ravaged south-east Asia and in and some mental impairment, may occur. Patients

the following 30 years was responsible for over 700 000 recover after a few days to a week and no permanent

children being hospitalised and over 20 000 fatalities sequelae have been reported.

(Halstead, 1984).

Diagnosis of infection may be achieved by isolation In the western hemisphere, the ﬁrst major epidemic of the virus from blood or serological tests. The HI test

of DHF/DSS struck Cuba in 1981 and was responsible is only useful in individuals with no previous ﬂavivirus

for 24 000 cases of DHF and 10 000 of DSS (Gubler infection history, because of the extensive cross-

and Costa-Valez, 1991) (sporadic cases of suspected reaction between Wesselsbron and other ﬂaviviruses.

DHF had been reported from Central America since The capture IgM assay is the test of choice for

1968). Only the energetic response of the Cuban health diagnosis of recent Wesselsbron infection.

authorities, with intensive education and mass hospitalisation, kept the mortality down to only 158. Following on this epidemic, conﬁrmed or suspected

DENGUE cases of DHF have been reported in the Americas almost every year, with the most severe outbreaks of dengue fever occurring in Peru in 1990, involving over

Dengue is, at present, the most important arboviral

76 000 cases (Centers for Disease Control, 1991a). cause of death and disease in man (Gubler and Costa- Dengue returned to Cuba in 1997. A summary of the Valez, 1991). The infection has spread widely from present status of dengue worldwide is that there are 2.5 south-east Asia to the Americas, the Paciﬁc and billion persons at risk, more than 20 million cases/year Africa, now involving several million people annually. and 30 000 deaths. Imported cases of dengue in In all major tropical areas of the world the incidence of travellers returning to temperate countries are reported dengue fever (DF) and dengue haemorrhagic fever in the USA annually and DHF was reported in two (DHF)/dengue shock syndrome (DSS) has increased cases in the UK in 1991 (Jacobs et al., 1991). Blood dramatically over the past few years, with an ever- donations collected during a dengue outbreak in Hong increasing frequency and extent of epidemics and a Kong were thought to have resulted in two cases of greater severity of cases. The spectre of the introduc- dengue from infected transfusion blood. tion of infection to Aedes aegypti populations in

non-endemic countries is of great international concern.

Virus Properties and Host Range History The dengue viruses form a subgroup of the genus Outbreaks of illnesses clinically resembling dengue

Flavivirus and although extensive cross-reactivity is fever have been recorded since 1779 and 1780 in Java

seen with serological tests such as haemagglutination (Indonesia), Cairo and Philadelphia. Similar epidemics

inhibition (HI) which cannot reliably distinguish of dengue-like illnesses occurred at 10–30 year inter-

dengue from many other ﬂaviviruses, neutralisation vals throughout tropical and subtropical regions of the

tests (NT) are able to deﬁne dengue virus as a distinct world. Although the precise aetiology could not be

antigenic subgroup. There are four serotypes of dengue established and infections such as chikungunya are

based on neutralisation tests. However, with all clinically and epidemiologically very similar, the

serological tests there is extensive cross-reactivity majority of these epidemics were probably dengue.

between the four serotypes, although they are distin- The spread of the disease has been markedly acceler-

guishable with the high speciﬁc plaque reduction ated by the advent of widespread air travel over the

neutralisation test. Following natural infection, pro- last three decades. Dengue fever was generally

tective immunity is homotypic. Dengue serotypes 1, 3

541 and 4 show a closer antigenic and genetic relationship

FLAVIVIRUSES

also on domestic animals, while the purely domestic A. to each other than dengue 2. However, within each of

albopictus randomly feeds on man, domestic animals the serotypes, considerable heterogeneity and strain

and feral animals and birds. To establish infection, A. variation is demonstrable on nucleic acid sequence

aegypti would need to feed on individuals with high analysis and DNA/RNA hybridisation studies as well

levels of viraemia, and this may select for viral strains as by antigen signature analysis using monoclonal

of higher virulence, which are more likely to produce antibodies. The importance of these strain variations

the severe epidemics associated with A. aegypti. In with respect to the epidemiology and virulence of the

addition, the biting habits of A. aegypti, which virus remains to be determined. Molecular analysis of

characteristically takes interrupted blood meals and isolates from the South Paciﬁc suggested that the

will thus feed on a number of individuals before recent epidemics (1988–1989) were due to a new

becoming engorged, enhances its ability to spread the genotype, rather than a previously circulated DEN-1

virus. It is thus not unusual for a single mosquito to strain.

spread infection amongst several individuals at a single The only vertebrate hosts of dengue virus in nature

feeding. In both mosquitoes, biting activities are are man and several species of Asian and African

maximal soon after daybreak and in late afternoon, subhuman primates. Other vertebrates can be infected

and are related to human activities and movements. experimentally only with diﬃculty, including baby

Outbreaks associated with both vectors are climatically mice, which usually require several blind passages of

inﬂuenced by heavy rainfall and high temperature. patient material to obtain an isolate. The invertebrate

The recent upsurge of dengue in existing endemic hosts of dengue are members of the genus Aedes,

zones and the spread to new areas has been attributed especially the subgenus Stegomyia. The most impor-

to modern phenomena of human and social behaviour. tant mosquito hosts as far as human infection is

Infestations of A. aegypti in tropical towns and concerned are Aedes aegypti, A. albopictus and A.

villages, which were controlled by extensive spraying polynesiensis . Other Aedes species, including A. africa-

and other vector control measures in the 1950s and nus , A. leuteocephalus and the A. furcifer group, are

1960s, have now resurged due to uncontrolled urbani- involved in the maintenance of the forest cycle of

sation and the mass migration of rural populations dengue in Africa, whereas in Asia the mosquitoes

into informal housing settlements and squatter camps concerned belong to the A. niveus complex.

on the peripheries of cities and towns. These population movements have been accentuated by famine, poverty and war. Stagnant water pools and lack of reticulated water supplies have provided ample oppor-

EPIDEMIOLOGY tunities for mosquito breeding, coupled with the breakdown of vector control in many tropical coun-

Dengue displays many epidemiological similarities to tries. Overcrowding and grossly inadequate housing, yellow fever and chikungunya viruses and there is

which is so characteristic of the sprawling slums of the considerable overlap in the ecologies of these three

tropical world, greatly facilitate the spread of vector- virus infections. Essentially there are three transmis-

borne diseases and is especially conducive to dengue sion cycles of dengue: a forest cycle in primates and

transmission resulting from the interrupted feeding involving forest species of Aedes; a rural or semi-rural

habits of A. aegypti. The spread of infection has also cycle in humans, with the peri-domestic Aedes species

been enhanced by the modern era of jet travel, which being the vectors; and an urban cycle in humans

facilitates the transportation of infected individuals to involving domesticated Aedes species. By far the most

non-infected areas, creating the threat of the introduc- important of these three for both endemic and

tion of infection if infestations of A. aegypti are epidemic human dengue is the urban cycle. It is, in

suﬃciently high. Modern air travel has also been fact, doubtful whether the forest cycle plays any

responsible for increasing the number of imported signiﬁcant direct role in human dengue.

cases of dengue into countries where physicians are, in The two major mosquito vectors of urban dengue

the main, ignorant of the infection, resulting in are A. aegypti and A. albopictus. Although A.

perplexing diagnostic diﬃculties until a history of albopictus is considered more sensitive to oral infection

travel to endemic zones is elicited. More recently, the with dengue viruses, A. aegypti is a more important

inﬂuence of international trade in the spread of dengue vector for human disease and is especially responsible

has been observed. Motor vehicle tyres and casings for explosive epidemics. The anthropophilic A. aegypti

from south-east Asia containing remnant pools of usually preferentially feeds on man and occasionally

water where infected A. albopictus mosquito larvae water where infected A. albopictus mosquito larvae

Clinical Features

The majority of infections with dengue virus, based on the extent of population seropositivity, are asymptomatic. Clinical manifestations of dengue occur in two forms—classical dengue fever and DHF/DSS. Classi- cal dengue fever is an acute disease characterised by a sudden onset of fever, severe headache which is typically frontal in distribution, together with retro- orbital pain, nausea and vomiting. Severe muscle and bone pain and arthralgia are characteristic of dengue and usually more pronounced in the back, which led to it being termed ‘break bone fever’ by Dr Benjamin Rush in 1778. He also aptly described the associated severe depression as ‘break heart fever’. There is frequently a diﬀuse, discrete maculopapular rash which usually heralds the recovery phase of the illness. In spite of the severity of symptoms, which may be incapacitating, the disease is temporary and full recovery takes place.

The grave complication of DHF/DSS is governed by two factors, prior infection and age. Thus, DHF and DSS occurs in approximately 0.18% and 0.007%, respectively, of cases of primary dengue fever compared to 2.0% and 1.1%, respectively, of dengue fever due to secondary infection (Halstead, 1981). It is also rare in individuals over the age of 15 years. DHF/DSS resembles yellow fever in its biphasic presentation. The ﬁrst phase is not unlike uncomplicated dengue fever. This is followed by a brief remission of symptoms, when the fever subsides to normal or close to normal. There is then a sudden deterioration in the patient’s condition, marked by profound prostration, hypotension, circulatory collapse and manifestations of bleeding and shock. Bleeding is seen commonly as petechiae in the skin and mucous membranes, especially in the oral cavity, ecchymoses, bleeding at injection and skin puncture sites, gastrointestinal bleeding and haemorrhagic pneumonia.

The cause of the haemorrhagic diathesis in DHF is complex. There is evidence of vascular injury with increased permeability and extravasation of ﬂuid from the vascular into the interstitial ﬂuid compartment, producing hypotension and DSS. Bleeding due to vascular damage is suggested by the presence of petechiae and a positive tourniquet test. In addition there is a marked thrombocytopaenia, although it is

not yet clear what the relative contributions of impaired platelet formation due to direct suppression of megakaryocyte production in the bone marrow or excess destruction due to endothelial damage. Evi- dence exists for both. Thirdly, there is haematological evidence of a consumptive coagulopathy with a moderate increase in ﬁbrin degradation products which, however, rarely reaches the stage of disseminated intravascular coagulation.

The deﬁnition of DHF is controversial. The WHO criteria for DHF are an acute onset of fever, haemorrhagic manifestations, which include at least a positive tourniquet test, a thrombocytopaenia of 100 000/ml or less and a haemoconcentration with a haematocrit increase by 20% or more. Manifestations of haemorrhagic fever, however, do diﬀer from country to country and amendments to the criteria have been proposed to include, amongst others, references to the age of the patient (516 years) (Halstead, 1989). The severity of DHF/DSS has also been graded by the WHO from I to IV (Anonymous, 1980):

Grade I Fever with non-speciﬁc constitutional symptoms, the only haemorrhagic manifestations being a positive tourniquet test.

Grade II As for Grade I, but with speciﬁc haemorrhagic manifestations. Grade III Signs of circulatory failure or hypotension. Grade IV Profound shock with pulse and blood

pressure undetectable.

Pathogenesis of DHF/DSS The pathogenesis of DHF/DSS has been studied

intensively for a number of decades. Nevertheless, the pathways to the development of severe dengue have not, as yet, been deﬁnitively established. Essentially, there are two hypotheses involving immunological or virological mechanisms.

The immunological theory of DHF/DSS is based on the phenomenon of antibody-mediated enhancement of infection. Investigations of antibody-mediated enhancement in dengue have been the hallmark studies of this phenomenon, which have since been shown to

be important facets in the pathogenesis of a number of viral diseases, from rabies to HIV (Kurane et al., 1991). The major cell infected by dengue virus in vivo is the monocyte/macrophage (even though a number of cell lines from a variety of tissues may be infected in vitro ). Three elements partake in the process of

542

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

antibody-mediated enhancement: antibody, virus and the receptor for the Fc portion of IgG.

Evidence for the involvement of pre-existing antibodies comes from epidemiological observations that DHF/DSS is much more common in individuals with pre-existing antibodies. These pre-existing antibodies may be derived from passively acquired maternal antibodies in infants less than 1 year of age, or from previous infection in children over 1 year of age. This was particularly evident in the Cuban epidemic of 1981 when DHF/DSS due to type 2 virus was usually

observed in individuals infected some 4 years

previously with type 1 virus. Experimental work by Halstead and colleagues demonstrated enhancement of infection in experimental dengue in monkeys using human serum as well as sera from hyperimmunised animals. It was later demonstrated that only IgG antibody and not IgM, which has potent neutralising activity, was able to produce enhancement. Using monoclonal antibodies, enhancing epitopes on the E and pre-M proteins of dengue virus could be demonstrated, some of which could also be neutralising and others not (Kurane et al., 1991).

The receptor on the monocyte/macrophage cells which form the third component of the enhancing complex is thought to be the FcgRI molecule which binds the Fc portion of IgG with great avidity and also, but probably to a lesser extent, the FcgRII molecule. The eﬀect of the attachment of the virus– antibody complex to the FcgR receptor is both to enhance the binding of virus to its target cell and also to induce a signal to the cell facilitating internalisation of the complex. There remain, however, many unanswered questions with the antibody-mediated enhancement hypothesis. First, DHF/DSS may well occur in the absence of pre-existing antibodies, even though it is far more common for them to be there. It is also not clear whether binding of virus to its speciﬁc receptor is essential for infectivity. The mechanism by which enhanced infection of cells produces the components of a DHF/DSS syndrome is not known.

The alternative hypothesis of Rosen (1986) and colleagues holds that the severe complications of DHF/DSS are the direct results of properties of the virus, that is, the consequence of infection with unusually virulent strains of dengue circulating in a particular area and giving rise to outbreaks of DHF/ DSS. However, as mentioned above, although nucleic acid sequencing techniques and antigen signature analysis have demonstrated strain variations within serotypes, no consistent relationship between strain variation and either virulence or heightened infectivity has been reliably demonstrated. There has also been no

consistent association with serotype and DHF/DSS. Earlier observations, in both Thailand and the 1981 Cuban epidemic, suggested that dengue type 2 may be more frequently associated with DHF/DSS. Subse- quent observations have now shown that all four serotypes may be responsible.

Neither of these two hypotheses is able to elucidate why the development of DHF/DSS is so dependent on age or why DHF/DSS is more likely to follow when dengue infection occurs in some areas but not in others.

Diagnosis

The clinical diagnosis of dengue, both the uncomplicated dengue fever form and DHF/DSS, if often unreliable. Dengue fever may resemble clinically a variety of acute febrile illnesses, although the severe muscle and bone pain is suggestive of dengue. Similarly, DHF may resemble other causes of haemorrhagic fever, although thrombocytopaenia with haemoconcentration and signs of a moderate consumptive coagulopathy is suggestive of dengue.

The most widely used serological test is the HI test, detecting antibodies as early as 4 days post-onset. A specific diagnosis of dengue can be made early in primary infections, but cross-reactions with other flaviviruses occur in late primary or secondary infections. The IgM antibody capture ELISA (MAC ELISA) is being used during outbreaks of dengue, and there are now rapid assays available for the detection of dengue IgG and IgM, although cross- reaction may occur with the IgG assay. The IgM antibodies may persist for over 3 months, so the test is also useful for retrospective studies, but this persistence may cause diagnostic problems in areas where dengue is endemic. A combined IgG and IgM assay, detecting high levels of IgG indicating secondary infection, is useful in dengue endemic areas. Immuno- fluorescent assays have been used successfully to detect dengue IgG and IgM antibodies. The CF test is more specific than the HI test, but the antibodies detected by this assay appear later and disappear earlier. The NT and PRNT tests are the most specific and sensitive but are difficult to perform and thus tend to be used only for specific purposes.

Virus isolation, the only deﬁnitive way of being able to type isolates, is diﬃcult and if mice are used, a number of blind passages are usually required. Intracerebral inoculation of adult or larval Toxo- rhynchites spp. is a sensitive and rapid method for the isolation of dengue virus, giving results in 2–3 days.

FLAVIVIRUSES

543

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

The use of a rapid centrifugation method may increase carried out in Puerto Rico, where blood samples are sensitivity and reduce time scales. Intrathoracic inocu-

collected by collaborating physicians on a regular basis lation of mosquitoes is easier and just as sensitive, but

and sent for analysis. Interest and cooperation is also head squashes cannot be made or tested for speciﬁc

diﬃcult to sustain. The use of blood samples sent to dengue antigen for at least 7 days post-inoculation.

laboratories for testing for acute febrile illnesses or The most commonly used system of virus isolation is

‘viral syndromes’ may be an easier way of recruiting the inoculation of mosquito cell lines, viz. A. albopictus

specimens for serological testing. Surveillance may also (C6-36), A. pseudoscutellaris (AP-61) and Toxorhynch-

utilise the routine investigation of all viral haemor- ites ambionensis (TRA-248), which are almost as

rhagic fever cases, which should include tests for sensitive as mosquito inoculation, which allows speciﬁc

dengue. Vector surveillance may be of beneﬁt to results to be obtained within 2–3 days using ﬂuor-

demonstrate low infestations (house index of 51%) escent-labelled monoclonal antibodies. A combination

or to look out for the introduction of exotic mosquito of MAC ELISA and RT-PCR on peripheral blood

species, e.g. A. albopictus.

leukocytes has been shown to give high levels of Vector control aims at the elimination of the main sensitivity and speciﬁcity.

mosquito vector, Aedes aegypti. While this is technically feasible, with both adulticide campaigns using ultra- low-volume spraying with malathion and also larvicide treatment of stagnant water, the costs are high and the

Control eﬀects are temporary. In many tropical countries where A. aegypti eradication had been achieved,

There is, at present, no licensed dengue vaccine. reinfestations have taken place to levels equalling, or Current strategy is to develop a vaccine against all

even exceeding, those in pre-control times. However, four serotypes. Successful trials of monovalent vac-

long-term community-based programmes need to be cines have been reported, but, as yet, not for

implemented in endemic countries. In non-endemic experimental tetravalent vaccines. Because of the lack

countries there should be a vigilant monitoring for the of availability of a vaccine, control of dengue depends

possible importation of dengue, e.g. through motor car on (a) surveillance to obtain early warning of epi-

tyres, or travellers or migrants from endemic areas. demics or preferably to be able to predict impending epidemics, and (b) eﬀective vector control.

Epidemiological surveillance may be of two types: ﬁrst, proactive surveillance in interepidemic periods in

JAPANESE ENCEPHALITIS endemic areas or in countries which are not yet

infected but are vulnerable to the introduction of Japanese encephalitis virus (JE) is the major arboviral new infections because of high infestations of A.

cause of encephalitis worldwide. First described as a aegypti ; second, reactive surveillance, where monitor-

clinical entity in Japan in 1870, it is now thought to be ing is instituted once suspected or conﬁrmed cases of

responsible for about 50 000 cases annually, half of dengue have already occurred. This form of surveil-

whom are left with permanent neurological or psychological handicap, and in a further quarter it is

lance is very insensitive as numerous cases may occur rapidly lethal. Infection has been demonstrated in 16 which are not suspected to be dengue, before autho- countries in south-east Asia, stretching to India in the rities are alerted to the outbreak. In the Comoros west and to southern Russia in the north—a popula- Islands, dengue type 1 was diagnosed in 62/116 clinical tion of over 2 billion people. cases in 1993. However, an investigation to determine

the prevalence of dengue infection in over-5 year-olds, using a dengue IgM assay, estimated that at least

60 000 recent cases had occurred at that time. Viral Features and Host Range

A number of instruments of surveillance may be used for proactive monitoring. Disease surveillance

JE virus shows some cross-reactivity with St Louis requires intensive educational eﬀorts, especially at

encephalitis virus , West Nile virus, and Murray Valley peripheral primary health care level, to alert health

encephalitis virus , and together they form the mos- care workers as to the possibility of dengue. It is,

quito-borne encephalitis complex. The virus is however, very diﬃcult to sustain interest, especially if

antigenically relatively homogeneous and recovery no cases materialise. Viral and serological surveillance

from infection results in solid protection. Nucleic involves the active recruitment of specimens, e.g. as

acid analysis, however, has revealed some signiﬁcant acid analysis, however, has revealed some signiﬁcant

The vertebrate hosts of JE virus are man and his domestic animals and birds; no wild animals and birds are known to be infected to any signiﬁcant extent. The major mosquito vector of the virus is Culex tritaeniorhynchus , although a number of other species of the genera Culex, Monsonia, Aedes and Anopheles have yielded isolates of JE virus.

Epidemiology Nestling birds, particularly of the heron family, play

an important epidemiological role in the dissemination of JE virus, with a second cycle involving domestic animals, particularly the pig. In seroprevalence studies, high NT antibodies were found in several other animal species, e.g. cattle, horses, dogs, monkeys and bats. As with the wild bird population, the high turnover of the domestic pig population resulting in a continuous supply of susceptible animals is a contributing factor to the pig being a major amplifying host for JE virus. JE rarely causes disease in domestic animals, although fatal encephalitis in horses and abortions in sows have been recorded. Studies on birds have implicated other species, whose main habitat is the rice paddies, as vertebrate hosts of JE virus—these water birds include water herons and bitterns. In India ardeid birds such as cattle egrets are implicated.

JE virus has been isolated from many species of mosquito but the principal vector in many areas is Culex tritaeniorhynchus . Other mosquitoes, mainly the Culex spp., are considered to be important in speciﬁc regions, e.g. C. gelidus in south-east Asia, for the transmission of JE virus.

Man is a dead-end host and plays little role in the ampliﬁcation of the virus. JE is thus predominantly a rural problem, with disease closely related to rainfall and irrigation.

Clinical and Pathological Features Infection with JE virus is considerably more wide-

spread than the incidence of encephalitis would

indicate. Infection may present non-speciﬁcally as a mild febrile illness or as an aseptic meningitis, or, rarely, as a variety of inﬂammatory manifestations in the viscera. The typical disease manifestations of acute meningomyeloencephaliis have been widely estimated to occur at between 1/20 (Rodrigues, 1984) to 1/600–800 persons infected (Halstead, 1981).

The major target cells for JE virus are the T lymphocyte and the peripheral blood mononuclear cells. In fatal cases of encephalitis, viral antigen is also demonstrable in the neurons. Factors determining the neuroinvasiveness of JE virus involve both viral and host factors. Nucleotide sequencing of non-neurovirulent mutants of JE virus have demonstrated single base changes in the coding region for E protein (Cecilia and Gould, 1991). Age is an important host factor determining neurovirulence, encephalitis being more common and more severe in the young as well as in elderly individuals. Experimental work in rats has shown a relationship between neurotropism and neuronal maturity in that the virus selectively infects immature neurons (Ogata et al., 1991). The reason for greater neuroinvasiveness in the elderly is unknown, but is consistent with the features of the related St Louis encephalitis and West Nile virus, which similarly display greater neuroinvasiveness in the elderly.

The typical case of Japanese encephalitis commences after an incubation period of 1–2 weeks, with fever and headache, followed rapidly by depression of the level of consciousness, progressing from stupor to coma. Localising cranial nerve and other neurological signs occur in about 30% of cases and in children generalised seizures are common—the frequency of seizures in patients increases with the severity of the encephalitis.

A quarter of cases of clinical encephalitis will recover with no permanent sequelae and a quarter will die rapidly. The remaining half will recover with varying degrees of permanent neuropsychiatric sequelae. In addition, especially in children, the virus may persist in lymphocytes and reactivate to give recurrent disease after recovery (Sharma et al., 1991).

Grave prognostic signs include a short prodromal period, deep coma, decerebrate posture, breathing abnormalities and the ability to isolate virus from the CSF. Recently a reduction of serum iron levels has been demonstrated in patients due to the sequestering of iron in the spleen, and a direct relationship between low serum iron and prognosis has been shown (Bharadwaj et al., 1991).

The pathology of the brain in fatal cases has revealed microfoci of necrosis scattered throughout the central nervous system, but especially involving the thalamus, the basal ganglia and the deep cerebral nuclei.

FLAVIVIRUSES

545

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

Diagnosis the potential to eliminate Japanese encephalitis. Reports of allergic reactions to the Biken vaccine

Routine diagnosis is usually carried out by serology have resulted in a cautionary warning being issued using HI, IF, CF or ELISA techniques. Test results

against routine administration of JE vaccine to should, however, be treated with caution because of

travellers, except to those who are likely to be at extensive cross-reactivity with other ﬂaviviruses and

high risk in endemic areas, particularly rural areas, for because up to a quarter of patients fail to demonstrate

a month or longer during the season of vector activity

a serological rise in titre due to their late presentation

(Nothdurft et al., 1996).

to medical attention. A preferable serological test is the Because man is only an incidental host, interruption IgM antibody capture ELISA on serum or CSF, or a

of transmission of the virus would be independent of the more recently developed particle agglutination assay,

extent of immunity in human populations, and eradica- which detects JE speciﬁc IgM. The detection of speciﬁc

tion of infection would need to aim for the elimination of IgM antibodies in CSF is diagnostic of acute Japanese

virus circulating in the vertebrate reservoir of domestic encephalitis.

animals. At present, economic realities preclude anything Virus isolation from blood is rarely successful during

approaching a suﬃciently widespread programme of the acute illness because the viraemic phase is probably

immunisation in domesticated pigs. over by the time central nervous system symptoms

Vector control is diﬃcult because of community appear. Virus isolation from the CSF is usually also

resistance to mosquito control programmes, which unsuccessful and, if positive, indicates a poor prog-

need to include and may signiﬁcantly tamper with nosis, as mentioned above. A variety of isolation

subsistence agriculture of rural populations. Thus, techniques are very sensitive to JE virus, including

methods which have been attempted include short- intracerebral inoculation of suckling mice, intrathor-

term drainage of rice ﬁelds and attempts to introduce acic inoculation of live mosquitoes, the use of common

more drought-resistant rice in order to reduce potential mammalian cell lines such as Vero and LLC-MK2 and

mosquito breeding sites. Ultra-low-volume insecticide mosquito cell lines, especially those of A. albopictus

spraying has had limited success, with increasing and A. pseudoscutellaris.

insecticide resistance, especially in Culex tritaeniorhynchus . Measures to prevent biting by mosquitoes, such as the use of insect repellents, mosquito netting, etc., should be encouraged. Most biting activity is

Control

concentrated at nightfall.

The major component of the control of Japanese encephalitis is widespread immunisation of both man and domestic animals, especially pigs. A number of

ST LOUIS ENCEPHALITIS human vaccines have been developed. A formalin-

inactivated lyophilised vaccine of mouse brain origin, St Louis encephalitis virus (SLE) is the major cause of derived from the Nakayima-NIH strain of JE virus, is

epidemic viral encephalitis in the USA. Although the distribution of the virus ranges from southern Canada

prepared by Biken, Japan, and widely used in Japan to Argentina and a few sporadic cases have been and Korea and also by travellers to endemic areas. In reported in South and Central America, virtually all China, a BHK-prepared inactivated vaccine is used. human disease has occurred in the USA. Both vaccines are highly immunogenic and protection

rates of over 90% are achieved. Experimental live attenuated vaccines are being developed and Chinese workers have successfully attenuated the SA14 strain

Viral Features and Host Range by 100 passages in BHK cell culture (Stephenson, 1988). The live attenuated vaccine (SA14-14-2) is now

SLE is antigenically closely related to the other widely used within the PRC. Recombinant DNA

members of the Japanese encephalitis subgroup of technology also holds promise for the future develop-

ﬂaviviruses. In addition, cross-immunity to other ment of safe, eﬀective JE vaccines (Konishi et al.,

ﬂaviviruses has also been demonstrated, including 1991), ChimeriVax-JE using YF 17D as a live vector

dengue virus type 2. Antigenic heterogeneity has been for the envelope genes of SA14-14-2 is in the trial

shown with monoclonal antibodies, although there is stage. Similarly, a number of veterinary vaccines have

no evidence that this has any epidemiological implica- been used in pigs and horses. Vaccination thus holds

tions. By means of T1 restriction mapping, a number

547 of genotypes of SLE have been deﬁned and have been

FLAVIVIRUSES

ataxia, mental confusion and disorientation. Cranial used as epidemiological markers.

nerve palsies may occur in about 20% of cases, but the The major vertebrate hosts of SLE are birds,

absence of focal ﬁndings or seizures may be useful especially domesticated sparrows (Passer domesticus),

diﬀerential features to distinguish SLE from cases of which act as the main amplifying host (Centers for

focal encephalitis, such as herpes simplex. In more Disease Control, 1991b). In addition, small mammals

severe cases there may be midbrain involvement and such as racoons, opossums and rodents are also

progressively severe coma. Overall case fatality is infected, as well as a variety of domestic animals.

approximately 9% of symptomatic cases, although in However, other than birds, there is no evidence that

outbreaks it may reach 20%. The likelihood of animals play any role as maintenance or amplifying

encephalitis, and also its severity, is related directly hosts.

to age, with case fatalities in the elderly during Invertebrate vectors of SLE are various species of

outbreaks reaching 30% (Monath, 1980). Culex mosquitoes, depending on location. In the rural west it is C. tarsalis, in the northern and southern regions of the central USA it is mainly C. pipiens and

C. quinquefasciatus , and in Florida C. nigripalpus. Diagnosis For rapid serological diagnosis of SLE infection, the

IgM antibody capture ELISA is the method of choice, Epidemiology

using either serum or CSF. Cross-reactions may occur if other ﬂaviviruses are active in the area, but this is not

In all parts of the USA the transmission cycle of SLE

a major factor in North America. The IF test on SLE- involves birds and mosquitoes. Man and probably

infected cells is a useful alternative to the IgM ELISA domestic animals are incidental and dead-end hosts.

for testing sera, and the HI test may be used for Epidemiological characteristics, however, diﬀer in the

surveys or diagnosis if acute and convalescent sera are three regions described above. Thus, in the central

available. In regions where ﬂavivirus activity is USA, where C. pipiens and C. quinquefasciatus are the

conﬁned mainly to one virus, the HI test would be a major vectors, epidemic outbreaks result from the

useful screening test, followed by the SLE IgM build-up of virus in domesticated house sparrows and

antibody capture ELISA test to determine recent mosquito larvae breeding in discarded containers and

SLE infection.

open house foundations, characteristic of older housing construction. Regular outbreaks have occurred at approximately 10 year intervals until 1977, followed by irregular, unpredictable outbreaks observed in large

Control urban localities. In the western USA, SLE occurs as

low-grade endemic activity transmitted by C. tarsalis Although Sabin developed an inactivated mouse-brain and associated with agricultural irrigation, although

vaccine during the Second World War, no licensed there are exceptions, such as the large focal outbreak in

vaccine is available at present because of the low Los Angeles in 1984, which was probably due to C.

priority accorded to the infection. Similarly, speciﬁc pipiens acting as an accessory vector.

vector control programmes directed at SLE are either impractical or thought not to be of suﬃcient urgency. Secondary measures to protect against mosquito bites, such as insect repellent and screening, may be of some

Clinical Features beneﬁt during reported outbreaks. The majority (over 90%) of infections with SLE virus

are asymptomatic. In clinically apparent cases, the disease is characterised by an abrupt onset of a febrile

WEST NILE VIRUS illness accompanied by constitutional symptoms of malaise, nausea, vomiting and headache. Occasionally

West Nile virus (WN) is one of the most ubiquitous of there may be a more slow insidious onset. Central

human arbovirus infections, being found throughout nervous system involvement may be in the form of

Africa, in Asia, parts of Europe, USA and Canada. aseptic meningitis or focal encephalitis. Encephalitis

The virus was ﬁrst isolated in the West Nile province of signs may be manifested as neck stiﬀness, dizziness,

Uganda in 1937.

Viral Features and Host Range WN is related by neutralisation tests to the other

members of the JE subgroup of ﬂaviviruses, particularly Kunjin virus which is believed to have evolved from, or is a variant of, WN virus.

The major vertebrate hosts of WN are birds, although in addition the virus is able to infect a variety of domesticated animals (particularly equines) and wild animals, as well as man and subhuman primates. There is a suggestion that WN is becoming more widespread throughout animal species, with isolates from dogs and around 100 species of birds. The death of a harbour seal due to WN has been recorded in the USA. The maintenance vectors of WN consist of a variety of mosquitoes, especially of the genus Culex. On the basis of a study of phylogenetic relationships of isolated viruses, WN can be broadly classiﬁed into two lineages, lineage I, consisting of isolates from Central and North Africa, Europe, Israel and North America. Isolates from Central and Southern Africa and Madagascar are classiﬁed as lineage II.

Epidemiology The transmission cycle of WN consists mainly of wild

birds as the vertebrate host and ornithophilic Culex mosquitoes as the maintenance vector. The major Culex mosquito vector in Africa and the Middle East is

C. univittatus ; in south-east Asia, C. tritaeniorhynchus; and in France, C. modestus. Culex species appear to be the main vectors in North America, with isolates having also been made from Ochlerotatus spp. and Aedes spp. Other vectors were also implicated with limited isolations.

Major epidemics of WN have been reported in Israel during the 1950s, in France in 1962 and the largest epidemic ever recorded took place in South Africa in 1974, which involved tens of thousands of individuals. Epidemics have occurred characteristically in relatively localised areas, e.g. close to Tel Aviv in the Israeli epidemic, in the Rhone delta in France and the semi- desert Karoo region and in and around major cities of the high veld of South Africa. The distribution of WN in South Africa is predominantly in the inland plateau region, where it shares the same geographical distribution and ecology as Sindbis virus, which also produces

a disease which is usually clinically indistinguishable from WN. The presence of WN in the USA was ﬁrst recognised in 1999, and had spread extensively to at

least 43 states by October 2002, and to provinces of Canada.

Outbreaks of WN, as with many other arboviruses, have been governed by climatic conditions, such as heavy rainfall particularly in early summer, and high summer temperatures. During outbreaks high attack rates in humans have been observed, e.g. in some worst-aﬀected towns in South Africa, 50–80% of the human population were infected due to the high rate of feeding by C. univittatus. This mosquito is probably also responsible for sporadic cases in interepidemic periods. In the early stages of the epidemic in the USA, WN virus infection was recognised in mid- to late summer. In 2002 the virus was detected as early as May. Because viraemia is low in man, epidemic activity is directly due to infection of mosquitoes from viraemic birds and human outbreaks are merely the ‘spill-over’ of extensive epizootic activity in birds—an important factor which facilitates epidemiological surveillance. Furthermore, no human-to-human transmission occurs, although there have been recent reports of transfusion and organ donor infection and a case of intrauterine transmission, and, as in the case of JE, human population immunity has no bearing on the suppression of epidemic activity itself, and susceptible individuals would remain vulnerable irrespective of the proportion of immune individuals in the population.

Clinical Features

In the majority of cases WN presents as a mild febrile illness. The onset of disease is characteristically sudden, following a short incubation period of 3–5 days. Fever is usually the ﬁrst sign, followed by headache, nausea and vomiting. Ocular pain is frequently reported, as is pharyngitis. Muscle pain occurs diﬀusely and there may be arthralgia.

During the ﬁrst few days after onset, a maculopapular rash usually appears, which is discrete, with each of the rash elements demarcated by a sharp halo. The rash usually ﬁrst appears on the trunk and then spreads to the face and extremities, and may persist for

a week. Unlike measles, there is no desquamation. Convalescence is rapid in children but may be

somewhat more prolonged in adults and characterised by weakness and malaise. The illness not infrequently recrudesces during the convalescent period. Up to October 2002, approximately 6% of reported cases in the USA were fatalities.

Although WN virus is classiﬁed virologically within the JE group of mosquito-borne encephalitides,

548

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

549 involvement of the CNS was believed to be very rare.

FLAVIVIRUSES

The disease was ﬁrst recognised during two epi- However, in the USA the incidence of CNS involve-

demics of a virulent encephalitis in Queensland and in ment was much more common, with approximately

the Murray Valley in 1917 and 1918. A number of 0.7% of patients showing severe neurological illness

epidemics were subsequently described until 1925, and up to 69% of reported cases showing some clinical

followed by an inexplicable gap until the 1950s. The evidence of meningoencephalitis. Acute ﬂaccid para-

last substantial epidemic took place in 1974 and since lysis (AFP) attributed to Guillain–Barre´ syndrome has

then only some individual sporadic cases have been associated with WN virus infection, several cases

occurred.

having been reported in the USA (Centers for Disease The infection is found in a patchy distribution from Control, 2002).

New Guinea through Darwin to the northern parts of Rarely, cases of visceral involvement including

Western Australia and down the east coast as far south severe hepatitis, occasionally with a haemorrhagic

as Brisbane, and in the basin of the Murray Darling presentation, have been reported.

River. The virus was reintroduced to Central Australia in 2000 following a period of unusually high rainfall.

Diagnosis Viral Features and Host Range

The HI, CF or NT tests can be used for serological diagnosis of WN infection. The HI test detects

The virus displays a close relationship with Kunjin virus antibodies within a few days after onset, but the IgM

and also JE. Two distinct strains can be demonstrated, antibody capture ELISA is recommended as the

a New Guinea and an Australian variant. Individual diagnostic test of choice for WN virus infection.

isolations within the two groups are, however, Cross-reactivity is not a problem in patients with no

remarkably conserved.

previous exposure to flavivirus infection or vaccination. The NT assay would be used to identify specific flavivirus antibodies.

Virus isolation may be readily achieved from blood Epidemiology from infected individuals, despite lower levels of viraemia. Suckling mice are particularly sensitive to

The major maintenance vector is Culex annulirostris intracerebral inoculation and, in addition, cell culture

and a number of vertebrate hosts, chieﬂy wild birds, of mammalian origin as well as insect cell lines are

are involved in the transmission cycle. Domestic commonly used for virus isolation. RT-PCR is a rapid

animals are infected, but they probably do not play method for the detection of WN viral RNA.

any signiﬁcant role as amplifying hosts. Outbreaks have characteristically occurred in the summer months.

Control With the impact of WN on human health in the USA,

Clinical Features there has been increasing interest in the development of

a vaccine. During outbreaks, vector control by As with most other arboviruses, the vast majority of insecticide spraying may be applied. Epidemiological

infections are asymptomatic—only about 1/800–1000 surveillance makes use of sentinel animals, such as

infections being clinically manifest (Anderson, 1954). hamsters, goats, guinea-pigs or pigeons, to detect early

Once clinical disease occurs, however, high case warning signs of impending outbreaks.

fatality rates of 18–42% have characterised the various outbreaks. The clinical features in symptomatic patients are a sudden onset of high fever (up to

MURRAY VALLEY ENCEPHALITIS 40.68C), nausea, vomiting and severe frontal headache. Signs of encephalitis vary from mild neurological

Murray Valley encephalitis virus (MVE) is a relatively involvement with disturbances of consciousness and uncommon cause of human disease—less than 1000

neck stiﬀness to rapid onset of coma with respiratory cases have been reported—all of them conﬁned to

failure. Patients with severe neurological involvement Australia and New Guinea.

and coma, who were kept alive on life-support systems

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

and subsequently survived, have all had remaining through Finland and Sweden in the north, through permanent and severe sequelae.

Germany to France in the west and down to Italy, Greece and Yugoslavia in the south, encompassing the central European countries in between. RSSE is found

Diagnosis predominantly in the taiga (the coniferous forest belt on the edge of the steppes and the tundra region of

Serology is generally carried out by HI or ELISA tests. Siberia) and in western Siberia. Their pathogenic The RT-PCR assay may detect MVE early after onset

potentials are also diﬀerent, CEE causing a very of illness, providing a rapid and speciﬁc diagnosis.

much milder disease in experimentally infected sheep and monkeys as compared to RSSE, and this is reﬂected also in the clinical expression of their respective infections in man. Domestic animals, such

TICK-BORNE ENCEPHALITIS as sheep, goats and cows, infected with CEE excrete virus in their milk. The virus is also relatively stable to

The tick-borne encephalitides (TBEs) are a closely low pH and, experimentally, animals can rarely be related subgroup of viruses within the genus Flavivirus.

infected by oral inoculation. In man, milk-borne Although serological tests such as HI and CF give

transmission of CEE through ingestion of goat, considerable cross-reactivity with members of the

sheep or cow’s milk or dairy products made from ﬂavivirus group, the tick-borne encephalitis (TBE)

them, such as cheese, is an important route of subgroup of ﬂavivirus are far more closely antigeni-

acquisition.

cally related to each other. In contrast to many of their mosquito-borne arbovirus counterparts, the tick-borne encephalitis subgroup is found almost exclusively (with some exceptions, such as louping ill in the UK and

Epidemiology Powassan in North America) in Asia and eastern and

central Europe. Within the subgroup is the entity of Both CEE and RSSE are endemic diseases with an TBE, which consists of two subtypes: TBE—Central

increased seasonal incidence in the summer months European subtype, or Central European encephalitis

related to climatic conditions of temperature and virus (CEE), and TBE—Far Eastern subtype, or

humidity, which aﬀect tick activity. Infections occur Russian Spring-Summer encephalitis virus (RSSE).

predominantly in rural populations, especially farmers Other viruses within the TBE subgroup are Omsk

and forest workers. In addition, some 10–20% of CEE haemorrhagic fever virus , Kyanasur forest disease virus

infections are transmitted through ingestion of goat, and Powassan virus, which will be considered separately.

sheep and cow’s milk and dairy products. Seropreva- lence studies of RSSE virus have shown population prevalences of up to 50% in inhabitants of the taiga.

Virological Features Seroprevalence to CEE depends on rural residence, occupation and age. Thus, in various studies in central Europe, seroprevalence ﬁgures of 11–20% have been

CEE and RSSE viruses are particularly closely related found in hunters, 5% in farmers and 1% in children to each other and are distinguishable only by mono-

(Gresikova et al., 1973).

clonal antibodies or specialised techniques such as antibody adsorption tests. However, monoclonal antibody studies of CEE, especially those directed against glycoprotein domains, demonstrate a degree of anti-

Clinical Features genic complexity. Also, studies of diﬀerent isolates of RSSE from diﬀerent geographical locations have

Disease due to CEE is relatively mild, with a low shown a heterogeneity of biological characteristics,

fatality of less than 5%. The incubation period is 1–2 such as mouse pathogenicity and plaque size. The two

weeks, followed by symptoms and signs of a non- viruses display clearly distinguishable biological diﬀer-

speciﬁc febrile illness, headache, nausea, vomiting, ences from each other. The distribution of the two

lassitude and occasionally some signs of neurological infections closely follows that of their arthropod

disorders may appear, especially related to visual vectors—Ixodes ricinus in the case of CEE and Ixodes

disturbances: blurring of vision and diplopia. This persulcatus in the case of RSSE. The geographical

initial viraemic phase of the illness usually lasts some distribution of CEE stretches from Russia in the east

4–6 days and is followed by a brief remission period.

551 The majority of individuals infected probably only

FLAVIVIRUSES

candidate vaccines have undergone trials. The most experience a monophasic illness, which is rarely

commonly used human vaccine, FSME-Immun, has diagnosed speciﬁcally.

been administered extensively in central Europe with The second phase of the biphasic illness commences

very few mild allergic-type side-eﬀects (Stephenson, after a brief remission period, and is heralded in by a

1988). The seed virus was derived from an Austrian recrudescence of fever and signs of meningitis. The

tick isolate cloned in chick embryo cells and the most important signs of encephalitis are extrapyrami-

vaccine has proved to be highly eﬀective. A non- dal and cerebellar syndromes, which may often persist

pathogenic related virus isolated from a bank vole, the for months after recovery. Localising neurological

Skalica virus , is being investigated as a possible signs, such as cranial nerve involvement, occur

candidate for a live attenuated vaccine. uncommonly and are usually mild. The mortality in

The development of vaccines for RSSE has been less various outbreaks which have been studied has varied

successful. The original RSSE vaccine, developed by (1–5%).

Silber soon after the recognition of the infection in

A more severe degree of encephalitis is usually seen 1937, consisted of an inactivated suspension of infected with RSSE virus. The incubation period is similar but

mouse brain. However, the presence of contaminating this is usually followed by sudden onset of fever and

myelin posed an unacceptable risk of encephalitogenic constitutional symptoms and, in the second phase of

side-eﬀects and its use in man was withdrawn. Several illness, a more intense meningitis. In some individuals

attempts at developing a tissue culture-grown vaccine an aseptic meningitis picture may be the sole clinical

strain have not yielded a successful human vaccine. As manifestation of disease. In others encephalitic signs

with CEE, a surrogate non-pathogenic virus is being and symptoms, such as disturbed consciousness, may

investigated as a possible live attenuated vaccine lead to stupor, coma and death. A characteristic

strain—the Langat TP21 virus, isolated in Malaya. feature is lower motor neuron paralysis, which may resemble poliomyelitis but usually predominantly aﬀects the upper limbs, spreading to the neck, and

OMSK HAEMORRHAGIC FEVER which may be followed by bulbar paralysis and death.

The reported case fatality rates have varied (8–54%). Viral Features and Host Range In addition, residual paresis and atrophy of muscles

of the upper limbs and neck may persist for long Omsk haemorrhagic fever virus (OHF) is closely related periods. Post-recovery epileptiform seizures may reﬂect

to TBE and cannot be diﬀerentiated using polyclonal permanent neural damage.

hyperimmune sera. They are diﬀerentiated on gel precipitation using cross-absorbed monospeciﬁc sera. The infection is conveyed by ixodid ticks, Dermocenter reticulates and D. marginatus. A number of animal

Diagnosis species are susceptible and the virus has been isolated from wild rodents. However, the most important

The IgM antibody capture ELISA on serum or CSF is vertebrate host is the muskrat (Ondatra zibethica), the serological test of choice for diagnosis of TBE virus

which is highly susceptible to infection, the virus infection. The HI test is useful but a signiﬁcant rise in

usually producing a rapidly fatal haemorrhagic dis- antibody level must be demonstrated before a diag-

ease. The virus is excreted in the urine and faeces of nosis can be made. Virus can be isolated from blood or

sick animals and horizontal infection as well as CSF but specimens must be taken early after onset of

arthropod infection is thought to play a role in symptoms. The virus may also be readily isolated from

transmission. The majority of human infections post mortem tissues such as brain and also infected

(60%) have occurred in hunters of muskrats, with tick pools. The virus is readily isolated by intracerebral

transmission occurring as a result of direct contact inoculation of suckling mice or cell cultures, such as

during the skinning of animals. A further 28% of Vero or chick embryo.

infections occur in adult family contacts of hunters.

Control Epidemiology Vaccines have been developed against both CEE and

Infection has been limited to the Omsk region in the RSSE viruses. In the case of CEE, a number of

forest-steppe landscape of western Siberia, adjacent to

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

TBE endemic zones. The majority of cases were Epidemiology recorded between 1945 and 1949. Between 1945 and

1958, a total of 1488 cases were recorded, with no In addition to monkeys, a number of rodents are typically transmitted cases occurring since then.

known to be infected, such as rats and shrews, as well Occasional laboratory-acquired infections have been

as bats and other animals. The main tick vector is reported (Jelinkova-Skalova et al., 1974).

Haemophysalis spinigera . Infection occurs predominantly in poor villagers working in forests.

Clinical Features Clinical Features

Clinically the disease presents with a sudden onset of fever, headache and myalgia. There is a recrudescence

The disease is characterised by a sudden onset of fever of fever followed by haemorrhagic manifestations,

after an incubation period of 3–8 days. The fever may especially epistaxis, but also gastrointestinal bleeding

rise rapidly to 408C, associated with headache and and bleeding at other sites. Bronchopneumonia is a

severe myalgia reminiscent of dengue fever. Muscle frequent complication and occasionally meningitis

pain is also predominantly found in the back and neck may occur with long-term complications, such as

regions. A regular ﬁnding in patients in the acute stage psychomotor retardation and depression.

is papulovesicular lesions on the soft palate. There is usually a cervical and axillary lymphadenopathy but occasionally this is generalised. Earlier reports of the disease laid great emphasis on the haemorrhagic

Diagnosis and Control manifestations, which occurred as early as the third day of illness. These consist of bleeding from the nose, gums and gastrointestinal tract. Associated with this

Infection may be diagnosed by isolation of virus from there is a marked thrombocytopenia and neutropenia, patients’ blood and intracerebral inoculation into

but no evidence of bone marrow suppression or suckling mice. Serological testing by ELISA, CF and

capillary damage. The cause of the haemorrhagic NT are also available. Control measures to prevent

diathesis has more recently been thought to be auto- infection involve the avoidance of ticks and care in

immune in nature (Pavri, 1989). Many of these earlier handling muskrat carcasses in endemic areas, as well as

cases were reported in poor villagers, who were often laboratory safety measures. Because of extensive cross-

infected with bacteria and parasites, and the associated reactivity, CEE vaccine would probably impart good

raised interferon levels and IgE antibodies may also protection.

have contributed to the original clinical picture. Later studies of the disease, including those of laboratory- acquired infections, have rather put emphasis on neurological complications, such as severe headache,

KYASANUR FOREST DISEASE neck stiﬀness, coarse tremors, abnormal reﬂexes and mental disturbances. Mortality in KFD is approximately 5–10%.

Viral Features and Host Range The ﬁrst isolation of Kyasanur forest disease virus

(KFD) was made in 1957 from a dead monkey found Diagnosis and Control near the Kyasanur State forest in Kamataka (formerly Mysore) State in India. A few months previously a

The virus can be readily isolated from patients’ blood lethal epizootic amongst monkeys had been reported in

using mice or cell culture and antibodies can be the adjacent forested areas, with human cases termed

detected by HI, CF, NT or ELISA tests. Vector ‘monkey disease’ by the villagers. So far all human

control programmes have been carried out in the cases have been limited to Kamataka State—on

forest, especially spraying in the vicinity of dead average, about 500/year, in addition to a number of

monkeys when these are encountered. A formalin- laboratory-acquired infections in both India and the

inactivated vaccine has been prepared in India and USA. There is no evidence of the disease having

immunisation programmes of villages in the aﬀected existed prior to 1957.

areas have been carried out.

553 POWASSAN VIRUS

FLAVIVIRUSES

Centers for Disease Control (1990) Yellow fever vaccine. Recommendations of the Immunization Practices Advisory Committee (ACIP). Morbid Mortal Wkly Rep, 39, 1–6.

Centers for Disease Control (1991a) Dengue epidemic—Peru, 1990. Morbid Mortal Wkly Rep, 40, 145–147. Powassan virus (POW) is named after the town of

Viral Features and Host Range

Centers for Disease Control (1991b) St Louis encephalitis Powassan in northern Ontario, where the ﬁrst human

outbreak—Arkansas, 1991. Morbid Mortal Wkly Rep, 40, virus isolate was made from a fatal encephalitis case in

a 5 year-old boy. The disease is rare, less than 50 cases Centers for Disease Control (2002) Acute ﬂaccid paralysis having been reported worldwide. Case reports have

syndrome associated with West Nile virus infection— come from Canada and the USA as well as Russia. Mississippi and Louisiana, July–August 2002. Morbid Mortal Wkly Rep , 51, 825–827. Chen WR, Tesh RB and Rico-Hesse R (1990) Genetic variation of Japanese encephalitis virus in nature. J Gen Virol , 71, 2915–2922.

Epidemiology

Gresikova M, Thiel W et al. (1973) Haemagglutinin- inhibition antibodies against arboviruses in human sera

A number of arthropod vectors have yielded isolates of from diﬀerent regions in Steiermark (Austria). Zentralbl the virus. In North America the major vector has been

Bakteriol Parasit, Infektionsk Hyg , 1(224), 298. Ixodes cookie Gubler DJ and Costa-Valez A (1991) A program for , with Dermacentor andersoni, Ixodes prevention and control of epidemic dengue and dengue

marxi and I. spinipalpus and, in Russia, Haemaphysalis haemorrhagic fever in Puerto Rico and the US Virgin neumanni , Ixodes persulcatus and Dermacentor

Islands. Bull PAHO, 25, 237–247. silvarum occasionally being infected. In addition,

Halstead SB (1981) Arboviruses of the Paciﬁc and South-East infected mosquito species, Aedes togoi and Anopheles

Asia. In Textbook of Paediatric Infectious Diseases (eds Feigin RD and Cherry JD), p 1132. WB Saunders,

hrycanus , have also been reported. Vertebrates infected

Philadelphia, PA.

with POW have occurred mainly in mammals, but also Halstead SB (1984) Selective primary health care: strategies birds, amphibians and reptiles.

for control of disease in the developing world. XI. Dengue. Rev Infect Dis , 6, 251.

Halstead SB (1989) Antibody, macrophages, dengue virus infection, shock, and hemorrhage: a pathogenic cascade.

Clinical Features

Rev Infect Dis , II(suppl 4), S830–S839. Iversson LB (1980) Aspects of the encephalitis epidemic

Clinical cases of POW have presented with encepha- caused by arbovirus in the Ribeira Velley, Sao Paulo, Brazil, during 1975–1978. Rev Saude Publ, 14(1), 9–35.

litis, meningoencephalitis and aseptic meningitis. In Jacobs MG, Brook MG, Weir WRC and Bannister BA (1991) some cases focal encephalitic signs have occurred and

Dengue haemorrhagic fever: a risk of returning home. Br in one case from Russia the patient died following

Med J , 302, 828–829.

bulbar paralysis. In a series of 19 cases in North Jelinkova-Skalova E, Tesarova J, Buresova V et al. (1974) America, two deaths occurred in the acute illness

Laboratory infection with virus of Omsk haemorrhagic fever with neurological and psychiatric symptomatology.

phase. Ceskoslovenska Epidemiol Microbiol Immunol (PRAHA),

23 (4,5), 290–293. Jennings AD, Gibson CA, Miller BR et al. (1994) Analysis of a yellow fever virus isolated from a fatal case of caccine- associated human encephalitis. J Infect Dis, 169, 512–518.

REFERENCES

Konishi E, Pincus S, Fonseca BAL et al. (1991) Comparison of protective immunity elicited by recombinant vaccinia

Anderson SG (1954) Murray Valley encephalitis and viruses that synthesize E or NS1 of Japanese encephalitis Australian disease. J Hyg, 52, 447.

virus. Virology, 185, 401–410. Anonymous (1980) Guide for Diagnosis, Treatment and

Kurane I, Mady BJ and Ennis FA (1991) Antibody- Control of Dengue Haemorrhagic Fever , 2nd edn. Technical

dependent enhancement of dengue virus infection. Rev Advisory Committee on DHF for the South-east Asian and

Med Virol , 1, 211–221.

Western Paciﬁc Regions. World Health Organization, Monath TP (1980) Epidemiology in St Louis Encephalitis (ed. Geneva.

Monath TP), chapter 6. American Public Health Associa- Bharadwaj M, Prakash V, Mathur A and Chaturvedi UC

tion, Washington, DC.

(1991) Prognostic signiﬁcance of serum iron levels in cases Nathan N, Barry M, Van Herp M and Zeller H (2001) of Japanese encephalitis. Postgrad Med J, 67, 247–249.

Shortage of vaccines during a yellow fever outbreak in Cecilia D and Gould EA (1991) Nucleotide changes

Guinea. Lancet, 358, 2129–2130. responsible for loss of neuroinvasiveness in Japanese

Nothdurft HD, Jelinek T, Marschang A et al. (1996) Adverse encephalitis virus neutralization-resistant mutants. Virology,

reactions to Japanese encephalitis vaccine in travellers. J 181 , 70–77.

Infect , 32, 119–122.

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

Ogata A, Nagashima K, Hall WW et al. (1991) Japanese and recurrence of infection in children. Clin Exp Immunol, encephalitis virus neurotropism is dependent on the degree

of neuronal maturity. J Virol, 65, 880–886. Pavri K (1989) Clinical, clinicopathologic and hematologic

Stephenson JR (1988) Flavivirus vaccines. Vaccine, 6, 471–482. features of Kyasanur forest disease. Rev Infect Dis, II,

World Health Organization (1990) Yellow fever in 1988. S854–S859.

Wkly Epidemiol Rec , 65, 213–220. Rodrigues RM (1984) Epidemiology of Japanese encephalitis

World Health Organization (1991) Yellow fever—epidemic in in India. In National Conference on Japanese Encephalitis,

Cameroon, 1990. Wkly Epidemiol Rec, 66, 76–77. 1982 . Indian Council of Medical Research.

Rosen L (1986) The pathogenesis of dengue haemorrhagic World Health Organization (1996) Inclusion of yellow fever fever. S Afr Med J (suppl), 40–42.

vaccine in the EPI. Wkly Epidemiol Rec, 71, 181–185. Sharma S, Mathur A, Prakash V et al. (1991) Japanese

World Health Organization (1996) Yellow fever in 1994 and encephalitis virus latency in peripheral blood lymphocytes

1995. Wkly Epidemiol Rec, 71, 313–318.

18 Bunyaviridae

Robert Swanepoel

National Institute for Communicable Diseases, Sandringham, South Africa

INTRODUCTION quently the demonstration of weak serological cross- reactions between the groups resulted in the viruses

At present the family Bunyaviridae comprises approxi- being included in a Bunyamwera supergroup (Anon- mately 300 animal viruses assigned to four genera:

ymous, 1967). The family Bunyaviridae, containing a Orthobunyavirus (named after Bunyamwera virus),

single genus Bunyavirus, was erected when members of Phlebovirus (named after phlebotomus/sandﬂy fever),

the supergroup and certain ungrouped viruses were Nairovirus (named after Nairobi sheep disease virus)

found to have similar morphology (Murphy et al., and Hantavirus (named after Hantaan virus). A further

1973; Porterﬁeld et al., 1974; Fenner, 1976) and later

40 inadequately characterised animal viruses are the genera Phlebovirus, Uukuvirus, Nairovirus and considered possible members of the family and there

Hantavirus were added to the family as morphological is a genus of plant viruses (Karabatsos, 1985; Calisher

and biochemical aﬃnities between the viruses and Karabatsos, 1989; Peters and LeDuc, 1991;

became evident (Bishop et al., 1980; Matthews, 1981; Calisher, 1991; Murphy et al., 1995). Most of the

Schmaljohn et al., 1985). More recently, the uuku- animal viruses were discovered in the course of surveys

viruses were reduced to a serogroup within the genus on haematophagous arthropods or wild vertebrates,

Phlebovirus , since they were found to share coding and and the fact that new members of the family are

replication strategies with the phleboviruses, while the constantly being encountered has been interpreted to

genus Bunyavirus has been renamed as genus Ortho- indicate that many remain to be discovered (Peters and

bunyavirus (Simons et al., 1990; Calisher, 1991; LeDuc, 1991). Some of the viruses are important

Murphy et al., 1995). Within genera, members are still pathogens of humans or livestock, but the majority

classified on the basis of antigenic affinities and they have no known medical or veterinary significance.

are arranged in serogroups, antigenic complexes, Sometimes a pathogenic role is discovered for a virus

viruses (or serotypes), subtypes and varieties, in order years after its initial isolation. Although most members

of increasing relatedness, i.e. members of a genus may of the family are thought to be arthropod-borne (i.e.

only exhibit distant antigenic relationship to each arboviruses), transmission by vectors has been demon-

other, whereas diﬀerences between varieties of a virus strated conclusively in comparatively few instances.

are consistent but minimal. However, the concept of Members of the rodent-associated genus Hantavirus

classiﬁcation of viruses by serotype has had to be are not considered to be arthropod-borne.

revised with respect to agents that fail to grow in The origins of the family can be traced to the initial

laboratory culture systems, with greater emphasis detection of close antigenic relationships within certain

being placed on the deﬁnition of genotypes through groups of viruses, including one containing Bunyam-

nucleotide sequencing of the genome. This applies wera virus (named after a place in Uganda) (Casals,

particularly to recently discovered hantaviruses 1957, 1961; Casals and Whitman, 1960, 1961). Subse-

(Monroe et al., 1999).

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

THE VIRUS S segment of the Phlebovirus genome consists of ambisense RNA, i.e. has bi-directional coding, a

Structure property which is shared only with the RNA of viruses of the family Arenaviridae. The ﬁrst 8–13 nucleotide The viruses of the family are spherical, 80–120 nm in

bases at the 3’ ends of the RNA segments tend to have diameter, and have a host cell-derived bilipid-layer

a sequence which is conserved within the viruses of envelope through which virus-coded glycoprotein

each genus, with a complementary (palindromic) spikes or peplomers project (Figure 18.1). The virions

consensus sequence occurring at the 5’ end; and the contain three major structural proteins: two envelope

ends of the segments are non-covalently linked, so that glycoproteins, G1 and G2, and a nucleocapsid protein

the RNA occurs in a loosely bound circular conﬁg- N, plus minor quantities of a large or L protein (145–

uration within the nucleocapsids. The segmented 259610 3 Da), believed to be the viral transcriptase, an

nature of the genome suggests that the potential exists RNA-dependent RNA polymerase (Table 18.1)

for reassortment to occur in co-infections, and it is (Bishop, 1990; Schmaljohn and Patterson, 1990; Peters

thought that this mechanism may have contributed to and LeDuc, 1991; Calisher, 1991) (Hazara virus of the

the evolution of diversity in the family, but experi- genus Nairovirus is believed to have three glycopro-

mental evidence indicates that there are genetic teins). Members of the family have a three-segmented,

restraints and that reassortment occurs with facility single-stranded RNA genome and each of the seg-

only between closely related members of bunyavirus ments, L (large), M (medium) and S (small), is

serogroups or diﬀerent strains of an individual contained in a separate nucleocapsid within the virion.

phlebovirus (Peters and LeDuc, 1991). The sizes of the structural proteins and RNA segments

The L RNA segment of the genome codes for the vary with genus (Table 18.1). The genomic RNA is in

viral transcriptase, and the M segment for the G the negative-sense (complementary to mRNA), but the

proteins, as well as a non-structural protein NS m in the

Figure 18.1

Table 18.1 Major biochemical properties of members of the Bunyaviridae. Information derived from sources cited in the text Viruses

Molecular weights of major structural proteins (610 3 )

Molecular weights of RNA species (610 6 )

MS Bunyaviruses

G1 G2 N

1.8–2.3 0.3–0.5 Sandﬂy fever group

1.8–2.2 0.7–0.8 Uukuniemi group

1.0–1.3 0.4–0.7 Nairoviruses

1.5–2.3 0.6–0.7 Hantaviruses

557 genera Orthobunyavirus and Phlebovirus. The S seg-

BUNYAVIRIDAE

of laboratory rodents. Members of the family can be ment RNA codes for the N protein, as well as a non-

grown in a variety of cell cultures (Vero cells have been structural protein NS s in the bunyaviruses and

most commonly used), but some of the viruses are non- phleboviruses. Non-structural proteins have not as

cytolytic, so that their presence has to be demonstrated yet been demonstrated in the nairoviruses or hanta-

by immunoﬂuorescence or similar means. Hanta- viruses. The viral glycoproteins are responsible for

viruses are diﬃcult to grow in vitro, and several of recognition of receptor sites on susceptible cells,

the more recently discovered members of the genus manifestation of viral haemagglutinating ability and

have not yet been adapted successfully to cell cultures for inducing protective immune response in the host.

(Monroe et al., 1999).

The N protein induces production of, and reacts with, An abridged classiﬁcation of the family, showing complement-ﬁxing antibody.

selected members known to cause infection of humans and livestock in relation to their vectors and distribution, is presented in Tables 18.2–18.6 (Karabatsos, 1985; Calisher and Karabatsos, 1989; Peters and

Biological Characteristics LeDuc, 1991; Calisher, 1991). In addition, high prevalences of antibody to many other viruses have

Viruses which attach to receptors on susceptible cells been found in particular human populations, but are internalised by endocytosis and replication occurs

conclusive evidence of infection or disease association in the cytoplasm. Virions mature primarily by budding

is lacking: antigenic cross-reactivity between viruses through endoplasmic reticulum into cytoplasmic vesi-

can complicate the interpretation of survey ﬁndings or cles, which are presumed to fuse with the plasma

render it diﬃcult to arrive at a serological diagnosis in membrane to release virus, but it appears that virus

individual cases of disease.

can also bud directly from the plasma membrane There is a broad tendency for antigenic grouping to (Figure 18.2) (Anderson and Smith, 1987). Most of the

correlate with geographic distribution and with the viruses have been isolated and propagated by intra-

type of vector involved in transmission (Tables 18.2– cerebral inoculation of suckling mice, but the

18.4). Although a greater variety of arthropod-borne hantaviruses produce chronic and inapparent infection

members occurs in tropical and subtropical countries

Figure 18.2

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

Table 18.2 Abridged classiﬁcation of the genus Orthobunyavirus showing members known to cause infection of humans and domestic animals. Information derived from sources cited in the text

Serogroup

Livestock Distribution ANTIGENIC COMPLEX

Putative vectors

Human infection

disease

Virus (synonym)

Natural

Laboratory

Subtype Variety

Bunyamwera BUNYAMWERA (21)a Bunyamwera

Africa Batai (Calovo)

Mosquitoes

Europe, Asia Cache Valley

Mosquitoes

N. America Maguari

Mosquitoes

S. America Fort Sherman

Mosquitoes

C. America Germiston

Mosquitoes?

Africa Ilesha

Mosquitoes

Africa Ngari

Mosquitoes

Africa, Madagascar Shokwe

Mosquitoes

Africa Tensaw

Mosquitoes

N. America WYEOMYIA (8) Wyeomyia

Mosquitoes

C. and S. America Three other complexes (3) Anopheles A TACAIUMA (6) Tacaiuma

Mosquitoes

S. America One other complex (6) Anopheles B One complex (2) Bakau One complex (5) Bwamba BWAMBA (2) Bwamba

Mosquitoes

Africa Pongola

Mosquitoes

Africa Group C CARAPARU (5) Caraparu

Mosquitoes

C. and S. America Ossa

Mosquitoes

C. America Apeu

Mosquitoes

S. America MADRID (1) Madrid

Mosquitoes

C. America MARITUBA (6) Marituba

Mosquitoes

S. America Murutucu

Mosquitoes

S. America Restan

Mosquitoes

S. America Nepuyo

Mosquitoes

C. and S. America ORIBOCA (2) Oriboca

Mosquitoes

S. America Itaqui

Mosquitoes

S. America California CALIFORNIA ENCEPHALITIS (6) California encephalitis

Mosquitoes

N. America Inkoo

Mosquitoes

Europe La Crosse

Mosquitoes

N. America Snowshoe hare

Mosquitoes

N. America Tahyna (Lumbo)

Mosquitoes

Europe, Asia, Africa

(continued)

559 Table 18.2 (continued)

BUNYAVIRIDAE

Serogroup

Livestock Distribution ANTIGENIC COMPLEX

Putative vectors

Human infection

disease

Virus (synonym)

Natural

Laboratory

Subtype Variety

MELAO (7) Jamestown Canyon

N. America Keystone

Mosquitoes

N. America GUAROA (1) Guaroa

Mosquitoes

C. and S. America One other complex (1) Capim Five complexes (10) Gamboa Two complexes (8) Guama GUAMA (4) Guama

Mosquitoes

C. and S. America CATU (1) Catu

Mosquitoes

S. America Three other complexes (7) Koongol One complex (2) Minatitlan One complex (2) Nyando NYANDO (2) Nyando

Mosquitoes

Africa Olifantsvlei Two complexes (5) Patois Two complexes (7) Simbu AKABANE (2) Akabane

Mosquitoes

Asia, Africa, Australasia MANZANILLA (12) Oropouche

Ceratopogonids

S. America Tinaroo

Ceratopogonids +

Australasia SHUNI (3) Shuni

Ceratopogonids

Africa Aino

Ceratopogonids +

Asia, Australasia Four other complexes (8) Tete One complex (6) Turlock Two complexes (5) Ungrouped (4)

Ceratopogonids

a Figures in parentheses indicate the total numbers of recognised members of the relevant taxon.

of Latin America and Africa, many viruses, including

be transmitted by culicine mosquitoes, including several important pathogens, occur in temperate

aedines, but some are transmitted by anopheline countries and the distribution of the family extends

mosquitoes. Simbu serogroup viruses are associated to the Arctic region. Moreover, there are many

particularly with ceratopogonid midges (Culicoides instances on record of residents of temperate countries,

spp.), while the sandﬂy fever serogroup of phlebo- which lack indigenous disease, acquiring infection

viruses (apart from Rift Valley fever and a few during travels abroad. Most of the viruses appear to

other mosquito-borne viruses), are associated with

560

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

Table 18.3 Abridged classiﬁcation of the genus Phlebovirus showing members known to cause infection of humans and domestic animals. Information derived from sources cited in the text

Serogroup

Livestock Distribution ANTIGENIC COMPLEX

Putative vectors

Human infection

disease

Virus (synonym)

Natural

Laboratory

Sandfly fever SANDFLY FEVER NAPLES (4) a Sandﬂy fever Naples

Europe, Africa, Asia Toscana

Phlebotomids

Europe CANDIRU (6) Alenquer

Phlebotomids

S. America Candiru

Phlebotomids?

S. America PUNTA TORO (2) Punta Toro

Phlebotomids?

C. America RIFT VALLEY FEVER (3) Rift Valley fever (Zinga)

Phlebotomids

Africa Four other complexes (8) Unassigned to complex (22)

Mosquitoes

Europe, Africa, Asia Chagres

Sandﬂy fever Sicilian

Phlebotomids

C. America Uukuniemi UUKUNIEMI (13) Uukuniemi

Phlebotomids

Europe a Figures in parentheses indicate the total numbers of members of the relevant taxon.

Ixodids

Table 18.4 Abridged classiﬁcation of the genus Nairovirus showing members known to cause infection of humans and domestic animals. Information derived from sources cited in the text

Serogroup

Livestock Distribution COMPLEX

Putative vectors

Human infection

disease Virus (synonym)

Natural

Laboratory

Crimean-Congo haemorrhagic fever CRIMEAN-CONGO HAEMORRHAGIC FEVER (3) a

E. Europe, Africa, Asia Dera Ghazi Khan One complex (6) Hughes One complex (10) Nairobi sheep disease NAIROBI SHEEP DISEASE (2) Nairobi sheep disease (Ganjam)

Crimean-Congo haemorrhagic fever

Ixodids

Africa, Asia Dugbe

Ixodids

Africa Qalyub One complex (3) Sakhalin One complex (7) Thiafora One complex (2)

Ixodids

a Figures in parentheses indicate the total numbers of members of the relevant taxon.

phlebotomids (sandﬂies). The Tete serogroup of Transmission bunyaviruses, the Uukuniemi serogroup of phlebo-

viruses and the nairoviruses are associated with ixodid It is characteristic of arthropod-borne viruses that they and argasid ticks. Some viruses have been isolated

produce viraemia in at least one species of vertebrate from more than one type of vector.

to allow the infection to be acquired by biological

vectors which take blood meals. During a so-called extrinsic incubation period, commonly lasting 1–2 weeks in dipterid vectors (mosquitoes, midges and sandflies), the virus replicates in the vector and spreads to produce infection of the salivary glands, thereby permitting transmission to occur to a second vertebrate host. Virus is thus maintained by circulation between the vector and a vertebrate host. The maintenance cycle may be cryptic, involving wild vertebrates which develop inapparent infection, with incidental spread of infection to susceptible domestic animals or humans which impinge on the cycle. It has been postulated that through selection pressure brought about by long association with the virus, natural maintenance hosts often develop transient viraemic infection without displaying susceptibility to the pathogenic effects of the virus concerned. Small mammals and birds, which occur in large numbers, breed prolifically to ensure a constant supply of non- immune individuals and are subject to periodic population explosions, constitute ideal maintenance hosts for arboviruses. Species susceptible to disease may themselves serve to amplify circulation of virus through infecting vectors, but humans serve this purpose for members of the Bunyaviridae in few instances only (Oropouche, sandfly fever) and are usually ‘dead-end’ hosts. Domestic animals which develop disease or undergo inapparent infection may serve as link hosts between the natural cycle and humans, which in turn gain infection from contact with infected tissues of livestock or products such as milk, or from vectors infected by feeding on livestock [Rift Valley fever, Crimean-Congo haemorrhagic fever (CCHF)].

Since the biting activity of arthropod vectors, and hence the infection of vertebrates, is seasonal, the fate of arthropod-borne viruses during winters or dry seasons of inactivity has long constituted a central enigma in the epidemiology of arbovirus diseases. Many plausible mechanisms for overwintering or hibernation of arboviruses have been described, including persistent infection of vertebrates, migration of infected birds or mammals, hibernation of infected adult vectors, and continuous vector activity in tropical locations (Reeves, 1974). Transovarial transmission of infection in arthropod vectors, however, theoretically constitutes an ideal mechanism for ensur- ing the perpetuation of the viruses, and comparatively early in the history of the investigation of arbovirus diseases convincing evidence was produced to indicate that the phenomenon occurs in phlebotomids and ixodid ticks (Tesh, 1984). The evidence for mosquito- borne viruses long remained in doubt, but in recent

years many investigators have demonstrated transovarial transmission of bunyaviruses, particularly members of the California encephalitis serogroup, as well as Rift Valley fever virus and members of the families Togaviridae and Flaviviridae in mosquitoes (Tesh, 1984; Linthicum et al., 1985; Peters and LeDuc, 1991; Swanepoel, 2003). Even in the absence of transovarial transmission of infection, the overwintering of viruses transmitted by ixodid ticks can be explained by the long intervals which occur between the feeding of successive instars of the vectors.

In general, viruses transmitted by dipterid ﬂies (mosquitoes, midges and sandﬂies) may cause sporadic infections but are capable of causing explosive epidemics at irregular intervals of years, when climatic conditions are particularly favourable for the breeding of vectors, or human manipulation of the environment results in large-scale juxtaposition of susceptible persons or livestock and vectors. Viruses transmitted by ixodid ticks tend to cause sporadic disease in locations where there is occupational or recreational exposure of humans to ticks, but human intervention can precipitate the occurrence of larger outbreaks of disease.

Clinical syndromes associated with members of the Bunyaviridae range from inapparent infections known from routine monitoring of laboratory workers, through moderate to severe inﬂuenza-like illness with or without a maculopapular rash and characterised by fever (often biphasic), headache, myalgia, arthralgia and malaise, to encephalitis or haemorrhagic disease with necrotic hepatitis; while the hantaviruses of Asia and Europe are associated with a group of diseases known collectively as haemorrhagic fever with renal syndrome (HFRS), and the hantaviruses of the Americas are associated with an acutely fatal respiratory disease known as hantavirus pulmonary syndrome (HPS). The information presented below on the epidemiology and disease associations of individual viruses is derived from a few collated sources, except where indicated otherwise (Karabatsos, 1985; Calisher and Karabatsos, 1989; Porterﬁeld, 1990; Gonzalez-Scarano and Nathanson, 1990; Peters and LeDuc, 1991; McKee et al., 1991).

LABORATORY DIAGNOSIS The appropriate specimens and laboratory methods

required for conﬁrming diagnoses of the more important diseases are indicated in the relevant sections dealing with the individual infections below.

BUNYAVIRIDAE

561

Procedures developed and applied over decades for the isolation and identification of arthropod-borne members of the family, or for demonstrating immune responses, remain valid (Shope and Sather, 1979). However, there are residual problems concerning the sensitivity, specificity and rapidity with which certain infections can be diagnosed, and these are being solved through increasing utilisation of newer serological and molecular biological techniques. Isolation and identification of virus remains the definitive way of making a diagnosis, and this is especially true for what is perceived as a novel or as an undifferentiated febrile illness: it is easier to arrive at a serological diagnosis in diseases which are recognisable from their clinical presentation or from the circumstances under which patients become infected, such as Oropouche fever, sandfly fever, CCHF, Rift Valley fever, or HFRS. Sporadic undifferentiated febrile illnesses, in contrast, have usually been identified in the course of surveys, and the diagnosis of individual cases requires clinical acumen and recourse to the services of a specialised laboratory able to screen for a range of viruses known or considered likely to occur in the area where the infection was acquired.

Virus Detection and Identification Most members of the family were discovered through

intracerebral inoculation of suckling mice, and this method is still widely used for isolating the viruses. A few of the viruses are also pathogenic for weaned mice or hamsters, some even by a peripheral route, and this constitutes a useful screening method for preliminary identiﬁcation of isolates, e.g. Rift Valley fever virus. Some viruses that are non-pathogenic for laboratory rodents, such as the hantaviruses (not known to be arthropod-borne), can nevertheless be isolated in rodents through demonstrating the presence of viral antigens in tissues. Many cytopathic and non-cytopathic viruses, such as CCHF, can be isolated in mammalian cell cultures and detected by immuno- ﬂuorescence. The method has the advantage that it is usually rapid and therefore clinically useful, but it is not invariably more sensitive than the use of suckling mice for isolating viruses that are present in low concentrations in pathological specimens. Certain viruses which are non-pathogenic for laboratory mice, such as some of the neotropical phleboviruses, were only discovered because they proved to be cytopathic in mammalian cell cultures. Although it has not been proved for members of the Bunyaviridae,

some arboviruses can be isolated most successfully by inoculation of mosquito cell cultures, which do not manifest a cytopathic eﬀect, or live mosquitoes, and in these instances the isolation of virus has to be demonstrated by immunological means.

In some diseases, such as Rift Valley fever or CCHF, rapid diagnoses can sometimes be achieved without culturing virus, by demonstrating the presence of viral antigens directly in infected blood or other tissues by enzyme-linked immunoassay, immunoﬂuorescence or

a variety of other immunological methods. This approach was used in the discovery of hantaviruses in rodent tissues. In certain diseases, such as La Crosse encephalitis, it appears that virus is seldom present in blood or other tissues in infective concentrations at the time that the disease is recognised, while other viruses, such as the hantaviruses, appear to be present but are extremely difficult to isolate and adapt to laboratory host systems. An alternative is to detect viral nucleic acids in tissue extracts or histological sections by hybridisation with specific radiolabelled nucleic acid probes. A more sensitive technique is to use reverse transcription and the polymerase chain reaction to detect viral nucleic acids, and this has been used with notable success on blood and other tissues of human patients and rodents infected with hantaviruses. A further advantage of the technique is that it may be possible to select consensus sequence primers which are specific for either individual viruses, groups of viruses or all potential members of a genus (Puthavathana et al. , 1992; Arthur et al., 1992; Nichol et al., 1993; Xiao et al., 1994; Monroe et al., 1999).

Isolates are generally identified by serological means, and by definition viruses react most specifically with antisera in neutralisation tests, but these are technically difficult to perform with some viruses, or may not yield results sufficiently rapidly to be clinically useful. Antisera tend to be more cross-reactive in the other serological tests commonly used for the investigation of the Bunyaviridae (viz. complement fixation, haemagglutination-inhibition,

immunofluorescence and enzyme-linked immunoassay) and this is particularly true within serogroups of the genus Bunyavirus. The problem can be overcome, and the process accelerated, by using monoclonal antibodies to achieve simultaneous detection and identification of isolates, as in immunofluorescence tests on cell cultures. In instances where potentially new viruses, or viruses associated with undifferrentiated illnesses, have to be identified, cross-reactivity can be useful. Unidentified isolates may have to be tested against antisera to all of the known viruses of the region involved, or even ultimately against antisera to hundreds of viruses

562

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

563 which occur elsewhere in the world, and the process is

BUNYAVIRIDAE

establishing a rapid diagnosis. Antibody titres tend to facilitated by preliminary screening of the isolates with

be highest against the homologous infecting virus, so pools of antisera, or antisera that have deliberately

that problems of cross-reactivity can sometimes be been rendered cross-reactive by immunising laboratory

overcome by screening patients’ sera with a range of animals sequentially with several viruses. Morpho-

antigens prepared from all members of the virus logical or partial biochemical characterisation of

serogroup known to occur in the area concerned, as isolates, e.g. by performance of electron microscopic

is done with members of the California encephalitis examination or tests for sensitivity to ether and bile

serogroup. An alternative is the preparation of puriﬁed salts, may also facilitate the process of identifying a

antigens that contain virus proteins or peptides which virus.

react speciﬁcally with serotype antibody, and this In instances where viral nucleic acids have been

includes preparation of antigens by recombinant DNA detected, these can be identiﬁed by hybridisation under

technology (Feldmann et al., 1993). The same eﬀect stringent conditions with labelled probes that are

may be obtained by selective capture of viral proteins speciﬁc for individual viruses, or by demonstrating

with monoclonal coating antibody in enzyme-linked speciﬁc endonuclease restriction enzyme digestion

immunoassays.

patterns with the products of a polymerase chain reaction. However, it is becoming more common to perform nucleotide sequencing on the products of polymerase chain reactions to obtain more exact

GENUS ORTHOBUNYAVIRUS information on the phylogenetic relationships of the

aetiological agents concerned (Monroe et al., 1999). Serogroup Bunyamwera Bunyamwera, Ilesha, Ngari, Germiston and

Serology

Shokwe Viruses Bunyamwera virus is widely distributed in Africa and

Serological diagnosis of infections is beset with the has been isolated from aedines and other culicine same problems of cross-reactivity that apply to

mosquitoes, and/or human blood in Uganda, South antigenic identiﬁcation of isolates, and the diﬃculties

Africa, Kenya, Nigeria, Central African Republic, are compounded where patients have previously been

Cameroon and Senegal. Antibody has been found in infected with an antigenically-related virus: antibody

humans and/or domestic animals, rodents, bats and response tends to be broadly cross-reactive within

subhuman primates in the same countries as well as in serogroups following sequential infections. Neutralis-

Mozambique, Tanzania, Angola, Congo, Egypt and ing antibody, which reacts most speciﬁcally for

Tunisia. However, some of the antibody reactions individual viruses, usually becomes demonstrable by

recorded in surveys may have been due to infection day 7–10 of illness (earlier in Rift Valley fever) and,

with related viruses. Despite the widespread occur- after an initial post-convalescent decline in titre, tends

rence of antibody, human disease has seldom been to remain demonstrable for life, but the response is

recognised and the few cases which have been usually weak and diﬃcult to demonstrate following

described include several laboratory infections. Clinical Nairovirus infections. Complement-ﬁxing antibody

ﬁndings included fever, maculopapular rash, arthral- becomes demonstrable in the second or third week of

gia, neck stiﬀness, vertigo and temporary loss of visual illness, declines after several months, and tends to be

acuity. Severe encephalitis occurred in experimental group-speciﬁc with bunyaviruses and nairoviruses, but

infection of a tumour patient. Infection was conﬁrmed more speciﬁc among phleboviruses. Antibody demon-

in patients by isolation of virus from blood or strable by haemagglutination-inhibition, indirect

demonstration of an immune response. It seems likely immunoﬂuorescence or enzyme-linked immunoassay

that disease may be more common than currently becomes detectable at about the same time as

realised.

neutralising antibody and, after a post-convalescent Ilesha virus has been isolated from the blood of decline in titre, remains demonstrable for a period of

febrile humans in Nigeria, Uganda, Cameroon and the several years at least, and varies in speciﬁcity in

Central African Republic, and from anopheline diﬀerent groups of viruses. Demonstration of IgM

mosquitoes in the latter country. In addition, there is antibody activity in indirect immunoﬂuorescence tests

serological evidence that the virus occurs in Senegal or enzyme-linked immunoassays is most useful for

and Ghana. Few cases of disease have been reported,

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

and these consisted of undiﬀerentiated febrile illness Romania, Hungary, Germany, Portugal and Finland, with a rash.

in the sera of humans and/or birds, rodents, domestic Ngari virus has been isolated from mosquitoes in

ruminants and deer. The ﬁndings in seroprevalence Senegal, Burkina Faso, the Central Africa Republic

studies suggest that human infection is seldom accom- and Madagascar, as well as from a sheep in Mauritania

panied by overt disease, but febrile illness with malaise, and the liver of a person who succumbed to cerebral

myalgia, anorexia and sometimes abdominal pain, malaria in Senegal. A virus isolated from the blood of

tonsillitis, cough and dyspnoea (associated with lung

a human patient during the 1997–1998 Rift Valley inﬁltration), has been reported from Czechoslovakia fever epidemic in north-east Kenya and adjacent

and Malaysia on the basis of serological diagnoses. Somalia was thought to be a new bunyavirus and given the name Garissa virus, but it has subsequently been found to be an isolate of Ngari virus (Bowen et al. , 2001; Nichol, 2003). There were many deaths from haemorrhagic disease which could not all be

Cache Valley, Maguari, Fort Sherman, Tensaw conﬁrmed as cases of Rift Valley fever, and antibody

and Wyeomyia Viruses

to Ngari virus was found in patients in both Kenya and Somalia, but the importance of Ngari virus as a human

Cache Valley virus has been isolated from culicine and pathogen remains to be determined.

anopheline mosquitoes from widely separated loca- Shokwe virus has been isolated from mosquitoes,

tions in the USA and from Jamaica, and antibody has mainly aedines but also from other culicines in South

been found in the sera of humans and/or horses, sheep, Africa, Senegal, Ivory Coast and Kenya, and from

cattle, wild rodents, raccoons, deer and monkeys in the rodents and the blood of a febrile human in Ivory

USA, Canada, Trinidad and Guyana. Despite the Coast. Little is known of the pathogenic potential of

occurrence of high antibody prevalence rates, human the virus. Germiston virus has been isolated in South

disease has not been reported. Recently, however, the Africa, Zimbabwe, Mozambique, Kenya and Uganda

results of serological studies and pathogenicity trials, from Culex rubinotus, a mosquito which selectively

and the isolation of virus from a sentinel sheep, feeds on rodents, and from myomorph rodents (rats

incriminated Cache Valley virus as the causative and mice) in Uganda. Antibody has been found in the

agent of an outbreak of congenital abnormalities sera of humans and/or cattle and rodents in South

(hydranencephaly–arthrogryposis syndrome) among Africa, Botswana, Namibia and Angola. Two labora-

sheep in Texas (Chung et al., 1990a, 1990b). Maguari tory infections have been reported; one with

virus , a subtype of Cache Valley virus, has been isolated undiﬀerentiated febrile illness with rash, and the

from mosquitoes, mainly aedines, in Brazil, Argentina, other with signs of mild encephalitis. Virus was

French Guiana, Colombia and Trinidad, and from isolated from the blood of the patients.

horse blood in Guyana and Colombia. Antibody has been found in the same countries, as well as in Peru, Surinam and Venezuela, in the sera of humans and/or horses, cattle, sheep, water buﬀalo and birds. Human

Batai Virus disease has not been reported, but the virus is suspected of causing disease in horses. Fort Sherman

Calovo virus , ﬁrst isolated from anopheline mosquitoes virus , yet another subtype of Cache Valley virus, was in 1960 in what was then Czechoslovakia, is believed to

isolated from the blood of a patient with fever,

be closely related or identical to Batai virus, which had malaise, myalgia and sore throat in Panama, but no previously been isolated from culicine mosquitoes in

further information on the virus is available. Tensaw Malaysia in 1955. Furthermore, the name ‘Olyka’ was

virus has been isolated from several species of provisionally used for Batai virus isolated from

anophelines in south-eastern USA, where antibody mosquitoes in the Ukraine, and Chittoor virus isolated

has been found in humans, dogs, cattle and raccoons. from anophelines in India is also considered to be

A single case of encephalitis was reported in 1973. closely related or identical to Batai virus. Altogether,

Wyeomyia virus has been isolated from a range of this cluster of viruses has been isolated from anophe-

culicine mosquitoes in Colombia, Panama, French line and culicine mosquitoes in Malaysia, Thailand,

Guiana and Trinidad, and antibody has been found in Cambodia, India, Yugoslavia, Austria, the former

human sera in Panama and Trinidad. The virus has USSR and Czechoslovakia. Antibody has been found

been isolated once from the blood of a patient with in the same countries, as well as in Sri Lanka,

febrile illness in Panama.

Serogroup Anopheles A Tacaiuma Virus

Tacaiuma virus was isolated from the blood of a sentinel monkey and from forest mosquitoes in the Amazon region of Brazil, as well as from mosquitoes in Argentina. Antibody has been found in humans in Brazil and French Guiana, and in horses, rodents, bats and birds in Brazil. The virus has been isolated once from the blood of a patient with febrile illness in Brazil.

Serogroup Bwamba Bwamba and Pongola Viruses

Bwamba virus was originally isolated from blood samples from nine road workers with febrile illness in Uganda, and subsequently from eight febrile patients in Nigeria, three in the Central African Republic and one in Kenya, and from anopheline mosquitoes in Uganda, Nigeria and Senegal. Antibody was found in human sera in Uganda, Tanzania, Mozambique, South Africa, Botswana, Angola, Congo, Nigeria and Guinea; generally with very high prevalence, up to 97% in some populations, and including both children and adults. Antibody was also found in donkeys and a bird in South Africa. Bwamba virus appears to be an important pathogen and the eight isolations in the Nigerian series represented 5% of all arbovirus isolations from febrile patients over a 7 year period, while 18 diagnoses (virological and serological) made in similar patients in the Central African Republic represented 25% of arbovirus infections diagnosed over a 13 year period. The patients suﬀered prostrating illness with fever, headache, conjunctivitis, rash, epigastric pain and myalgia, and many had meningeal signs.

There have been numerous isolations of Pongola virus from mosquitoes, mainly aedines and other culicines, in South Africa, Mozambique, Kenya, Uganda, Ethiopia, Central African Republic and Ivory Coast. There has been one isolation of the virus from a febrile patient with headache and myalgia in Uganda (Kalunda et al., 1985). Neutralising antibody has been found in humans in South Africa, Mozambique, Botswana, Namibia and Angola, and in cattle, sheep, goats and donkeys in South Africa, but interpretation of the ﬁndings is complicated by the fact that there is unidirectional cross-neutralisation of Pongola virus by antibody to its close relative, Bwamba

virus . Moreover, the fact that most human isolates have reacted as Bwamba serotype, while most mosquito isolates have reacted as Pongola serotype, merits further investigation, particularly in view of a report that passage of a Bwamba isolate in mosquitoes led to selection of virus reacting as Pongola serotype (Johnson et al., 1978).

Serogroup C

Apeu, Caraparu, Ossa, Madrid, Marituba, Murutucu, Restan, Nepuyo, Itaqui and Oriboca Viruses

Apart from one virus, which occurs in Florida, USA, all of the known members of serogroup C occur in Central and South America and tend to be associated with tropical forests. The viruses named above have all been isolated from the blood of febrile humans, and variously from sentinel monkeys, rodents, occasional marsupials and fruit bats, and from a range of culicine mosquitoes in Brazil, Surinam, French Guiana, Guatamala, Honduras, Trinidad, Panama or Mexico. Serological evidence suggests that some of these viruses may also occur in Venezuela, Colombia and Peru. Pairs of the viruses which are closely related antigenically may circulate in the same geographic location yet occupy separate ecological niches, e.g. one virus circulates in arboreal monkeys and mosquitoes which feed in the canopy layer, while a closely related virus circulates in rodents and mosquitoes which feed at the level of the forest ﬂoor. Other pairs of closely related viruses coexist in the same habitat simply by utilising diﬀerent vectors. Sporadic infections occur in persons who enter forests. No large outbreaks of disease have been reported, but disease is observed when susceptible outsiders, such as military personnel, enter endemic regions. Laboratory infections are relatively common. Disease, which lasts for up to a week and runs a benign course, is characterised by fever, rigors, headache, photophobia, conjunctivitis, tachycardia, myalgia, arthralgia, prostration, leukopenia and, occasionally, pain in the right upper quadrant of the abdomen and jaundice.

Serogroup California

California encephalitis, La Crosse, Snowshoe hare, Jamestown Canyon and Keystone Viruses

California encephalitis virus , which is distributed across the western USA and into Canada, was isolated from

BUNYAVIRIDAE

565

mosquitoes in the early 1940s and shortly thereafter serological evidence was produced to indicate that it causes encephalitis. However, from the mid-1960s onwards it became clear that most cases of what are loosely termed ‘California encephalitis’ are in fact due to infection with the La Crosse subtype of virus, and this agent is responsible for the majority of the approximately 100 cases of arbovirus encephalitis diagnosed in the USA annually, except in years when there are epidemics of St Louis encephalitis (a ﬂavivirus). It must also be borne in mind that since its introduction into North America in 1999, the mosquito-borne ﬂavivirus West Nile virus has been responsible for a proportion of cases of encephalitis recognised each year, particularly in elderly or immunocompromised patients.

Most cases of disease due to La Crosse virus are recorded in the mid-west states of Wisconsin, Iowa, Indiana, Minnesota and Ohio, but the virus is widely distributed and the infection is probably underdiag- nosed elsewhere in the USA. The principal vector is Aedes triseriatus , a tree hole-breeding mosquito, and accordingly the virus tends to be focally distributed in woodlands, but also occurs in suburban situations where water that collects in discarded containers, such as motor vehicle tyres, aﬀords mosquito breeding sites. The virus is passed transovarially in the vector and overwinters in mosquito eggs; infection is ampliﬁed in the succeeding spring and summer in small mammals such as chipmunks and squirrels. The vector is a diurnal feeder and the infection is seen most commonly in forest workers and children who enter woodlands for recreational purposes, but also occurs focally in rural and suburban residents. Males are more commonly infected than females, among both children and adults. Seroprevalence surveys and prospective studies indicate that most infections are inapparent or benign. Probably less than 1% of infected adults develop encephalitis, but the incidence may be up to four times greater in young children.

After an incubation period of 3–7 days there is sudden onset of fever, headache, lethargy, nausea and vomiting, pharyngitis and sometimes respiratory illness. There is seldom a cutaneous rash. In mild cases of overt disease there may be transient meningismus and disorientation and recovery within 1 week. In severe disease there may be greater disturbance of consciousness, aphasia, tremors, chorea, positive Babinski signs and other abnormal reﬂexes, and hemiparesis in about 20% of patients. Seizures may occur from the second day of illness onwards: in about half of severely ill patients there may be generalised, life-threatening convulsions, and in a further 25% there are focal

convulsions associated with frontal or parietal brain lesions. Approximately one-third of severely ill patients become comatose. There may be marked leukocytosis, and examination of cerebrospinal fluid reveals elevated mononuclear and polymorphonuclear cell counts, but protein and glucose levels tend to remain normal. Electroencephalograms show general- ized slow-wave activity or localised changes and sometimes epileptiform discharges. Treatment is symptomatic and includes monitoring and control of intracranial pressure and vigorous anticonvulsant therapy as indicated. Less than 1% of patients with severe disease succumb and most are discharged from hospital after about 2 weeks of illness, but may remain irritable and emotionally labile for a few weeks. There are seldom residua, but patients who suffer seizures in the acute illness may have recurrent convulsions over a period of 1 or more years, and lasting hemiparesis occurs in about 1% of patients. Virus has not been isolated from throat swabs, blood, stools or cerebrospinal fluid, and only with difficulty from brain specimens. Histopathological lesions are not pathognomonic and include cerebral oedema, perivascular cuffing and focal gliosis in grey matter. A variety of methods are used for making a serological diagnosis, but demonstration of IgM antibody activity by means of enzyme-linked immunoassay holds greatest promise as a rapid diagnostic technique. There is no vaccine.

Snowshoe hare virus is a mosquito-borne subtype of California encephalitis virus with a distribution extending from north-western USA across most of Canada to Alaska. The natural host appears to be the snowshoe hare, Lepus americanus, and serological evidence suggests that the virus is occasionally associated with encephalitis in children and adults.

Jamestown Canyon virus is a mosquito-borne virus which occurs widely in the USA, and high prevalence rates of antibody are found in white-tailed deer, and in humans where there are high concentrations of the deer. Serological evidence of an association with encephalitis in humans has been found from the early 1980s onwards (Grimstad et al., 1986). Since antigens and tests commonly used for the serodiagnosis of ‘California encephalitis’ fail to allow response to Jamestown Canyon virus to be distinguished from response to other members of the serogroup, it is felt that the infection has probably been missed or under- diagnosed as a cause of encephalitis in the past, both in the mid-west states and elsewhere where the deer occurs. In contrast to La Crosse virus, Jamestown Canyon virus appears to cause encephalitis more frequently in adults than in children, and nervous disease is often preceded by respiratory illness.

566

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

567 Keystone virus is associated with swamp-breeding

BUNYAVIRIDAE

unknown. Virus can be isolated from blood early in the aedines and cotton rats and rabbits in south-eastern

illness, but the infection is usually diagnosed by USA, and has been known to cause inapparent

demonstration of an antibody response. There is no accidental infection in the laboratory.

vaccine.

Inkoo virus was isolated from aedine mosquitoes in Finland. Distribution of the virus extends to the Tahyna and Inkoo Viruses

Lapland region in the north of the country, and antibody occurs in the sera of humans, cattle, deer and

Tahyna virus is widely distributed in countries of hares. Antibody prevalence rates of up to 25% have central Europe, including Yugoslavia, Germany and

been recorded in humans, and a few cases of febrile Italy, with antibody prevalence rates being particularly

illness have been conﬁrmed serologically. high in the Rhone Valley of France, the Danube basin in Austria and in the southern Moravia region of the former Czechoslovakia, where up to 95% of adults

Guaroa Virus

may be immune in some communities. Viruses described as being Tahyna-like have been isolated in

Guaroa virus has been isolated from 12 febrile patients the former USSR, while Lumbo virus, which was

in Colombia and the Amazon region of Brazil, and isolated from saltwater-breeding mosquitoes on the

from anopheline mosquitoes in Panama and Colombia. coast of Mozambique, is considered to be indistin-

Antibody has been found in human sera in Colombia, guishable from Tahyna virus. Antibody to Tahyna virus

Brazil, Argentina and Peru. Disease ascribed to has also been found in southern China and Sri Lanka.

infection with the virus was characterised by fever, It is not yet clear whether a single virus occurs

headache, myalgia, arthralgia, prostration and leuko- throughout this range or whether, as seems more

penia. Virus was isolated from the blood of the likely, a cluster of closely related viruses, subtypes or

patients, and from a liver biopsy in one instance. varieties is involved.

The epidemiology of the disease has been studied most intensively in Moravia where seasonal ﬂooding of level woodlands provides extensive breeding sites for

Serogroup Guama mosquitoes. The virus is transmitted transovarially in

Aedes vexans , which overwinters as eggs, and in

Guama and Catu Viruses

Culiseta annulata , which overwinters as larvae. Catu virus has been isolated from sentinel monkeys in Ampliﬁcation of infection in spring occurs in small

Brazil, febrile humans and forest rodents in Brazil and mammals such as hedgehogs, hares and rabbits, as well

Trinidad, and from culicine and anopheline mosqui- as in domestic animals such as horses. Hedgehogs may

toes in the same two countries plus French Guiana. themselves serve as reservoir hosts for overwintering of

Guama virus has been isolated from febrile humans and virus in instances where they undergo chronic infection

sentinel monkeys in Brazil, and from rodents and/or during hibernation, with viraemia which persists for a

culicine mosquitoes in Brazil, Trinidad, Surinam, few days after awakening. Once infection of vertebrates

French Guiana and Panama. The two viruses cause occurs in summer, other species of mosquito also

isolated cases of benign disease characterised by fever, become infected and serve as vectors for transmission

headache, myalgia and leukopenia, in persons who of the virus.

enter tropical forests. Catu virus has also been Seroprevalence surveys indicate that infection is

associated with laboratory infection. much more common than overt disease in rural residents of Moravia. Nevertheless, the infection accounts for up to 20% of patients hospitalised with

Serogroup Nyando febrile illness in the region, including both adults and

children. Patients may present with undiﬀerentiated febrile illness with leukocytosis, but pharyngitis,

Nyando Virus

cough, and chest pain and inﬁltration (demonstrable Nyando virus has been isolated from anopheline and by X-ray imaging) may predominate, or gastrointest-

aedine mosquitoes in Kenya, Central African Republic inal symptoms such as nausea, vomiting and

and Senegal. Antibody has been found in human sera abdominal pain may be dominant. Aseptic meningitis

in Kenya and Uganda, and the virus was isolated from occurs in a minority of patients but fatal disease is

the blood of a single human patient with biphasic the blood of a single human patient with biphasic

Serogroup Simbu Oropouche Virus

Oropouche virus was originally isolated from the blood of a febrile patient in Trinidad, but large epidemics involving thousands of people have occurred exclusively in northern Brazil over the past 20 years. In addition to humans, the virus has been isolated from a sloth, a few species of culicine mosquitoes and the midge Culicoides paraensis. Antibody has been found in the sera of humans and/or monkeys and birds in Brazil, Trinidad and Colombia. Virus is thought to be maintained in nature by circulation in forest primates, sloths or birds and an unidentiﬁed vector, and is introduced into urban settings by infected travellers or by extension from the sylvatic cycle. Humans serve as ampliﬁer hosts in the urban cycle, and the vector is the Culicoides midge, which breeds in decaying waste from tropical agricultural products. Epidemics occur when there are large concentrations of vectors and susceptible humans. Aerosol infection is suspected to have occurred in laboratory workers. The incubation period is 4–8 days, and there is sudden onset of fever, chills, headache, myalgia, arthralgia and prostration. There may be a rash, and occasionally signs of meningitis or encephalitis, but there are no deaths or sequelae. Viraemia lasts 2–5 days, as does illness, but myalgia persists for a further 3–5 days, and strenuous exertion in early convalescence can precipitate a relapse of symptoms.

Akabane, Aino, Tinaroo and Shuni Viruses Most members of the Simbu serogroup do not occur in

the Americas, but are widely distributed in Africa, Asia and Australasia. Of these latter viruses, only Shuni virus has been marginally implicated in causing human disease, but a few have been incriminated as causative agents in large outbreaks of abortion, stillbirth and congenital defects (hydranencephaly–arthrogryposis syndrome) in domestic ruminants, particularly sheep and cattle, and the suspicion exists that most members of the serogroup have the potential for producing this type of disease. Akabane virus has been incriminated in outbreaks of the disease in Japan, Australia and Israel, Aino virus in Japan and Australia, Tinaroo virus in

Australia and Peaton virus has been shown to be teratogenic in experimental infections in Australia. The viruses are transmitted by species of Culicoides midges which mostly breed in dung, independently of available surface water, but which are favoured by the humid conditions created by heavy rainfall. The natural hosts of the viruses are unknown, but antibodies have been found in wild herbivores. Infection is usually inapparent in domestic ruminants. However, if infection occurs in pregnant animals at critical stages of gestation, there may be embryonal or fetal death, or arrestation of brain development (hydranencephaly), and the consequent lack of trophic effect of nervous stimulation on skeletal muscles of the fetus results in postural defects of the limbs, with joints locked in flexion (arthrogryposis). Once the stage of organogen- esis in gestation is past, fetuses are less susceptible to the harmful effects of infection (the timing varies with length of gestation in different species).

Shuni virus has been isolated from cattle, sheep, midges and once from the blood of a febrile human in Nigeria, and from cattle and mosquitoes in South Africa. It was also isolated from the brain of a horse with histopathological lesions of meningoencephalitis, which was submitted for laboratory examination for suspected rabies in Zimbabwe (Foggin and Swanepoel, 1977).

GENUS PHLEBOVIRUS Serogroup Sandfly fever

Sandfly fever Naples, Sandfly fever Sicilian and Toscana Viruses

Sandﬂy fever (also known as phlebotomus fever or pappataci fever from the vector, Phlebotomus papatasi) has been known for at least two centuries as a febrile illness encountered by armies invading the Mediterra- nean basin. It was demonstrated shortly after the start of the twentieth century that the disease was caused by

a virus transmitted by sandﬂies, but it was only during the Second World War that it was shown that there are in fact two distinct viruses and that there is no cross- immunity. Between them, the two viruses are known to occur in Morocco, Tunisia, Egypt, Sudan, Somalia, Italy, Greece, Yugoslavia, Turkey, the former USSR, Israel, Saudi Arabia, Iraq, Iran, Pakistan, India and Bangladesh, and are probably present in many intervening countries. Throughout this range, Phle- botomus papatasi is the vector and it breeds in moist soil in dark niches such as in rubble, drains, cracks in

568

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

soil and in animal burrows, equally successfully in large cities and remote rural locations. The viruses are transmitted transovarially in the vector, which overwinters in the larval stage, and adult flies emerge to assume biting activity in summer. High infection rates have been recorded in newly emerged sandflies and it is not known whether amplification in vertebrates is essential to ensure perpetuation of the viruses, but humans develop sufficiently intense viraemia to serve as source for the infection of the vector. No wild vertebrate hosts of the viruses are known, but antibody has been found in gerbils (whose burrows are utilised by the vector). Sandflies are nocturnal feeders, but will feed in dark rooms where they rest during daylight hours. Human infection rates recorded in outbreaks range from 3–75% but the attack rate varies focally and is influenced by background immunity in the human population. Large epidemics have often occurred in association with socio-economic uphea- vals, wars or natural disasters, such as earthquakes, which create ideal breeding conditions for sandflies and/or lead to widespread exposure of susceptible humans to sandflies.

Experimental evidence suggests that most sandfly fever infections are symptomatic. Typically, there is sudden onset of fever of 2–4 days duration, severe headache, sore eyes and photophobia, myalgia, arthralgia, anorexia and malaise. Occasionally there may be sore throat, nausea and vomiting, abdominal pain and diarrhoea, epistaxis and dizziness. Patients may have injected conjunctivae and a flushed appearance, but there is seldom a rash and meningeal signs are rare. The disease may be milder in children. Treatment is symptomatic. Recovery is complete and no deaths have been recorded. There appears to be lifelong immunity to the homologous virus. The diagnosis can be confirmed by isolation of virus from blood taken in acute illness, or demonstration of IgM antibody activity, or rising antibody titres in convalescence. Prevention of infection includes the use of insect repellants, but the treatment of walls (sandfly resting sites) with residual insecticides is highly effective.

Toscana virus was ﬁrst isolated in 1971 in Tuscany, Italy, from Phlebotomus perniciosus, a sandﬂy that breeds in forest litter. Antibody was found to be common in the sera of rural and suburban residents of the region, and an association was established between the occurrence of aseptic meningitis and serological evidence of infection with Toscana virus. Since then several cases of meningitis due to the infection have been encountered regularly each year in summer months in the endemic region, and these can sometimes be diagnosed by isolation of virus from

cerebrospinal fluid in acute illness, but usually by demonstrating IgM antibody activity or rising antibody titres in sera taken during convalescence. The virus is transmitted transovarially in the vector, P. perniciosus , and no vertebrate maintenance host has been identified, but antibody has been found in rodent sera, and the virus has been isolated from an insectivorous bat. The vector is widely distributed in Europe and the virus has been isolated from cerebrospinal fluid from a meningitis patient in Portugal, so the disease may occur more widely than at present recognised.

Alenquer, Candiru, Punta Toro and Chagres Viruses

Alenquer virus and Candiru virus were isolated from the blood of febrile patients in the Amazon region of Brazil, but otherwise little is known of their biology, and it is surmised that they are transmitted by phlebotomid flies. Punta Toro virus and Chagres virus were isolated from febrile patients and from phlebotomids (Lutzomyia spp.) in Panama. These two viruses are known to be transmitted transovarially in phlebotomids, and antibodies to them have been found in primates, sloths, porcupines and other rodents. Disease thus far associated with all four viruses fits the description of classical sandfly fever, with the difference that epidemics are unknown and only isolated cases have been seen in persons who entered tropical forests for occupational or recreational purposes.

Rift Valley Fever Virus The literature on Rift Valley fever has been reviewed

extensively (Henning, 1956; Weiss, 1957; Easterday, 1965; Peters and Meegan, 1981; Shimshony and Barzilai, 1983; Meegan and Bailey, 1989; Swanepoel and Coetzer, 2003). It is an acute disease of domestic ruminants in mainland Africa and Madagascar, caused by a mosquito-borne virus and characterised by necrotic hepatitis and a haemorrhagic state, but infections are frequently inapparent or mild. Large outbreaks of the disease in sheep, cattle and goats are distinguished by heavy mortality among newborn animals and abortion in pregnant animals. Humans become infected from contact with tissues of infected animals or from mosquito bite, and usually develop mild to moderately severe febrile illness, but severe complications occur in a small proportion of patients.

BUNYAVIRIDAE

569

The virus recently escaped from the African region to cause a major outbreak of disease on the Arabian Peninsula in 2000–2001.

The disease was ﬁrst recognised in the Rift Valley in Kenya early in the twentieth century, but the causative agent was not isolated until 1930. Since then large outbreaks of the disease have been recorded in Kenya, South Africa, Namibia, Mozambique, Zimbabwe, Zambia, Sudan, Egypt, Mauritania and Senegal, while lesser outbreaks, periodic isolations of virus or serological evidence of infection have been recorded in Angola, Botswana, Burkina Faso, Cameroon, Central African Republic, Chad, Gabon, Guinea, Madagascar, Malawi, Mali, Nigeria, Somalia, Tanzania, Uganda and Zaire. Epidemics may be extremely severe and, for example, it is estimated that 500 000 ewes aborted and

a further 100 000 sheep died in the ﬁrst outbreak of the disease to be recognised in South Africa in 1950–1951. Prior to the 1970s, epidemics were seen only in eastern and southern Africa, where they tend to occur at irregular intervals of 5–15 years or longer when above-average rainfall favours the breeding of the mosquito vectors. Meteorological conditions conducive to the occurrence of epidemics usually prevail over large tracts of Africa, so there has been some tendency for outbreaks in adjacent territories, such as Zimbabwe and Mozambique, Kenya and Tanzania or South Africa and Namibia, to coincide. The fate of the virus during inter-epidemic periods was unknown for decades, but on the basis of observations made in Uganda, Kenya and South Africa, it was widely accepted that the virus was endemic in indigenous forests, which extend in broken fashion from East Africa to the coastal regions of South Africa. The virus was thought to circulate in Eretmapodites spp. mosquitoes and unknown vertebrates in the forests, and to spread in seasons of exceptionally heavy rainfall to livestock- rearing areas, where the vectors were believed to be ﬂoodwater-breeding aedine mosquitoes of the sub- genera Aedimorphus and Neomelaniconion, which attach their eggs to vegetation at the edge of stagnant surface water. In contrast to other culicine mosquitoes, it is obligatory that the eggs of aedines be subjected to

a period of drying as the water recedes before they will hatch on being wetted again when next the area ﬂoods. Thus, the aedine mosquitoes overwinter as eggs, which can survive for long periods in dried mud, possibly for several seasons if the area remains dry.

On the inland plateau of South Africa, where sheep rearing predominates, surface water gathers after heavy rains in undrained shallow depressions (pans) and farm dams which aﬀord ideal breeding environments for aedines. On the watershed plateau of

Zimbabwe, where cattle farming predominates, aedines breed in ‘vleis’, low-lying grassy areas which constitute drainage channels for surrounding high ground and which are ﬂooded by seepage after heavy rains. Vleis correspond to what are termed ‘dambos’ in the livestock-rearing areas of central and eastern Africa. Sustained monitoring in Zimbabwe revealed that a low level of virus transmission to livestock occurred each year in the same areas where epidemics occurred. The generation of epidemics, therefore, was associated with the simultaneous intensiﬁcation of virus activity over vast livestock-rearing areas where it was already present, rather than lateral spread from cryptic endemic foci. Comparison of the distributions of canopy forests and vleis in Zimbabwe, plotted from satellite images and aerial photographs, with the distribution determined for endemic Rift Valley fever, revealed remarkable overlap between the endemic areas and areas where vleis were common.

A major advance in the understanding of the epidemiology of the disease was made when the virus was isolated from unfed Aedes mcintoshi mosquitoes (=Aedes lineatopennis sensu lato) hatched in dambos on a ranch in Kenya during inter-epidemic periods in 1982 and 1984, conﬁrming that the virus is endemic in livestock-rearing areas and indicating that it appears to

be maintained by transovarial transmission in aedines. The available evidence suggests that in Zimbabwe, as in Kenya, A. mcintoshi is the most important maintenance vector of the virus, while A. dentatus is probably also a maintenance vector; the same two species, and possibly A. unidentatus and A. juppi, are maintenance vectors on the inland plateau of South Africa. Heavy rainfall and the humid conditions which prevail during epidemics favour the breeding of other biting insects besides aedine mosquitoes. Following extensive flooding of aedine breeding sites, significant numbers of livestock become infected and circulate high levels of virus in their blood during the acute stage of infection. Other culicines and anopheline mosquitoes then become infected and serve as epidemic vectors, particularly Culex theileri in southern Africa, and biting flies such as midges, phlebotomids, stomox- ids and simulids serve as mechanical transmitters of infection. Although contagion has been demonstrated on occasion under artificial conditions, non-vectorial transmission is not considered to be important in livestock, as opposed to humans. Epidemics generally become evident in late summer, after there has been an initial increase in vector populations and in circulation of virus, and terminate in late autumn, when the onset of cold weather depresses vector activity, or when most animals are immune following natural infection, or

570

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

571 after there has been successful intervention with

BUNYAVIRIDAE

known to be identical to Rift Valley fever virus. vaccine.

Various theories were advanced to account for the Antibody surveys and laboratory studies have failed

ﬁrst known appearance of the virus in Egypt in 1977, to prove that the virus is maintained in transmission

including the carriage of infected mosquitoes from the cycles in rodents, birds, monkeys, baboons or other

Sudan at high altitude by prevailing winds associated wild vertebrates, although it is felt that wild ruminants

with the inter-tropical convergence zone. The intro- could play a role similar to their domestic counterparts

duction of the virus through the transportation of in areas where they predominate. It is also believed

infected sheep and cattle on the Nile or overland from that the possibility of endemicity of the virus in forests

northern Sudan to markets in southern Egypt was cannot be dismissed entirely, and merits further

considered to have been the strongest possibility, and investigation.

the movement of slaughter animals by sea could It was recognised from the time of the original

account for the evidence of infection detected in the investigations in Kenya that febrile illness in humans

northern and eastern coastal areas of Egypt. Although accompanied outbreaks of disease in livestock, and

transportation on some routes would take a long time that some patients experienced transient loss of visual

in relation to the course of the infection, Rift Valley acuity, but the occurrence of serious ocular sequelae

fever virus has been shown to persist for prolonged was ﬁrst reported in the 1950–1951 epidemic in South

periods in various organs of sheep, particularly the Africa. Human deaths following natural infection were

spleen, for up to 21 days after infection. The same ﬁrst recorded in South Africa during the epidemic of

could be true for goats and cattle, or even the camels 1974–1976, when seven patients are known to have

brought in by overland caravan routes. It is believed died of encephalitis and haemorrhagic fever associated

that humans slaughtering or handling the tissues of with necrotic hepatitis. Subsequently, deaths were also

such animals could have become infected and served as observed in Zimbabwe.

the amplifying hosts for the infection of mosquitoes Outbreaks of Rift Valley fever were reported in the

since the main vector in the Egyptian epidemic, Culex Sudan in 1973 and 1976. In 1977 and 1978 a major

pipiens , is known to be peridomestic and anthropo- epidemic occurred along the Nile delta and valley in

philic. In at least one instance there were indications Egypt, causing an unprecedented number of human

that human infections centred on a location where infections and deaths, as well as numerous deaths and

introduced camels were slaughtered. abortions in sheep and cattle and some losses in goats,

The occurrence of the epidemic in Egypt raised the water buﬀaloes and camels. Estimates of the number

spectre that Rift Valley fever could be introduced to of human cases of disease range from 18 000 to

the mainland of Eurasia, and the possibility was

4 200 000, with at least 598 deaths occurring from underscored by the fact that the virus is apparently encephalitis and/or haemorrhagic fever. A severe

capable of utilising a wide range of mosquitoes as epidemic occurred in 1987 in the Senegal River basin

vectors. Extensive preventive vaccination of livestock of southern Mauritania and northern Senegal. In

was undertaken at the time in the Sinai peninsula and Mauritania alone an estimated 224 human patients

Israel. However, only isolated outbreaks of Rift Valley died of the disease, and there was a high rate of

fever were recorded in Egypt in 1979 and 1980, and abortion in sheep and goats. Further outbreaks of Rift

thereafter the country remained free of the disease for Valley fever occurred in Mauritania in 1993 and 1998,

12 years, until ocular complications of the infection in with smaller numbers of human casualties, while a

humans and abortions in cattle and water buﬀalo were minor outbreak of disease in livestock occurred in

noted in the Aswan Governate in May 1993. On this Senegal in 1994–1995.

occasion there was not the same tendency for an The outbreaks of Rift Valley fever which occurred in

explosive outbreak of the disease to occur as in 1977– North and West Africa diﬀered in many respects from

1978, but by October 1993 infections of humans and the pattern of disease which had hitherto been

livestock, including sheep, had been recognised across observed in sub-Saharan Africa; in particular, they

the length of the country in Sharqiya, Giza and El occurred independently of rainfall in arid countries,

Faiyum Governates, and further infections were apparently in association with vectors which breed in

observed in 1994 (Anonymous, 1993, 1994; Arthur et large rivers and dams. The presence of the virus in the

al. , 1993; Botros et al., 1997). Sudan and certain West African countries had long

From late October 1997 to February 1998, a large been known from antibody studies, and there had been

outbreak of Rift Valley fever occurred in north-eastern periodic isolations of the virus in West Africa, where it

Kenya and adjoining southern Somalia, following the was sometimes reported as Zinga virus, which is now

occurrence of heavy rains and extensive ﬂooding in occurrence of heavy rains and extensive ﬂooding in

In September 2000, Rift Valley fever broke out simultaneously in Jizan Province in south-west Saudi Arabia and in adjoining Yemen. The outbreaks lasted until early 2001, and resulted in 245 human deaths and the loss of thousands of sheep and goats. There had been heavy rains in the inland mountain range which runs parallel to the coast, with drainage from the mountains resulting in the creation of ideal mosquito- breeding habitats (Jupp et al., 2002). There was speculation that the virus may have been imported from Africa with slaughter animals, or carried from Africa by wind-borne mosquitoes in 2000, but there were no known epidemics in the Horn of Africa at the time. It is much more likely that infected animals were imported during the 1997–1998 epidemic in East Africa, and that infection had smouldered on the Arabian Peninsula until ideal circumstances for an epidemic occurred following heavy rains in 2000. It remains to be determined whether the virus has become endemic on the Arabian Peninsula.

In contrast to the main vector in the Egyptian epidemic of 1977–1978, the principal mosquito vectors of Rift Valley fever virus in sub-Saharan Africa tend to

be zoophilic and sylvatic, with the result that humans become infected mainly from contact with animal tissues, although there are instances where no such history can be obtained and it must be assumed that infection has resulted from mosquito bite. Occasional infections diagnosed in tourists from abroad who visited countries in Africa are also thought to have resulted from mosquito bites. Generally, persons who become infected are involved in the livestock industry,

such as farmers who assist in dystocia of livestock, farm labourers who salvage carcases for human consumption, veterinarians and their assistants, and abattoir workers. There are numerous reports of humans becoming infected while investigating the disease in the ﬁeld or laboratory, and the ﬁrst known human fatality was recorded in 1934 in a laboratory worker, but since the infection was complicated by thrombophlebitis and the patient died from pulmonary embolism, the potential lethality of the virus for man was overlooked until fatal infections were recognised during the 1974–1976 epidemic in South Africa. The results of surveys following epidemics in southern Africa indicated that 9–15% of farm residents became infected, with a slight preponderance of adult males, although it appeared that housewives also gained infection from handling fresh meat.

No outbreaks of the disease have been recognised in urban consumer populations and it is surmised that the fall in pH associated with the maturation of meat in abattoirs is deleterious to the virus. Moreover, highest infection rates were found in workers in the by- products sections of abattoirs in Zimbabwe, and the implication is that the carcases of infected animals which reach abattoirs are generally recognised as being diseased and are condemned as unﬁt for human consumption.

Human infection presumably results from contact of virus with abraded skin, wounds or mucous membranes, but aerosol and intranasal infection have been demonstrated experimentally and circumstantial evidence suggests that aerosols have been involved in some human infections in the laboratory, and in the ﬁeld during the Egyptian epidemic. Many infections in Egypt are thought to have resulted from the slaughter of infected animals outside of abattoirs, and the fact that the mosquito vector was anthropophilic is thought to explain the high incidence of infection which occurred in people of all ages and diverse occupations. Low concentrations of virus have been found in milk and body ﬂuids, such as saliva and nasal discharges of sheep and cattle, and it appears that there may have been a connection between human infection and consumption of raw milk in Mauritania. In view of the intense viraemia which occurs in humans and the fact that virus has been isolated from throat washings, it is curious that there are no records of person-to-person transmission of infection.

Despite the sudden and dramatic change perceived in the nature of the human disease in the mid-1970s, it was deduced from the 598 reported deaths and 200 000 estimated cases of disease that Rift Valley fever had a case fatality rate of less than 1% in Egypt, where a

572

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

high prevalence of schistosomiasis may have predisposed the population to severe liver disease. The fatality rate may even have been lower in relation to total infections, since an antibody prevalence rate of 30% was detected and the human population was estimated at 1–3 million in the areas aﬀected by the epidemic. On the other hand, remarkably high estimates of 5% and 14% were made for case fatality rates in two separate populations in the 1987 epidemic in Mauritania, on the basis of the proportion of IgM antibody-positive persons who actually reported illness considered to be compatible with Rift Valley fever, but it can be deduced that the fatality rates in terms of total IgM antibody-positive persons are much closer to the corresponding fatality rate in Egypt.

The majority of Rift Valley fever infections in humans are inapparent or associated with moderate to severe, non-fatal, febrile illness. After an incubation period of 2–6 days, the onset of the benign illness is usually very sudden and the disease is characterised by rigor, fever that persists for several days and is often biphasic, headache with retro-orbital pain and photophobia, weakness, and muscle and joint pains. Sometimes there is nausea and vomiting, abdominal pain, vertigo, epistaxis and a petechial rash. Deferves- cence and symptomatic improvement occur in 4–7 days in benign disease and recovery is often complete in 2 weeks, but in a minority of patients the disease is complicated by the development of ocular lesions at the time of the initial illness or up to 4 weeks later. Estimates for the incidence of ocular complications range from 51% to 20% of human infections, and possibly the diﬀerences stem from failure to record mild cases in populations where illiterate persons are less likely to report minor disturbances of vision. The ocular disease usually presents as a loss of acuity of central vision, sometimes with development of scoto- mas. The essential lesion appears to be focal retinal ischaemia, generally in the macular or paramacular area, associated with thrombotic occlusion of arterioles and capillaries, and is characterised by retinal oedema and loss of transparency, caused by dense white exudate and haemorrhages. Sometimes there is severe haemorrhage and detachment of the retina. The lesions and the loss of visual acuity generally resolve over a period of months with variable residual scarring of the retina, but in instances of severe haemorrhage and detachment of the retina there may be permanent uni- or bilateral blindness.

Probably 51% of human patients develop the haemorrhagic and/or encephalitic forms of the disease. Underlying liver disease may predispose to the haemorrhagic form of the illness. The haemorrhagic

syndrome starts with sudden onset of febrile illness similar to the benign disease, but within 2–4 days there may be development of a petechial rash, purpura, ecchymoses and extensive subcutaneous haemorrhages, bleeding from needle puncture sites, epistaxis, haematemesis, diarrhoea and melaena, sore and inﬂamed throat, gingival bleeding, epigastric pain, hepatomegaly or hepatosplenomegaly, tenderness of the right upper quadrant of the abdomen and deep jaundice. This is followed by pneumonitis, anaemia, shock with racing pulse and low blood pressure, hepatorenal failure, coma and cardiorespiratory arrest. Factors contributing to fatal outcome in the hepatic form of the disease include anaemia, shock and hepatorenal failure, with the kidney lesions possibly being as important as shock in producing anuria. A proportion of the less severely aﬀected patients may make a protracted recovery without sequelae.

Encephalitis may occur in combination with the haemorrhagic syndrome. Otherwise, signs of encephalitis in humans may supervene during the acute illness or up to 4 weeks later and include severe headache, vertigo, confusion, disorientation, amnesia, meningismus, hallucinations, hypersalivation, grinding of teeth, choreiform movements, convulsions, hemiparesis, lethargy, decerebrate posturing, locked-in syndrome, coma and death. A proportion of patients may recover completely, but others may be left with sequelae, such as hemiparesis.

Abortion is the usual, if not invariable, outcome to infection of pregnant ruminants, but an attempt to relate the occurrence of abortion in humans to evidence of Rift Valley fever infection in Egypt produced inconclusive results.

By analogy with the course of events believed to follow natural infection with other arthropod-borne viruses, it can be surmised that the pathogenesis of the disease may involve some replication of virus at the site of inoculation, conveyance of infection by lymphatic drainage to regional lymph nodes, where there is further replication with spillover of virus into the circulation to produce primary viraemia, which in turn leads to systemic infection, and that intense viraemia then results from release of virus following replication in major target organs. Wild Rift Valley fever virus, which has not been subjected to serial passaging in laboratory host systems, is described as being hepato-, viscero- or pantropic, and immunoﬂuorescence studies in laboratory animals indicate that replication occurs in littoral macrophages of lymph nodes, most areas of the spleen except T-dependent peri-arteriolar sheaths, foci of adrenocortical cells, virtually all cells of the liver, most renal glomeruli and some tubules, lung

BUNYAVIRIDAE

573

tissue and scattered small vessel walls, as well as in necrotic foci in the brains of individuals who develop the encephalitic form of the disease. These sites correspond to the lymphoid necrosis in lymph nodes and spleen, hepatic necrosis and adrenal, lung and glomerular lesions seen in humans and livestock, and the brain lesions in humans (encephalitis has not been described in natural disease of ruminants). Titration of infectivity in organ homogenates indicates that the liver and spleen are the major sites of virus replication. Cell damage is ascribed directly to the lytic effects of the virus, but the inflammatory response seen in human brain tissue suggests that there may also be an immunopathological element to the pathogenesis of encephalitis. The same may be true for ocular lesions. Recovery is mediated by non-specific and specific host responses, and the clearance of viraemia correlates with the appearance of neutralising antibody. No significant antigenic differences have been detected between isolates of the virus, although differences in pathogenicity for laboratory rodents have been demonstrated and immunity appears to be lifelong.

The haemostatic derangement which occurs in Rift Valley fever has been investigated in rhesus monkeys, but the mechanisms involved remain speculative. Impairment of coagulation occurs even in benign infection of monkeys, and moderate thrombocytopenia has been observed in benign infection in sheep, but haemostatic derangement is most severe in the fatal hepatic syndrome. It is postulated that the critical lesions are vasculitis and hepatic necrosis. Destruction of the antithrombotic properties of endothelial cells is thought to trigger intravascular coagulation, and the widespread necrosis of hepatocytes and other aﬀected cells to result in the release of procoagulants into the circulation. Severe liver damage presumably limits or abolishes production of coagulation proteins and reduces clearance of activated coagulation factors, thereby further promoting the occurrence of disseminated intravascular coagulopathy, which in turn augments tissue injury by impairing blood ﬂow. Vasculitis and haemostatic failure result in purpura and widespread haemorrhages.

Clinical pathology ﬁndings in humans are compatible with observations made in haematological and coagulation studies on monkeys, except that leukocytosis and anaemia may be more marked in severe human disease (Al Hazmi et al., 2003). In most species there is an initial leukopenia followed by leukocytosis, and the same may be true for humans. Monkeys may have prolonged activated partial thromboplastin times and prothrombin times even in benign infection, and in severe liver disease there may be depletion of

coagulation factors II, V, VII, IX, X and XII, thrombocytopenia and platelet dysfunction, increased schistocyte counts and depletion of ﬁbrinogen, together with raised ﬁbrin degradation product levels. Raised serum aspartate aminotransferase and alanine aminotransferase levels have been recorded even in benign disease in humans.

Treatment is essentially symptomatic, and supportive therapy in the haemorrhagic disease includes replacement of blood and coagulation factors. Results obtained in animal models suggest that the administration of immune plasma from recovered patients may be beneﬁcial. The antiviral drug ribavirin inhibits virus replication in cell cultures and laboratory animals, and it has been suggested that it could be used even in benign disease in order to obviate the potentially serious complications which may occur in humans.

Specimens to be submitted for laboratory conﬁrma- tion of the diagnosis include blood from live patients, and tissue samples, particularly liver, but also spleen, kidney, lymph nodes and heart blood of deceased patients. Tissue samples should be submitted in duplicate in a viral transport medium, and in 10% buﬀered formalin for histopathological examination.

Viral antigen can frequently be detected rapidly in tissues and/or blood by a variety of immunological methods, including immunodiffusion, complement- fixation, immunofluorescence and enzyme-linked immunoassay. Viraemia lasts for up to a week. The virus is cytopathic and can be isolated readily in almost all cell cultures commonly used in diagnostic laboratories, and identified rapidly by immunofluorescence. Virus can also be isolated in suckling or weaned mice, or hamsters, inoculated intracerebrally or intraperitoneally, and antigen can be identified in harvested brain or liver by the immunological methods mentioned above. Definitive identification of isolates is achieved by performing neutralisation tests with reference antiserum.

Histopathological lesions, particularly those in the liver, are considered to be pathognomonic, and are essentially similar in humans and domestic ruminants. The severity of the lesions varies from primary foci of coagulative necrosis, consisting of clusters of hepatocytes with acidophilic cytoplasms and pyknotic nuclei, multifocally scattered throughout the parenchyma, to massive liver destruction in which the primary foci comprising dense aggregates of cytoplasmic and nuclear debris, some ﬁbrin and a few neutrophils and macrophages, can be discerned against a background of parenchyma reduced by nuclear pyknosis, karyor- rhexis and cytolysis to scattered fragments of

574

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

575 cytoplasm and chromatin, with only narrow rims of

BUNYAVIRIDAE

disease. The remaining members of the serogroup have degenerated hepatocytes remaining reasonably intact

been isolated from ticks associated with passerine or close to portal triads. Intensely acidophilic cytoplasmic

sea birds, and have no known medical or veterinary bodies, which resemble the Councilman bodies of

signiﬁcance.

yellow fever, are common, and rod-shaped or oval eosinophilic intranuclear inclusions may be seen in intact nuclei. Icterus may be evident.

Antibody to Rift Valley fever virus can be demonstrated in complement ﬁxation, enzyme-linked

GENUS NAIROVIRUS immunoassay, indirect immunoﬂuorescence, haemag-

Serogoup Crimean-Congo Haemorrhagic Fever Diagnosis of recent infection is conﬁrmed by demonstrating seroconversion or a four-fold or greater rise in

glutination-inhibition or

neutralisation

tests.

Crimean-Congo Haemorrhagic Fever Virus titre of antibody in paired serum samples, or by

The literature on CCHF is the subject of several demonstrating IgM antibody activity in an enzyme-

comprehensive reviews (Chumakov, 1974; Hoogstraal, linked immunoassay.

1979, 1981; Watts et al., 1989). A disease given the Benign Rift Valley fever in humans must be

name ‘Crimean haemorrhagic fever’ was ﬁrst observed distinguished from other zoonotic diseases, such as

on the Crimean peninsula in 1944, and it was brucellosis and Q fever, while the fulminant hepatic

demonstrated through the inoculation of human disease must be distinguished from the so-called

subjects that the disease was caused by a tick- formidable viral haemorrhagic fevers of Africa: Lassa

transmitted virus, but the virus itself was only isolated fever, Crimean-Congo haemorrhagic fever, Marburg

in laboratory hosts (mice) in 1967. In 1969, it was disease and Ebola fever. The occurrence of HFRS

shown that the agent of Crimean haemorrhagic fever associated with hantavirus infections, is also a

was identical to a virus named ‘Congo’ which had been theoretical possibility in Africa.

isolated in 1956 from the blood of a febrile child in An inactivated and a live attenuated vaccine are

Stanleyville (now Kisangani) in what was then the available for immunisation of livestock, but it is

Belgian Congo (now Zaire), and since that time the usually diﬃcult to persuade farmers to vaccinate

two names have been used in combination. livestock during long inter-epidemic periods. The

The distribution of CCHF virus extends over eastern attenuated vaccine confers lifelong immunity in

Europe, Asia and Africa: the presence of the virus or sheep, but is abortigenic and teratogenic in a small

antibody to it has been demonstrated in the former proportion of pregnant ewes. The attenuated vaccine is

USSR, Bulgaria, Greece, Turkey, Hungary, Yugosla- poorly immunogenic in cattle and they are immunised

via, France, Portugal, Kuwait, Dubai, Sharjah, Iraq, annually with the inactivated vaccine. A formalin-

Iran, Afghanistan, Pakistan, India, China, Egypt, inactivated cell culture vaccine produced in the USA is

Ethiopia, Mauritania, Senegal, Burkina Faso, Benin, used on an experimental basis to immunise persons,

Nigeria, Central African Republic, Zaire, Kenya, such as laboratory and ﬁeld workers, who are regularly

Uganda, Tanzania, Zimbabwe, Namibia, South Africa exposed to Rift Valley fever infection.

and Madagascar. However, the evidence for France and Portugal is based on limited observations and needs to be conﬁrmed.

Serogroup Uukuniemi In many instances virus or antibody was discovered in deliberate surveys, but in some countries of eastern Uukuniemi virus Europe and Asia the presence of CCHF ﬁrst became evident in nosocomial outbreaks of disease, or in Uukuniemi virus was originally isolated in Finland in

epidemics which arose in circumstances where humans 1960 from Ixodes ricinus ticks, which parasitise live-

were exposed to ticks and livestock on a large scale, stock but also bite humans. The virus has subsequently

such as in major land reclamation or resettlement been isolated from ticks, birds and ﬁeld mice in

schemes in Bulgaria and parts of the former USSR. Finland, Norway, Poland, Lithuania and the former

Notable outbreaks of the disease in Eurasia during USSR and Czechoslovakia. Antibody to the virus has

recent years have resulted from the exposure of people been found in human sera in Finland, Hungary and

to blood and ticks from slaughter stock imported from former Czechoslovakia, but no evidence has been

Africa and Asia to Saudi Arabia in 1990, the United presented to indicate that infection is associated with

Arab Emirates in 1994–1995, and Oman in 1995, plus

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

large-scale exposure of war refugees to outdoor trans-stadial transmission of virus by adult ticks to conditions in Kosovo in 2000 and Albania in 2001

large vertebrates.

(El Azazy and Scrimgeour, 1997; Khan et al., 1997; Young ruminants generally acquire natural infection Papa et al., 2002a, 2002b; Scrimgeour et al., 1996;

early in life and are viraemic for about a week. Williams et al., 2000). Prior to 1981, a total of 15 cases

Humans become infected when they come into contact of the disease had been reported in Africa, eight of

with the viraemic blood of young animals in the course them laboratory infections, and only one patient had

of performing procedures such as castrations, vaccina- developed haemorrhagic manifestations and died.

tions, inserting ear tags or slaughtering the animals. Since then sporadic cases of haemorrhagic disease

Animals which are raised under tick-free conditions and deaths have been diagnosed regularly each year in

and moved to infested locations later in life may southern Africa, probably as a result of increased

acquire tick-borne diseases of livestock at the same awareness among clinicians, and severe disease has

time that they become infected with CCHF virus, and also been recorded elsewhere in Africa.

consequently humans become infected from contact The virus has been isolated from at least 29 species

with viraemic blood in the course of treating sick of ixodid ticks, but for most species there is no

animals or butchering those that die. The available deﬁnitive evidence that they are capable of serving as

evidence suggests that the infection in humans is vectors, and in some instances the virus recovered from

acquired through contact of viraemic blood with engorged ticks may merely have been present in the

broken skin, and this accords with the fact that bloodmeal imbibed from a viraemic host. Members of

nosocomial infection in medical personnel usually three genera of ixodid ticks, Hyalomma, Dermacentor

results from accidental pricks with needles contami- and Rhipicephalus, have been shown to be capable of

nated with the blood of patients, or similar mishaps. transmitting infection trans-stadially and transova-

Common source outbreaks involving more than one rially, but Hyalomma ticks are considered to be the

case of the disease can occur when several people are principal vectors, and with the exception of

exposed to infected tissues. Infection appears to be Madagascar the known distribution of CCHF virus

limited to those who have contact with fresh blood or coincides with the world distribution of members of

other tissues, probably because infectivity is destroyed this genus of ticks. Moreover, the prevalence of

by the fall in pH which occurs in tissues after death, antibody to CCHF virus detected in the sera of wild

and there has been no indication that CCHF virus vertebrates in southern Africa was highest in large

constitutes a public health hazard in meat processed herbivores, known to be the preferred hosts of adult

and matured according to normal health regulations. Hyalomma ticks, and in small mammals such as hares,

Many human infections result directly from tick bite, which are the preferred hosts of immature Hyalomma.

and it has been observed that people can also become Mammals of intermediate size, and passerine and

infected from merely squashing ticks between the water birds, generally lacked evidence of infection, but

ﬁngers. Some patients are unable to recall contact antibody was found in ostriches, which are known to

with blood or other tissues of livestock, or having been

be parasitised by adult Hyalomma ticks. Virus or bitten by ticks, but live in or have visited a rural antibody has also been demonstrated elsewhere in the

environment where such exposure to infection is sera of small mammals of Eurasia and Africa, such as

possible. Town dwellers sometimes acquire infection little susliks, hedgehogs, hares and certain myomorph

from contact with animal tissues or tick bite while on rodents, and in some instances it has been shown that

hunting or hiking trips.

these hosts develop viraemia of suﬃcient intensity to The majority of patients tend to be adult males infect ticks.

engaged in the livestock industry, such as farmers, High prevalences of antibody occur in domestic

herdsmen, slaughtermen and veterinarians. Seropreva- ruminants in areas infested by Hyalomma ticks and the

lence studies indicate that infection of humans is virus causes inapparent infection or mild fever in

uncommon despite the widespread evidence of infec- cattle, sheep and goats, with viraemia of suﬃcient

tion in livestock, and this may be explained by the facts intensity to infect ticks. It is doubtful whether

that viraemia in livestock is short-lived, and of low transovarial transmission occurs with suﬃcient fre-

intensity compared to that in other zoonotic diseases, quency in ticks to ensure indeﬁnite perpetuation of the

such as Rift Valley fever, and that humans are not the virus in the absence of ampliﬁcation of infection in

preferred hosts of Hyalomma ticks. The low preva- vertebrate hosts, and in particular, it is believed that

lences of antibody generally found in populations at the infection of small vertebrates constitutes an

risk, and the paucity of evidence of inapparent important amplifying mechanism, which facilitates

infection encountered among the cohorts of cases of infection encountered among the cohorts of cases of

Incubation periods are generally short, usually 1–3 days (maximum 9) following infection by tick bite, and are usually 5 or 6 days (maximum 13) in persons exposed to infected blood or other tissues of livestock or human patients. Onset of the disease is usually very sudden. Patients develop fever, rigors, chills, severe headache, dizziness, neck pain and stiﬀness, sore eyes, photophobia, myalgia and malaise, with intense backache or leg pains. Nausea, sore throat and vomiting commonly occur early in the illness and patients may experience non-localised abdominal pain and diarrhoea at this stage. Fever is often intermittent and patients may undergo sharp changes of mood over the next 2 days, with feelings of confusion and aggression. By day 2–4 of illness, patients may exhibit lassitude, depression and somnolence, and have a ﬂushed appearance with injected conjunctivae or chemosis. By this time, tenderness may be localised in the right upper quadrant of the abdomen, and hepatomegaly may be discernible. Tachycardia is common and patients may be slightly hypotensive. There may be lymphadenopathy, and enanthem and petechiae of the throat, tonsils and buccal mucosa.

A petechial rash appears on the trunk and limbs on day 3–6 of illness, and this may be followed rapidly by the appearance of large bruises and ecchymoses, especially in the anticubital fossae, upper arms, axillae and groin. Epistaxis, haematemesis, haematuria, melaena, gingival bleeding and bleeding from the vagina or other oriﬁces may commence on day 4 or 5 of illness, or even earlier. Sometimes a haemorrhagic tendency is evident only from the oozing of blood from injection or venepuncture sites. There may be internal bleeding, including retroperitoneal and intracranial haemorrhage. Severely ill patients may enter a state of hepatorenal and pulmonary failure from about day 5 onwards and progressively become drowsy, stuporous and comatose. Jaundice may become apparent during the second week of illness. The mortality rate is approximately 30% and deaths generally occur on days 5–14 of illness. Patients who recover usually begin to improve on day 9 or 10 of illness, but asthenia, conjunctivitis, slight confusion and amnesia may continue for a month or longer.

Changes in clinical pathology values recorded during the ﬁrst few days of illness include leukocytosis or leukopenia, and elevated aspartate transaminase, alanine transaminase, g-glutamyl transferase, lactic dehydrogenase, alkaline phosphatase and creatine kinase levels, while bilirubin, creatinine and urea levels increase and serum protein levels decline during the

second week. Thrombocytopenia, elevation of prothrombin ratio, activated partial thromboplastin time, thrombin time and fibrin degradation products, and depression of fibrinogen and haemoglobin values are also evident during the first few days of illness, indicating that the occurrence of disseminated intravascular coagulopathy is probably an early and central event in the pathogenesis of the disease. Changes are more severe in fatal than in non-fatal infections, and the occurrence of certain markedly abnormal clinical pathology values during the first 5 days of illness are predictive of fatal outcome (Swanepoel et al., 1987, 1989).

It is surmised that peripherally introduced CCHF virus undergoes some replication at the site of inoculation, and that haematogenous and lymph- borne spread of infection occurs to organs such as the liver, which are major sites of replication. Although it has not been shown conclusively that there is infection of endothelium, capillary fragility is a feature of the disease and there is evidence of formation of circulating immune complexes with complement activation, and this would contribute to damage of the capillary bed and the genesis of renal and pulmonary failure. Endothelial damage would account for the occurrence of a rash and would contribute to haemostatic failure through stimulating platelet aggregation and degranulation, with consequent activation of the intrinsic coagulation cascade. It is clear from the results of therapeutic administration of platelets to patients that they are consumed, and evidence of depression of thrombopoiesis in bone marrow has been reported. Widespread tissue damage in organs such as the liver would result in further release of procoagulants, such as tumour necrosis factor, into the bloodstream and impairment of the circulation through the occurrence of a disseminated intravascular coagulopathy would contribute to further tissue damage. Damage to the liver would also impair synthesis of coagulation factors to replace those which are consumed.

Lesions in the liver vary from disseminated foci of coagulative necrosis, mainly mid-zonal in distribution, to massive necrosis involving over 75% of hepatocytes, and a variable degree of haemorrhage, with little or no inﬂammatory cell response. Lesions in other organs include congestion, haemorrhage and focal necrosis in the central nervous system, kidneys and adrenals, and general depletion of lymphoid tissues. Fibrin deposits may be seen in small blood vessels in parenchymatous organs including the liver, and thrombus formation and infarction may contribute to the pathogenesis of the necrotic lesions in these organs.

BUNYAVIRIDAE

577

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

Where possible, patients are treated by specially antibody activity declines to undetectable levels by 4 trained staﬀ in institutions equipped for handling

months after infection, and IgG titres may begin to formidable viral haemorrhagic fevers, and barrier-

decline gradually at this stage, but remain demon- nursing techniques are used for the protection of

strable for at least 5 years. Recent or current infection medical personnel. Therapy appropriate for dissemi-

is conﬁrmed by demonstrating seroconversion, or a nated intravascular coagulopathy, such as the use of

four-fold or greater increase in antibody titre in paired heparin, may be contemplated early in the course of

serum samples, or IgM antibody activity in a single the disease by clinicians well versed in the treatment

sample. Patients who succumb rarely develop a of haemostatic failure, but the procedure is con-

demonstrable antibody response and the diagnosis is sidered to be risky, and generally only patients who

conﬁrmed by isolation of virus from serum, or from acquire nosocomial infection come to medical

liver specimens. Observation of necrotic lesions com- attention at a suﬃciently early stage. Standard

patible with CCHF infection in sections of liver treatment consists of replacement of red blood

provides presumptive evidence in support of the cells, platelets, other coagulation factors, protein

diagnosis.

(albumin) and intravenous feeding, as indicated by The disease must be distinguished from the other so- clinical pathology ﬁndings. Immune plasma from

called formidable viral haemorrhagic fevers: Lassa recovered patients has been used in therapy, but

fever, Marburg disease, Ebola fever and HFRS there is no ﬁrm evidence from controlled trials of

(hantavirus infections), other febrile illnesses such as the value of the treatment, and there has been a

Rift Valley fever, Q fever and brucellosis, which can be lack of a uniform product with proven virus-

acquired from contact with animal tissues, as well as neutralising activity. Ribavirin has been found to

tick-borne typhus (Rickettsia conorii infection, com- inhibit virus replication in cell cultures and in

monly known as tickbite fever), but many other suckling mice, and preliminary results of a trial in

conditions including bacterial septicaemias may human patients are promising.

resemble CCHF.

On account of the propensity of the virus to cause The control of CCHF through the application of laboratory infections, and the severity of the human

acaricides to livestock is impractical, particularly disease, investigation of CCHF is generally undertaken

under the extensive farming conditions which prevail in maximum-security laboratories in countries which

in the arid areas where Hyalomma ticks are most have biosafety regulations. Specimens to be submitted

prevalent. Pyrethroid preparations are available which for laboratory conﬁrmation of the diagnosis include

can be used to kill ticks which come into contact with blood from live patients and, in order to avoid

human clothing. Veterinarians, slaughtermen and performing full autopsies, heart blood and liver

others involved with livestock should be aware of the samples taken with a biopsy needle from deceased

disease and take practical steps, such as the wearing of patients. Virus can be isolated from blood and organ

gloves, to limit or avoid exposure of naked skin to suspensions in a wide variety of primary and line cell

fresh blood and other tissues of animals. Inactivated

mouse brain vaccine for the prevention of human identiﬁed by immunoﬂuorescence. Isolation and iden-

cultures, including Vero, CER and BHK 21 cells, and

infection has been used on a limited scale in eastern tiﬁcation can be achieved in 1–5 days, but cell cultures

Europe and the former USSR. Development of a safe lack sensitivity and usually only detect high concentra-

and eﬀective modern vaccine is inhibited by the limited tions of virus present in the blood of severely ill

potential demand for such a vaccine. patients during the ﬁrst 5 days or so of illness. Intracerebral inoculation of suckling mice is more sensitive and can be used to demonstrate low con-

Serogroup Nairobi Sheep Disease centrations of virus present in blood up to 13 days

after the onset of illness. Virus antigen can sometimes

be demonstrated in the blood of severely ill patients Nairobi Sheep Disease Virus with intense viraemia, or in liver suspensions, by

Nairobi sheep disease virus was ﬁrst isolated from sheep enzyme-linked immunoassay.

blood in Kenya in 1910 and is known to be associated Antibodies, both IgG and IgM, become demon-

with disease of small ruminants, speciﬁcally sheep and strable by indirect immunoﬂuorescence from about

goats, in a narrow band straddling the equator from day 7 of illness (slightly earlier by enzyme-linked

Kenya in the east to Congo in the west. Antibody, but immunoassay), and are present in the sera of all

not disease, has also been found to the north of Kenya survivors of the disease by day 9 at the latest. The IgM

in Ethiopia and Somalia, and southwards along the in Ethiopia and Somalia, and southwards along the

Ganjam virus , ﬁrst isolated from ixodid ticks in India in 1954, is considered to be identical to Nairobi sheep disease virus . It has been isolated from the blood of sheep and humans with febrile illness in India, where it is associated with ticks of the genus Haemaphysalis. There is speculation that the virus may have been translocated from India to Africa with ectoparasites on sheep and goats, which have been traded along sea routes for centuries.

Dugbe Virus There have been approximately 600 isolations of

Dugbe virus from ixodid ticks, mainly Amblyomma variegatum , in Nigeria, Central African Republic and Ethiopia. The virus has also been isolated frequently from cattle blood in surveys, and from a giant rat (Cricetomys gambianus ), aedine mosquitoes and Culicoides midges in Nigeria, and there is serological evidence of the occurrence of the virus in Senegal and Uganda. There have been seven isolations of the virus from the blood of persons with benign febrile illness in Nigeria and Central African Republic (including a laboratory infection). One patient had mild meningitis and virus was isolated from cerebrospinal ﬂuid. Surprisingly, serosurveys have not revealed widespread human infection.

GENUS HANTAVIRUS

Hantaan, Dobrava, Seoul, Puumala, Sin Nombre and Related Viruses

Hantaviruses are associated with a range of nephrotic diseases in Asia and Europe, known variously as haemorrhagic nephrosonephritis, Korean haemorrhagic fever, Songo fever, epidemic haemorrhagic fever and nephropathia epidemica, but use of the generic term ‘haemorrhagic fever with renal syndrome’ (HFRS) is advocated, while the term ‘hantavirus pulmonary syndrome’ (HPS) is preferred for respiratory disease associated with hantaviruses in the Americas.

The existence of a febrile disease with haemorrhagic and renal manifestations has been recognised in Eurasia at least since the early years of the twentieth century and, in fact, descriptions of similar disease can

be traced back to antiquity. A disease known by various names, including haemorrhagic nephrosonephritis, and which caused outbreaks among civilians and soldiers, was investigated independently in the far eastern region of the former USSR and in Manchuria prior to the Second World War, and by the early 1940s it was established that the condition could be transmitted to human volunteers by inoculation of ﬁltrates of patients’ blood or urine, or tissue extracts from Apodemus ﬁeld mice (it had been observed that the incidence of disease was greatest at the end of summer, when the mice were most numerous). At the same time a febrile syndrome with abdominal pain, backache and renal manifestations was recognised in Scandinavia and numerous cases of this disease, later named nephropathia epidemica (NE), were observed in soldiers during the Second World War.

Thousands of cases of a disease named Korean haemorrhagic fever were observed in soldiers and civilians during the Korean war of the early 1950s, and the disease continued to be seen after the war. In 1976 it was found that convalescent sera from patients in Korea could be used to demonstrate the presence of an antigen by immunofluorescence in the tissues of Apodemus agrarius field mice caught near the Hantaan river. The antigen was shown to be associated with a virus that could be subcultured in field mice. Named Hantaan virus , it was successfully grown in cell cultures in 1981, and shortly thereafter characterised as a member of the family Bunyaviridae and placed in a new genus, Hantavirus. The virus is widely distributed as the causative agent of HFRS in Asia, particularly in the eastern portion of the former USSR, China and

BUNYAVIRIDAE

579

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

Korea. Virus associated with a severe form of HFRS in tissues of patients. The nucleotide sequence of the the Balkans (Albania, Greece, former Yugoslavia and

entire genome of the virus was determined even before Bulgaria) was found to be distinct from Hantaan virus,

it could be isolated and grown in cell cultures. The and was named Dobrava virus. It is associated with the

outbreak was apparently associated with a population

explosion of the deer mouse, Peromyscus maniculatus, recently, a second virus has been found in association

yellow-necked ﬁeld mouse, A. ﬂavicollis. More

incriminated as the natural host of the virus. Sporadic with A. agrarius, the reservoir host of Hantaan virus,

cases of similar disease were recognised elsewhere in and named Saaremaa virus; it causes milder disease

the USA, some retrospectively, and by the end of 1993 than Hantaan virus.

a total of 53 cases had been conﬁrmed (Nichol et al., In 1980 the presence of the causative agent of

1993; Bremner, 1994; Duchin et al., 1994). After nephropathia epidemica was demonstrated

objections were raised to various names proposed for immunoﬂuorescence in the tissues of Clethrionomys

the new virus, the name Sin Nombre (Spanish for ‘without glareolus voles in Finland, and subsequently the agent,

name’) virus was adopted and the disease was referred to named Puumala virus, was grown in cell cultures and

as HPS (for hantavirus pulmonary syndrome). shown to be related to Hantaan virus. Although

Isolated cases and outbreaks of HPS were subse- evidence obtained in former Yugoslavia, Germany,

quently recognised beyond the distribution range of Belgium, France and Britain suggests that Puumala

Peromyscus maniculatus in the USA, in Canada, and in virus occurs widely in Europe, it is most prevalent at

several countries of South America. A succession of northerly latitudes, extending into the Arctic circle in

new hantaviruses was discovered in association with Scandinavia and the adjoining western portion of the

HPS, or in rodents tested speculatively in surveys, and former USSR, where highest concentrations of the

many of these have been associated with HPS (Table bank vole occur.

18.5). Several new viruses were also discovered in Seoul virus was isolated in 1980 in Korea from the

rodents in Europe and Asia, and unidentiﬁed viruses tissues of peridomestic rats, Rattus rattus and R.

which had previously been isolated from bandicoots in norvegicus , in association with human disease which

Thailand and from suncid shrews in India were found occurred in urban as opposed to rural environments. It

to be hantaviruses. In general, the new viruses were has been incriminated of causing human disease in

discovered through the detection of cross-reactive Japan, China and Korea, but has been isolated from

antibody activity to hantavirus antigens, followed by rats in Egypt, the USA and elsewhere, and probably

the application of the polymerase chain reaction to has a worldwide distribution. Isolation of the virus

detect viral nucleic acid, and genetic characterisation. from rats led to speculation that hantaviruses in

Adaptation to cell cultures followed the initial general may have been disseminated worldwide with

identiﬁcation of the viruses, but in vitro culture has ship-borne rodents. However, the distribution patterns

not yet been achieved in all instances. It is suspected of most hantaviruses within the interiors of continents,

that, in addition to the hantaviruses currently known and the evolution of particular host relationships,

to be human pathogens, some of the remaining viruses constitute evidence against recent spread of the viruses.

may also prove to be associated with disease (Table The ﬁndings in Asia and Europe prompted interest

18.5) (Schmaljohn and Hjelle, 1997; Kanerva et al., elsewhere and, as a result, Prospect Hill virus was

1998; Monroe et al., 1999; Schmaljohn et al., 2002). isolated from Microtus pennsylvanicus voles in the

Serological classiﬁcation of hantaviruses has lagged USA, but no disease associations have been described

behind genetic characterisation, primarily because the for this virus.

lack of in vitro culture systems for some of the viruses In May 1993, an outbreak of an acute respiratory

has prevented the performance of deﬁnitive cross- disease in adults, with a high fatality rate, was

neutralisation tests, but the extant information on recognised in the Four Corners region of south-

antigenic aﬃnities is in agreement with the genetic western USA, where the borders of the states of

clustering of the viruses, which in itself conforms with Utah, Colorado, Arizona and New Mexico meet, but

the phylogeny of the rodent hosts (Table 18.5) the initial occurrence of cases could be traced back to

(Puthavathana et al., 1992; Arthur et al., 1992; Chu late 1992. Antibody cross-reactive with the antigens of

et al. , 1994, 1995; Schmaljohn and Hjelle, 1997; known hantaviruses was found in the sera of patients,

Kanerva et al., 1998; Monroe et al., 1999). In brief, and by means of reverse transcription and the

all hantaviruses are antigenically related, with the polymerase chain reaction with consensus sequence

greatest aﬃnities existing within clusters designated as hantavirus primers, it was possible to demonstrate the

Hantaan-like, Puumala-Prospect Hill-like, and Sin presence of nucleic acid of a novel hantavirus in the

Nombre-like, while Thottapalayam virus from shrews

581 Table 18.5 Abridged list of members of the genus Hantavirus. Information derived for sources cited in the text

BUNYAVIRIDAE

Vertebrate host Order: Subfamily (Virus genotype)

Disease Distribution Subtype/variety

Known/suspected host

Rodentia: Murinae (Hantaan-like viruses)

Hantaan

HFRS Asia Dobrava

Apodemus agrarius

HFRS Europe Saaremaa

Apodemus ﬂavicollis

HFRS Europe Amur

Apodemus agrarius

HFRS Asia Seoul

Apodemus peninsulae

HFRS Worldwide Thailand

Rattus rattus ; R. norvegicus

Bandicota indica

Asia

Rodentia: Arvicolinae (Puumala-Prospect Hill-like viruses)

Puumala

HFRS (NE) Europe Tobetsu

Clethrionomys glareolus

Japan Topografov

Clethrionomys rufocanus

Siberia Khabarovsk

Lemmus sibiricus

Siberia Tula

Microtus fortis

HFRS Europe Prospect Hill

Microtus arvalis ; M. rissiaemeridionalis

N. America Bloodland Lake

Microtus pennsylvanicus

N. America Isla Vista

Microtus ochrogaster

Microtus californicus

W. USA, Mexico

Rodentia: Sigmodontinae (Sin Nombre-like viruses)

Sin Nombre

W. and C. USA, Canada Monongahela

Peromyscus maniculatus (Grassland form)

HPS

E. USA, Canada New York

Peromyscus maniculatus nubiterrae (Forest form)

HPS

E. USA Blue River

Peromyscus leucopus (Eastern haplotype)

HPS

C. USA Limestone Canyon

Peromyscus leucopus (S.W./N.W. haplotypes)

HPS

S.W. USA, central Mexico Bayou

Peromyscus boylii

S.W. USA Black Creek Canal

Oryzomys palustris

HPS

S.E. USA Muleshoe

Sigmodon hispidus (Eastern form)

HPS

S. USA Can˜o Delgadito

Sigmodon hispidus (Western form)

Venezuela Andes

Sigmodon alstoni

Argentina, Chile Oran

Oligoryzomys longicaudatus

HPS

N.W. Argentina Lechiguanas

Oligoryzomys longicaudatus

HPS

C. Argentina Bermejo

Oligoryzomys ﬂavescens

HPS

Oligoryzomys chacoensis

N.W. Argentina

C. Argentina Pergamino

Hu 39694 a Unknown

HPS

C. Argentina Maciel

Akadon azarae

C. Argentina Laguna Negra

Necromys benefactus

Paraguay, Bolivia Juquitiba

Calomys laucha

HPS

Brazil Castelo dos Sonhos

Unknown

HPS

Brazil Araraquara

Unknown

HPS

Brazil Rio Mamore

Unknown

HPS

Bolivia, Peru El Moro Canyon

Oligoryzomys microtis

HPS

W. USA, Mexico Rio Segundo

Reithrodontomys megalotis

Costa Rica Choclo

Reithrodontomys mexicanus

Panama Calabazo

Oligoryzomys fulvescens

HPS

Zygodontomys brevicauda

Panama

Insectivora: Crocidurinae Thottapalyam

India a Virus will be named when the rodent host or distribution is identiﬁed.

Suncus murinus

HFRS, haemorrhagic fever with renal syndrome; HPS, hantavirus pulmonary syndrome.

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

in India is more distantly related to the others. Viruses while all of the viruses may cause infections associated of the Hantaan-like group are associated with rodent

with laboratory rodents. Rodents are subject to hosts of the subfamily Murinae and with HFRS;

periodic population explosions and crashes, and the Puumala-Prospect Hill-like viruses are associated with

incidence of human infection with Hantaan-like, the subfamily Arvicolinae (voles and lemmings) and

Puumala-like and Sin Nombre-like viruses increases with NE, and Sin Nombre-like viruses with the

in years when the rodents are most numerous. Person- subfamily Sigmodontinae and with HPS (Table 18.5).

to-person spread of hantavirus infection has been Despite the fact that Seoul virus appears to be very

observed in outbreaks of HPS caused by Andes virus in widely distributed, incontrovertible evidence of disease

Argentina, but it has not been established whether associated with hantaviruses (detection of virus) has

transmission is associated with direct contact, droplets, been obtained only for Asia, Europe and the Americas,

aerosols or contaminated fomites (Wells et al., 1997). while elsewhere there has only been inconclusive

Numerous cases of HPS have been reported in the serological evidence of infection. However, Seoul

Americas, and up to 200 000 hospitalised cases of virus , Hantaan virus and Puumala virus have been

HFRS are recorded in Eurasia each year, with more encountered as contaminants of laboratory rodent

than half occurring in China (Schmaljohn and Hjelle, colonies and are known to have caused infections in

1997; Monroe et al., 1999).

laboratory workers in the former USSR, Korea, Four clinical forms of HFRS are recognised and Japan, Belgium, France and England. In one instance

these vary in order of increasing severity from virus was inadvertently preserved for years in rat

nephropathia epidemica associated with Puumala tissues kept in frozen storage. Hence, the potential

virus infection, through mild or rat-borne HFRS exists for inadvertent dissemination of the viruses.

associated with Seoul virus infection, to Far Eastern The distributions of the hantaviruses, insofar as they

HFRS associated with Hantaan virus carried by A. are known, tend to overlap, but conform to the

agrarius ﬁeldmice, and so-called Balkan HFRS distributions of the rodent hosts. Individual viruses

associated with Dobrava virus carried by A. ﬂavicollis have been isolated from more than one type of rodent,

mice.

but each tends to have a particular association with a Far Eastern HFRS occurs in China, the eastern part single species of rodent. The viruses appear to be

of the former USSR and Korea, mainly in adult males apathogenic for their reservoir hosts. After the rodents

with occupational exposure to the outdoors, such as become infected there is an initial viraemia followed by

farmers, forest workers and soldiers stationed in the the persistence of infection, probably for life, in lungs,

ﬁeld, and seldom occurs in persons under 10 years of kidneys and possibly other organs, with chronic

age. Most cases are seen in autumn and early winter excretion of virus in urine, faeces and saliva, despite

when crops are harvested and the rodents are most the occurrence of a demonstrable immune response.

numerous, and subsequently when the agricultural There does not appear to be intrauterine transfer of

products are stored in proximity to homesteads. The infection, and transmission between rodents is thought

incidence of asymptomatic infection is unknown, but it to occur by bite, aerosol or contamination of dust,

was noted that American soldiers participating in an food and other fomites with excreted virus. Gamasid

exercise in Korea who seroconverted had all been ill, mite parasites of rodents are suspected to be capable of

while in parts of Korea high antibody prevalence rates transmitting infection, but transmission occurs in the

without corresponding levels of disease have been laboratory in the absence of mites. Foci with very high

observed.

infection rates are observed among rodents in nature. The classical form of Far Eastern HFRS described Humans become infected by the same means as

in Korea has well marked phases, but these may rodents, but airborne infection from dust contami-

overlap and be obscured in mild cases (Lee, 1982). An nated with rodent urine and faeces appears to be the

incubation period of 2–3 weeks is followed by the principal mechanism, and has been observed to occur

abrupt onset of a febrile phase, which lasts 3–7 days even in persons who brieﬂy visited infected colonies of

and is marked by high fever, chills, malaise, myalgia, laboratory rodents. Infection occurs in three main

anorexia, headache, dizziness, ocular pain and abdom- situations: rural or sylvatic infection with Hantaan-

inal and back pain, which is felt particularly in the like, Puumala-like or Sin Nombre-like viruses occurs

renal area as a result of peritoneal and retroperitoneal in persons who have occupational, residential or

oedema. Proteinuria is marked during this phase. recreational exposure to rodent-infested buildings or

Towards the end of the phase there is characteristic to the outdoors, urban infection with Seoul virus

ﬂushing of the face, neck and anterior chest, with the occurs indoors in association with rat infestations,

conjunctivae, palate and pharynx assuming an injected conjunctivae, palate and pharynx assuming an injected

be epistaxis, conjunctival, cerebral and gastrointestinal haemorrhages and extensive purpura. There may be severe nausea and vomiting, lung oedema and symptoms referable to the central nervous system. Most deaths occur at this stage. A diuretic phase which follows may last days or weeks, and marks the start of clinical recovery. Diuresis of 3–6 L/day is common, but is influenced by dehydration, electrolyte imbalance or secondary infections. Severely ill patients are at risk in this phase and may lapse into shock. A convalescent phase with progressive recovery of glomerular filtra- tion rate, renal blood flow and urine-concentrating ability, may last 2–3 months. Mortality has been reduced from the 10–15% observed during the Korean war to 5%, with intensive supportive therapy and renal dialysis.

Balkan HFRS, associated with Dobrava virus, is also seen mainly in adult males, including woodcutters, shepherds and military personnel, but cases generally occur in spring and summer, possibly because there is not the same type of cereal crop farming as in the Far East, and the reservoir host is encountered in outdoor activities and at campsites during the warmer months of the year. The disease is essentially similar to Far Eastern HFRS, but is more severe, with a higher proportion of patients requiring renal dialysis, and with a greater tendency for the development of disseminated intravascular coagulopathy and haemorrhages. Reported death rates range from 5% to 35%.

Natural outbreaks of mild or rat-borne HFRS, as opposed to outbreaks associated with laboratory rodent colonies, have been recorded in cities in Japan, China and Korea. The disease occurs in urban residents who have no contact with ﬁeld rodents, and most cases are seen in spring and early summer. The disease is less severe and runs a shorter course than disease associated with Hantaan virus

infections, and has less distinct clinical phases. There is also less tendency for haemorrhages and renal failure to occur, and frequently signs of liver involvement are dominant: abdominal pain, hepatomegaly and hepatic dysfunction. There are few deaths and mortality has been estimated at 1% or less.

Infection with Puumala-type virus occurs widely in Europe, but the disease, nephropathia epidemica, is recognised most frequently in Scandinavia and the neighbouring western region of the former USSR. The disease aﬀects mainly adult males and infection appears to be associated principally with outdoor activities. Disease is seen most commonly in late autumn and early winter, but many cases occur in late summer following the traditional vacation season. Cases seen during the colder months are ascribed to the invasion of homes and barns by voles at the onset of winter. The incubation period is thought to be about

1 month, but a range of 3 days to 6 weeks has been reported. There is abrupt onset of fever, headache and malaise. By the third or fourth day of illness there is nausea, vomiting and abdominal and lumbar pain. At this stage there may be azotemia, oliguria and proteinuria, which peaks 1 week after the onset of illness and declines over the next 3–6 days. Patients are extremely ill during the oliguric phase, and may manifest somnolence, restlessness, confusion and meningismus. Transient myopia or blurred vision is regarded as pathognomonic. Facial ﬂushing and maculopapular rash of the neck and trunk are seen occasionally, as are hepatomegaly, cervical lymphadenopathy and haemorrhages, such as epistaxis and gastrointestinal bleeding. Patients seldom require renal dialysis. The oliguria is followed by polyuria of 3–4 L daily for 7–10 days. At one stage it was thought that HFRS/NE and HPS were entirely distinct syndromes, but it is now recognised that there is some overlap, and in particular a proportion of NE patients may develop pulmonary inﬁltration similar to HPS, and some may even exhibit respiratory distress. Clinical improvement begins with the onset of polyuria, and 2 weeks after the onset of fever patients are subjectively well, but backache and lassitude may recur over weeks, and hyposthenuria may persist for months. Recovery is usually complete, and mortality is consistently 51%. The relatively high prevalence of antibody found in surveys suggests that inapparent infections may out- number cases of overt disease by up to 20-fold.

It should be stressed that the hantaviruses overlap in distribution and in the severity of HFRS which they induce and for instance, neither rural or urban domicile of patients nor season of occurrence of disease, allow Hantaan virus and Seoul virus infections

BUNYAVIRIDAE

583

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

to be distinguished with certainty in Asia. Infections pulmonary oedema and serous pleural eﬀusions, with with Dobrava- and Puumala-type viruses in the

scant lymphoid inﬁltration of the lung tissue. Some Balkans may be equally diﬃcult to distinguish on

survivors manifested transient diuresis, but otherwise occasion.

they make an uneventful recovery without sequelae Persons who develop HPS are often healthy young

(Nichol et al., 1993; Bremner, 1994; Duchin et al., adults, but may be of any age and either sex, although

1994; Schmaljohn and Hjelle, 1997; Kanerva et al., the disease occurs infrequently in children. Infection is

acquired in similar manner to HFRS from occupa- The underlying lesion in the pathogenesis of tional, residential or recreational exposure to the

hantavirus syndromes appears to be vascular damage, outdoors or rodent-infested buildings, and in many

and this is thought to be mediated by both viral instances infected rodents have been found in the

invasion of endothelial cells and immunopathological homes of victims. Incubation periods are similar to

mechanisms. Capillaries and small blood vessels dilate HFRS, generally falling into the range 2–3 weeks, but

and there is extravasation of plasma and cellular the disease is characterised by severe cardiopulmonary

elements into tissues, and the pathological changes dysfunction rather than renal failure and haemor-

observed in multiple systems all appear to be referable rhage, despite the fact that there is similar underlying

to the vascular damage (Kanerva et al., 1998). capillary permeability and marked thrombocytopenia.

Treatment of HFRS involves complex, phase- Onset of the prodromal phase of the disease is marked

speciﬁc monitoring and support of homeostasis, by sudden development of fever, headache, severe

including ﬂuid and electrolyte balances. Trials of myalgia and a cough which may be productive in some

ribavirin for the treatment of the disease in China instances. Gastrointestinal manifestations in some

have been complicated by diﬃculties such as lack of patients include abdominal pain, nausea, vomiting

uniformity of clinical status of patients at the time of and diarrhoea. After 3–6 days of illness there is

protocol entry, but there are indications that use of the progressive tachypnoea, tachycardia and hypotension

drug reduces mortality and leads to improvement of preceding the onset of acute respiratory distress with

objective markers of patient well-being and clinical pulmonary oedema. Patients are generally hospitalised

pathology values. Epidemiological evidence suggests at this stage, but some may die before they can be

that there is lifelong immunity to hantaviruses, at least admitted. In addition to tachypnoea, tachycardia and

to the homologous serotype. There has been research hypotension, on admission patients may be found to

on recombinant vaccines, but these are likely to ﬁnd have proteinuria, leukocytosis with neutrophilia, plus

application only in Asia where suitable populations at increased myeloid precursors and atypical lympho-

risk can be identiﬁed.

cytes, haemoconcentration, and thrombocytopenia, Investigation of hantavirus infections is usually plus increased prothrombin and partial-thromboplas-

undertaken in high-security laboratories to minimise tin times, although there is no rash and seldom a

the exposure of staﬀ to infection. Isolation and tendency towards overt or internal bleeding. Within 2

identiﬁcation of hantaviruses is a notoriously diﬃcult days of being admitted to hospital most patients

and time-consuming procedure, and is rarely successful develop diﬀuse bilateral interstitial and alveolar

on serum and urine specimens from patients. Demon- pulmonary inﬁltration and pleural eﬀusions demon-

stration of viral antigens in sera and urine is equally strable on radiographs, with hypoxaemia which has

unsuccessful. Viral nucleic acids of hantaviruses can be necessitated intubation, mechanical ventilation and

detected in the tissues of human patients and experi- oxygen supplementation in up to 88% of patients in

mentally and naturally infected rodents by means of some outbreaks. Renal insuﬃciency can occasionally

reverse transcription and the polymerase chain reac- follow prolonged hypoperfusion, but early renal

tion with appropriate primers (Xiao et al., 1992; insuﬃciency and increased serum creatine kinase levels

Arthur et al., 1992; Grankvist et al., 1992; Nichol (evidence of skeletal muscle inﬂammation) are not

et al. , 1993; Monroe et al., 1999). Detection of IgM uncommon in infection with Andes virus, Bayou virus

antibody by enzyme-linked immunoassay holds great- and Black Creek Canal virus. Death generally occurs

est promise as a rapid diagnostic technique. Antibody 6–8 days after the onset of illness, often within 48 h of

activity appears to be present in the sera of HFRS admission to hospital, but can range from 2 days after

patients from the time of hospitalisation, and titres the observed onset of illness to more than 2 weeks.

increase rapidly over the next 2 weeks. Owing to the Fatality rates often exceed 40%, and incurable shock

antigenic cross-reactivity between hantaviruses, it may and myocardial dysfunction may contribute to the

be diﬃcult to determine the serotype of the virus high mortality. Autopsies reveal non-cardiogenic

responsible for the infection from antibody tests, but

585 this can sometimes be inferred by using a range of

BUNYAVIRIDAE

Central African Republic. Mild febrile illness was antigens in enzyme-linked immunoassays (Feldmann

observed in two human patients who acquired et al. , 1993) or by performing neutralisation tests with

laboratory infection, and serological evidence of the full range of serotypes: antibody titres tend to be

infection was obtained in a patient who suﬀered highest against the homologous infecting serotype. In

meningoencephalitis in Yugoslavia. attempts to diagnose infection by an unknown

Kasokero virus was isolated from fruit bats in hantavirus, particularly in an locations where local

Uganda and caused four laboratory infections marked viruses are unknown, it is advisable that antigens

by febrile illness, headache, myalgia, arthralgia, representative of all four antigenic types be included in

abdominal pain, diarrhoea, chest pain, cough, as well the tests: Hantaan-like, Puumala-Prospect Hill-like,

as hyperactive reﬂexes in one patient. Bangui virus was Sin Nombre-like and Thottapalyam viruses.

isolated from the blood of a patient with febrile illness, headache and rash in the Central African Republic, and antibody was found in the sera of local residents.

POSSIBLE MEMBERS OF THE FAMILY Issyk-Kul virus was isolated from several species of BUNYAVIRIDAE

insectivorous bat and from argasid tick parasites of bats, birds and anopheline and aedine mosquitoes in the Central Asian Republics of the former USSR. It

Bhanja, Kasokero, Bangui, Issyk-Kul, Tataguine was demonstrated that aedine mosquitoes and argasid and Wanowrie Viruses

ticks are able to transmit the virus. Antibody was found in human sera and virus was isolated on at least

Bhanja virus has been isolated from ixodid ticks of ﬁve

19 occasions from the blood of persons suﬀering from genera—Haemaphysalis, Amblyomma, Dermacentor,

febrile illness with headache, dizziness, cough, nausea Boophilus and Hyalomma—variously in India, the

and vomiting. The cases included laboratory infec- former USSR, Yugoslavia, Bulgaria, Slovakia, Somalia,

tions. Keterah virus, isolated from argasid tick Central African Republic, Nigeria and Senegal. It has

parasites of bats and from bat blood in Malaysia, been suggested that the wide distribution of the virus

has been shown to be closely related or identical to could have resulted from the carriage of immature

Issyk-Kul virus . It is diﬃcult to be certain whether the ticks on migrating birds, although birds do not

natural vectors of Issyk-Kul/Keterah virus are argasid themselves appear to be susceptible to the virus.

ticks or mosquitoes.

Isolations of the virus have also been made from a Tataguine virus has been isolated from anopheline hedgehog, ground squirrel and blood samples from

mosquitoes in Senegal, Nigeria, Cameroon and Cen- cattle and sheep in Nigeria. Antibody has been found

tral African Republic. It appears to be a potentially in cattle, sheep and goats parasitised by the ticks, and

important pathogen: antibody has been found in in human sera in Slovakia, Yugoslavia, Italy and the

human sera in Senegal and Nigeria, and there have Table 18.6 Abridged classiﬁcation of possible members of the family Bunyaviridae. Information derived from sources cited in the

text Serogroup

Distribution ANTIGENIC COMPLEX Virus

Putative vectors

Human infection

Natural

Laboratory

Bhanja BHANJA (3) a Bhanja

Europe, Africa, Asia Yogue YOGUE (2) Kasokero

Ixodids

Africa Six other serogroups (17) Ungrouped (22)

Unknown

Bangui

Africa Issyk-Kul (Keterah)

Unknown

Asia Tataguine

Mosquitoes?

Africa Wanowrie

Mosquitoes

Africa, Asia a Figures in parentheses indicate the total numbers of recognised members of the relevant taxon.

Ixodids

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

been at least 31 isolations of the virus from the blood Casals J (1961) Procedures for identiﬁcation of arthropod- of febrile humans in Senegal, Nigeria, Central African

borne viruses. Bull WHO, 24, 727–734. Republic and Cameroon. The infections were char-

Casals J and Whitman L (1960) A new antigenic group of acterised by febrile illness with headache, rash and arthropod-borne viruses. The Bunyamwera group. Am J

Trop Med Hyg , 9, 73–77.

arthralgia. Casals J and Whitman L (1961) Group C. A new serological Wanowrie virus has been isolated from Hyalomma

group of hitherto undescribed arthropod-borne viruses. ticks in India, Iran and Egypt. Little else is known

Immunological studies. Am J Trop Med Hyg, 10, 250–258. about the virus except that it was isolated in Sri Lanka

Chu Y-K, Jennings G, Schmaljohn C et al. (1995) Cross- neutralization of hantaviruses with immune sera from

from the brain of a human patient who succumbed to experimentally infected animals and from hemorrhagic febrile illness with abdominal pain, vomiting, haema-

fever with renal syndrome and hantavirus pulmonary temesis and passing of blood per rectum.

syndrome patients. J Infect Dis, 172, 1581–1584. Chu Y-K, Rossi C, LeDuc J et al. (1994) Serological relationships among viruses in the in the Hantavirus genus, family Bunyaviridae. Virology, 198, 196–204.

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19 Arenaviruses

Colin R. Howard

Royal Veterinary College, University of London, UK

INTRODUCTION resurgence of interest in the link between zoonoses and persistent virus infections of rodents (see Chapter

The arenaviruses are a group of enveloped, single-

18 for a description of the hantaviruses). The Four stranded RNA viruses, the study of which has been

Corners outbreak of hantavirus pulmonary syndrome pursued for two quite separate reasons. First,

in 1993 served to heighten awareness that fevers of lymphocytic choriomeningitis virus (LCM) has been

hitherto unknown origin might equally be the result of used as a model of persistent virus infections for over

infection with agents normally maintained in rodent half a century; its study has contributed a number of

reservoirs. This is particularly so in Argentina, where cardinal concepts to our present understanding of

virologists and clinicians specialising in Argentinian interactions between viruses and the host immune

haemorrhagic fever have been in the vanguard of system. Although LCM infections of humans are rare,

national eﬀorts linking respiratory disease with the this virus remains the prototype of the Arenaviridae

discovery of new hantaviruses that can coexist with and is a common infection of laboratory mice, rats

arenaviruses in the same rodent populations. and hamsters. Second, certain arenaviruses cause

With the current concern about the so-called severe haemorrhagic diseases in man, notably Lassa

‘emerging viruses’, the arenaviruses are a good fever in Africa, and Argentinian haemorrhagic fever in

illustration of how environmental changes may result South America. More recently, several new arena-

in an altered balance between man and natural animal viruses have been described from South America, two

hosts, leading to unexpected diseases which can of which are associated with human infections. In

severely challenge local and national public health common with LCM, the natural reservoir of these

resources. In addition, there is a wide spectrum of infections is a limited number of rodent species

pathological processes associated with these viruses (Howard, 1986). Although the initial isolates from

that give useful insights into other zoonotic infections. South America were at ﬁrst erroneously designated as

All the evidence is that the morbidity of Lassa fever newly deﬁned arboviruses, there is no evidence to

and South American haemorrhagic fevers due to implicate arthropod transmission for any arenavirus.

arenavirus infection results from the direct cytopathic However, similar methods of isolation and the

action of these agents. This is in sharp contrast to the necessity of trapping small animals have meant

immunopathological basis of ‘classic’ lymphocytic historically that the majority of arenaviruses have

choriomeningitis disease seen in adult mice infected been isolated by workers in the arbovirus ﬁeld. A

with LCM virus.

good example of this is Guanarito virus, which For a general overview of the arenaviruses, see emerged during investigation of a dengue virus

Oldstone (2002a, 2002b). A comprehensive overview of outbreak in Venezuela (Salas et al., 1991). The

the arenaviruses causing human disease can be found discovery of Sin Nombre virus as a cause of

on http://www.cdc.gov/ncidad/dvrd/spb and http:// hantavirus pulmonary syndrome has led to a www.who.int/health-topics/index

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

Table 19.1 The arenaviruses: host and geographical distribution Virus

Distribution Worldwide

Natural host

Human disease

Lymphocytic Mus musculus ,

Europe, North and South choriomeningitis

Aseptic meningitis

Mus domesticus America Old World

Central African Republic Lassa

Ippy Arvicanthus sp.

Not recorded

West Africa Mobala

Mastomys natalensis

Lassa fever

Central African Republic Mopeia

Praomys jacksoni

Infection possible

Mozambique, Zimbabwe New World

Mastomys natalensis

Infection possible

Peru Amapari

Allpahuayo Oecomys sp.

Not recorded

Brazil Neocomys guianae Bear Canyon

Oryzomys gaedi ,

Not recorded

USA Flexal

Peromyscus californicus

Infection possible

Brazil Guanarito

Neocomys spp.

Not recorded

Venezuela Zygodontomys brevicuda Junı´n

Sigmodon alstoni

Venezuelan haemorrhagic fever

Calomys musculimus ,

Argentinian haemorrhagic fever

Argentina

C. laucha , Akadon azarae

Latino Calomys callosus

Bolivia Machupo

Not recorded

Bolivia Parana´

Calomys callosus

Bolivian haemorrhagic fever

Paraguay Pichinde

Oryzomys buccinatus

Not recorded

Colombia Pirital

Oryzomys albigularis

Not recorded

Venezuela Oliveros

Sigmodon alstoni

Not recorded

Argentina Sabia´

Bolomys obscurus

Not recorded

Brazil Tacaribe

Unknown

Brazilian haemorrhagic fever

Trinidad Tamiami

Artibeus literatus (bat)

Infection possible

Florida, USA Whitewater Arroyo

Sigmodon hispidus

Not recorded

Neotoma albigula

Not recorded

New Mexico, USA

PROPERTIES OF THE VIRUS microscope (Latin arena=sand). With the exception of LCM, all are referred to by names that reﬂect the

Nomenclature and Natural History geographical area in which they were isolated (Figure 19.1). Various strain designations are also The morphological, physicochemical and serological

commonly used, in particular for LCM and arena- properties of the arenaviruses were ﬁrst summarised by

viruses isolated from man. Multiple isolations of non- Pfau (1974). The members of the family currently

pathogenic viruses that infect New World rodents are identiﬁed are listed in Table 19.1. The various strains

made less frequently, with the exception of Pichinde and isolates of LCM are now considered to be a genus

virus, in which a large number of ﬁeld isolates from within the family Arenaviridae. A close serological

Colombia have been characterised. However, the relationship exists between LCM, Lassa virus and

recent resurgence of interest in these viruses has other arenaviruses from Africa. For this reason, they

uncovered a number of new arenaviruses that have are loosely referred to as the ‘Old World’ arenaviruses,

tentatively been described as new members of the in contrast to those from the Americas, although now

Arenaviridae . Several of these may be new variants of LCM can be found worldwide except in Australia. The

existing family members.

‘New World’ arenaviruses show varying degrees of All but one of the 21 1 members of the Arenaviridae serological relationships with Tacaribe virus, ﬁrst

so far described have rodents as their natural reservoir isolated in Trinidad. For this reason, viruses from

hosts. Although rodents are divided into over 30 the Americas are frequently regarded as members of the Tacaribe complex.

1 At the time of writing (January 2003), two further putative The Arenaviridae take their name from the sand-

arenaviruses have been recorded (Cupixi and Rio Carcarana sprinkled appearance when viewed in the electron

viruses).

ARENAVIRUSES

591 the genus Mastomys. This is also a member of the

Muridae and, in common with the host of LCM, frequents human dwellings and food stores. In contrast, nearly all arenaviruses isolated from the South American continent are associated with cricetid rodents, whose members frequent open grasslands and forest. The exception is Tacaribe virus, which was originally isolated from the fruit bat, Artibeus literatus .

ULTRASTRUCTURE OF ARENAVIRUSES AND INFECTED CELLS

Negative-staining electron microscopy of extracellular virus shows pleomorphic particles, of diameter 80– 150 nm (Figure 19.2). The virus envelope is formed from the plasma membrane of infected cells. A significant thickening of both bilayers of the membrane together with an increase in the width of the electron-translucent intermediate layer is characteristic of arenavirus maturation. Little is known about the internal structure of the arenavirus particle, although thin sections of mature and budding viruses clearly show the ordered, and often circular, arrangements of host ribosomes that are typical of this virus group and confer the ‘sandy’ appearance from which its name is derived. Distinct well-dispersed filaments 5–10 nm in diameter are released from detergent-treated virus. Two predominant size classes are present, with average lengths of 649 nm and 1300 nm, respectively; these lengths do not show a close relationship with the two virus-specific L and S RNA species. Each is circular and beaded in appearance. Convoluted filamentous strands up to 15 nm in diameter can be seen in preparations of spontaneously disrupted

Figure 19.1 Geographical distribution of Old World (a) and New World (b) arenaviruses. Lymphocytic choriomeningitis

Pichinde virus. These appear to represent globular virus (LCM) can be found on all continents except Australasia

condensations which arise from an association between neighbouring turns of the underlying helix. The basic conﬁguration of the ﬁlaments shows a linear

families distributed worldwide, arenaviruses are pre- array of globular units up to 5 nm in diameter, dominantly found within two major families: Muridae

probably representing single molecules of the viral (e.g. mice and rats) and Cricetidae (e.g. voles,

polypeptide. These ﬁlaments progressively fold lemmings, gerbils). The nature of the original reservoir

through a number of intermediate helical structures for LCM virus remains obscure, but it appears to be

to produce the stable 15 nm diameter forms (Young, mainly in species of the Muridae which evolved in the

Old World and subsequently spread to most parts of Arenaviruses replicate in experimental animals in the globe. Interestingly, there is a wide range of

the absence of any gross pathological eﬀect. However, tropism and virulence among those strains of LCM

cellular necrosis may accompany virus production, not virus originally isolated from laboratory mouse

unlike that seen in virus-infected cell cultures. The colonies.

variable pathological changes associated with arena- The natural reservoirs of Lassa virus and the

virus infections are further complicated by the remaining Old World arenaviruses are members of

occasional appearance of particles in tissue sections

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

Figure 19.2 Electron microscopy of arenaviruses. (a) Negatively stained Lassa fever particle showing the whole surface covered in projections. Few particles are less than 100 nm, and many are twice this size (6300 000). (b) Mopeia virus from southern Africa. Here the negative stain has disrupted the particle, the contents of which (X) have been extruded. 6150 000. (c) Lassa fever particles budding from an infected Vero cell. The thick arrow shows a mature particle, the thin arrow a maturing particle at the plasmalemma. Nucleocapsids and ribosomes line up immediately below the thickened membrane (white arrow) (639 000). Micrographs courtesy of Dr D. S. Ellis

that react strongly with ﬂuorescein-conjugated anti- both in vitro and in vivo. These usually appear early in sera. Granular ﬂuorescence with convalescent serum in

the replication cycle and consist largely of single the perinuclear region of acutely infected Vero cells is

ribosomes which later become condensed in an often seen. In addition, intracytoplasmic inclusion

electron-dense matrix, sometimes together with ﬁne bodies are a prominent feature in virus-infected cells,

ﬁlaments (Murphy and Whitﬁeld, 1975).

CHEMICAL COMPOSITION Proteins

All arenaviruses contain a major nucleocapsid associated protein of molecular weight 60–68 kDa with two glycoproteins in the outer viral envelope. These envelope glycoproteins are not primary gene products but arise by proteolytic cleavage of a larger, 75 kDa glycoprotein precursor polypeptide (GPC). Matura- tion and release of virus do not seem to be markedly inhibited in the presence of tunicamycin, an inhibitor of glycosylation but glycoprotein processing is essential for infectivity.

The major glycoprotein species (GP2) in the molecular weight range of 34–42 kDa represents the C-terminal cleavage product of the GPC envelope glycoprotein precursor. The ﬁrst 59 amino acids at the N-terminus of GPC act as a signal sequence, containing two distinct hydrophobic domains that could function during glycoprotein transport and virus assembly. GP1 is cleaved from the N-terminus at a unique cleavage site that is conserved among all arenaviruses except Tacaribe. GP1 assembles into tetramers linked by disulphide bonds. GP2 is also thought to form tetrameric structures proximal to GP1 in the glycoprotein peplomer, penetrating the viral membrane to form electrovalent bonds with the underlying N-RNA nucleocapsids.

A major antigenic site recognised by antibodies has been located between amino acids 390 and 405, and cross-reactive monoclonal antibodies bind epitopes in this region. The corresponding N-terminal product of GPC cleavage (GP1) is probably highly glycosylated with at least four antigenic domains. Neutralising monoclonal antibodies to LCM virus map to two of these regions and there is less sequence homology between the GP1 than between the GP2 molecules of diﬀerent arenaviruses. Polyclonal neutralising antibody

appears to react

predominantly

with

conformation-dependent structures within one of these domains.

The internal nucleocapsid-associated (N) protein accounts for over 70% of the protein present in puriﬁed virus and infected cells, and remains bound to the virus genome after solubilisation of the virus to form structures resembling a string of beads seen by electron microscopy. Cleavage products of the N protein are a consistent feature of both virus and virus-infected cells. Cleavage is not noticeable in Vero cells; yields of arenaviruses are lower in these cells, perhaps due to reduced availability of N for packaging. N protein accumulates in the cytoplasm of

infected cells, with a fragment of the N protein often seen in the nuclei, the exact function of which is not clear. Molecular cloning studies have shown a surprisingly high degree of homology between the 558– 570 amino acid N proteins of Old and New World arenaviruses, with structural motifs and RNA-binding domains particularly conserved. This would account for the serological cross-reactions seen using certain monoclonal antibodies raised against such epidemiologically distinct viruses and may indicate precise functional roles in virus replication for certain domains of the N polypeptide.

A minor component with a molecular weight in excess of 150 kDa is often observed in infected cells and is found with puriﬁed nucleocapsids. This L protein is coded by the larger RNA genome segment as shown by the study of reassortment viruses and represents the virus-speciﬁc RNA polymerase (Fuller- Pace and Southern, 1989; Lukashevich et al., 1997). Amino acid sequences common to all RNA-dependent RNA polymerases are present along the open reading frame coding for the L protein, which suggest the conservation of certain functional domains. An additional two sequences are shared with the RNA polymerases of bunyaviruses. A small, 11 kDa viral polypeptide, the Z protein, is considered to play a role in controlling the replication and expression of the genome owing to its zinc-binding properties. The Z protein may also modulate the interferon response to infection in vivo by binding to the nuclear oncoprotein PML (Djavani et al., 2001).

Nucleic Acid

The genome of arenaviruses consists of two single- stranded RNA segments of diﬀerent sizes, designated L and S, with S RNA being more abundant. Analysis of RNA is complicated by the presence of ribosomal 18S and 28S RNA, although these cellular RNA species are not essential for virus replication. The total ribosomal RNA content may in turn be inﬂuenced by the varying proportions of infectious to non-infectious particles present in virus stocks, particularly if cells are infected at a multiplicity above 0.1. In addition, there are small quantities of both cell and viral low molecular weight RNA. One of these species, mRNA coding for the viral Z protein, may have a role in the initial stages of infection. There is no obvious role for these host RNA molecules in either replication or the establishment of persistent infections (see Replication, below).

ARENAVIRUSES

593

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

Extracted virion RNA is not infectious and the least one strain of all arenaviruses known at that time, detection of a viral RNA polymerase led to the belief

using maximum parsimony to generate an unrooted that arenaviruses adopt a negative-strand coding

phylogenetic tree (reviewed by Clegg, 2002; Figure strategy with respect to viral protein synthesis. How-

19.3). The results conﬁrmed that the distant relation- ever, the actual coding strategy from the L and S RNA

ship between Old World and New World arenaviruses strands is not entirely in accord with all negative-

are broadly consistent with the previously determined strand RNA viruses, as some genetic information can

serological relationships, using polyclonal and mono- only be expressed by a genomic sense mRNA. This

clonal antibodies. The New World arenaviruses are ‘ambisense’ strategy is also a characteristic of some

divisible into three lineages: clade A contains the bunyavirus genomes (see Chapter 18). Such a coding

viruses Pichinde, Tamiami, Parana´ and Flexal; clade B strategy allows for the independent regulation of

the viruses Sabia´, Tacaribe, Amaparı´, Guanarito and arenavirus envelope and nucleocapsid proteins.

the human pathogens Machupo and Junı´n; and clade The S strand codes for the nucleoprotein (N) and the

C consists of Oliveros and Latino viruses. Interest- envelope glycoprotein precursor (GPC) in two main

ingly, Whitewater Arroyo virus, isolated in the USA, open reading frames located on RNA molecules of

appears closely related to Tamiami, until recently the opposite polarity. The 3’ half of the S RNA codes for

only arenavirus found in North America. However, the N protein by production of an mRNA with a viral-

full-length analysis of Whitewater Arroyo virus S sense sequence speciﬁc for the GPC protein. Thus,

RNA strand has shown a quite separate ancestry for expression of the genome is by synthesis of subgenomic

the nucleocapsid (N) and envelope (GP1, GP2) RNA from full-length templates of opposite polarities.

proteins, almost certainly the result of recombination The reading frames for the two major gene products

between two ancestral arenaviruses. There is less are separated by a hairpin structure of approximately

variability among the Old World members. As may

be expected from their natural history, Mopeia and as a control mechanism for genome expression by

20 paired nucleotides. This intergenic region may act

Mobala viruses are closely related to Lassa Fever forming stable stem–loop structures that in turn

virus. LCM virus occupies the distinctive position of regulate transcription. The S RNA of Tacaribe and

being closely related to the probable ancestral virus. Junı´n viruses are thought to form a second stem–loop

The propensity to cause serious human illness structure.

appears to have evolved on two distinct occasions. The L RNA strand represents about 70% of the

The South American pathogens are all conﬁned to viral genome; reassortment studies with virulent and

clade B, suggesting these viruses have acquired the avirulent strains of LCM virus have shown that the

capacity to infect humans as a result of a common lethality of the disease in guinea-pigs is associated with

mutational event. Lassa fever virus, by contrast, has the properties of the L RNA. The L protein is encoded

likely acquired its ability to cause serious haemor- by a large open reading frame covering 70% of the L

rhagic disease in humans by a separate evolutionary RNA strand: it is expressed via mRNA complemen-

event.

tary in sense to the viral genome. The mRNA for the Z For a thorough discussion of the phylogenetic protein is also expressed from the L RNA strand.

relationships among the arenaviruses, the reader is All arenavirus genomes have a conserved 3’-termi-

referred to Clegg (2002).

nus at the ends of the L and S RNA; this sequence is inversely complementary to the 5’-terminus of the same RNA strand. There is some evidence by electron

REPLICATION microscopy of intramolecular and intramolecular complexes promoted by this arrangement of 3’- and

Arenaviruses replicate in a wide variety of mammalian 5’-termini nucleotide complentarity.

cells, although either BHK-21 cells or monkey kidney cell lines are preferred for molecular studies (Howard, 1986). Arenaviruses can also infect a number of

Phylogenetic Analysis primary human cell lines and macrophages, including some members of the family that do not otherwise

Sequencing of PCR products can give useful quanti- cause human infections. Most arenaviruses also grow tative comparisons between newly discovered isolates

well in mouse L cells but the simultaneous production and those already characterised, providing caution is

of C-type retroviruses restricts the usefulness of such exercised both in the choice of primer sets and the

cells. The widely conserved cell protein a-dystroglycan method of analysis. Bowen et al. (1997) analysed at

has been identiﬁed as the cellular receptor for Old

ARENAVIRUSES

Figure 19.3 Phyogenetic analysis of Old and New World arenaviruses using nucleocapsid (N) gene sequences. The New World viruses are divisable into at least three clades (A–C). Reproduced from Clegg (2002) by permission of Springer-Verlag GmbH & Co.

World arenaviruses, such as Lassa fever and Mobala, types. Plaque assays are possible, but only under well- and those New World arenaviruses Latino and

deﬁned conditions. Cultures of persistently infected Oliveros in clade C (Cao et al., 1998). However,

cells are readily established, with morphology and other cell surface proteins and co-factors may also be

growth kinetics similar to those of uninfected cells. involved.

The major feature of an ambisense coding strategy is At low multiplicities of infection (i.e. below 0.1) the

that it allows for independent expression and regula- latent period is approximately 6–8 h, after which cell-

tion of the N and GPC genes from the S RNA associated virus increases exponentially. The titre of

segment. The N protein is expressed late in acute extracellular virus reaches a maximum 36–48 h after

infection and continues to be expressed in persistently infection. The passage history of any particular virus

infected cells in the absence of glycoprotein produc- stock is probably one of the most critical factors in

tion. This is explained by the production of a determining the kinetics of arenavirus replication.

subgenomic mRNA from a negative polarity, virus- Infected cells undergo only limited cytopathic

sense template. A control mechanism must therefore changes in the cell lines commonly employed, with

exist which determines the fate of nascent RNA of little or no change in the total level of host cell protein

negative polarity, destined either for encapsidation or synthesis; virus yields vary in diﬀerent susceptible cell

as a template for N protein-speciﬁc mRNA. In

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

contrast, the template for glycoprotein-speciﬁc mRNA case of the New World arenaviruses, serological cross- is of complementary sense to viral RNA and, as such,

reactions in the IF test, e.g. with sera from patients would not be required for nascent virus production.

with Bolivian (Machupo) and Argentinian (Junı´n) The lack of glycoprotein late in the replicative cycle or

haemorrhagic fevers, are found with ﬁxed cultures. in persistently infected cells would therefore imply

Substrates prepared from other members of the selective transcriptional or translational control of this

Tacaribe complex, which includes Junı´n and Machupo gene product.

viruses, also react with sera taken from these patients Both viral RNA and its complementary strand

during the acute phase and into early convalescence. contain at least one hairpin sequence, which may

Greatest cross-reactivity is seen between the closely provide recognition points for termination of tran-

related Junı´n and Machupo antigens, closely followed scription by viral RNA polymerase. The nucleotide

by Tacaribe virus-infected cells. ELISA has been used sequence in the hairpin region is of coding sense and

as an alternative to IF for early and rapid diagnosis; its may be transcribed, either as a discrete mRNA species

use was restricted by the small amounts of antigen or as a result of extended transcription of N or GPC

available for coating the solid phase but this has messengers through this region. The reading frames for

changed with the availability of recombinant antigens. viral gene products transcribed from LCM and

As a ﬁrst step towards diagnosis, the use of PCR can Pichinde viral genomes would ﬁt this hypothesis. In

be considered, provided that primer sets have been addition, a sequence for ribosomal 18S subunit

rigorously tested beforehand and the temperature binding is present on both mRNA molecules, although

cycling conditions optimised. The need is often to its signiﬁcance remains uncertain.

give a ﬁrst indication as to which of the various causes of viral haemorrhagic fever may be present, and thus it is often the case that PCR reactions need to be

DIAGNOSIS OF HUMAN ARENAVIRUS conducted in parallel using a range of primer sets INFECTIONS

speciﬁc for as many as six diﬀerent agents, whether these be arenaviruses or other suspected causes, e.g.

The diagnosis of arenavirus infections may be made by ﬁloviruses. Drosten et al. (2002) have shown that this is demonstration of a four-fold rise in speciﬁc antibody

possible; overcoming the common problems of low titre, the presence of speciﬁc IgM antibodies, or

sensitivity and non-speciﬁc ampliﬁcation often asso- isolation of the virus. Although arenaviruses can easily

ciated with such multiplex PCR tests. The advantages

be grown in a variety of mammalian cell cultures, it of PCR include the opportunity to obtain sequence must be remembered that clinical specimens from

information, of increasing relevance to the identiﬁca- patients suspected as having a viral haemorrhagic fever

tion of new family members. Also PCR is useful for should always be handled in biologically secure

diagnosis in the early stages of disease when antibodies containment facilities. For this reason tests for anti-

have yet to develop. The drawback, however, is that body are more useful, since inactivated viral antigens

PCR does not discriminate between the presence of for serology can prepared easily. For routine isolation,

RNA fragments and infectious virus. Thus, isolation the E6 clone of Vero cells is the cell line of choice,

of virus using cell cultures in a high-security facility although all arenaviruses grow well in primate and

should be attempted whenever possible. rodent-derived fibroblast cell lines. However, a cytopathic effect (CPE) is often difficult to see, and inoculated cultures often require examination by

ANTIGENIC RELATIONSHIPS immunoﬂuorescence (IF) or immunoperoxidase assay in order to detect viral antigens.

Monoclonal antibodies are used to distinguish between IF-based speciﬁc viral antibody tests are often the

virus strains because they can be prepared against preferred method for the diagnosis of human arena-

epitopes which go unrecognised when polyclonal virus infections. In the case of Lassa fever, infected cell

antisera are used. Buchmeier et al. (1981) summarised substrates are used that have been treated by ultra-

the patterns of reactivity with a panel of monoclonal violet (UV) light, acetone and cobalt irradiation to

antibodies directed against laboratory strains of the ensure safety. Drops of cell cultures dried onto glass

homologous LCM virus, and Lassa and Mopeia slides can be prepared in a central laboratory and these

viruses. Antibodies directed against the smaller, GPC preparations remain stable for many months. Most of

envelope glycoprotein cross-reacted by immuno- the antigen detected within acetone-ﬁxed infected cells

ﬂuorescence with all substrates examined, whereas represents cytoplasmic nucleocapsid protein. In the

antibodies directed against the larger GP1 glycoprotein antibodies directed against the larger GP1 glycoprotein

The plaque reduction neutralisation test is highly specific for all members of the Arenaviridae; it is notable that the few examples of cross-reactivity were obtained with high-titre animal antisera raised against Junıń, Tacaribe and Machupo viruses. However, the ease with which neutralising antibodies can be quantified varies greatly. No cross-reactions have been observed between Junıń and Machupo viruses in plaque reduction tests with human convalescent sera despite sharing a close antigenic relationship. A similar marked specificity of neutralisation has been demonstrated with LCM and Lassa sera, although neutralising antibodies to Lassa virus can be detected only with great difficulty. The sensitivity of the neutralisation test for LCM virus can be increased by incorporating either complement or anti-g-globulin into the test system.

CLINICAL AND PATHOLOGICAL ASPECTS Immune Response

The classic example of virus-induced immunopathological disease is LCM virus infection of adult mice (Casals, 1975) in which intracerebral inoculation causes severe disease and death. In contrast, if mice are infected before or shortly after birth they develop a non-pathogenic lifelong carrier state. The newborn mouse is immunologically immature and the virus does stimulate an immune response; in these circumstances the virus causes no illness. The immunologically mature mouse mounts an immune response following

LCM virus infection and a fatal choriomeningitis results, but without evidence of neuronal damage (Lehmann-Grube, 1971). Immunosuppression, either by neonatal thymectomy or by use of antilymphocytic serum, protects adult mice against fatal LCM infection; the pathological damage thus appears to be immune-mediated.

The immune responses are best understood in acute infection of mice. Intraperitoneal injection of adults gives rise to an asymptomatic acute infection of 2–3 weeks’ duration. Studies of such infections have resulted in a number of findings with implications beyond the field of arenavirus research. First, the description by Rowe (1954) of the immune-mediated pathology of acute LCM infection was the first demonstration that the pathogenicity of the viruses may not be solely related to their cytolytic effects. The observation that LCM virus infected cells were lysed by cytotoxic T cells led to the concept that recognition of a target cell requires the presence of both viral antigen and class I antigen of the host’s major histocompatibility complex (Zinkernagel and Doherty, 1979). Second, the persistence of virus in mice infected shortly after birth has provided a model for both host and viral factors involved in the establishment and maintenance of chronic infection. The finding of virus antigen–antibody complexes in persistently infected animals shows that B cell tolerance is not involved. Finally, activation of natural killer cell activity early in acute infection, which coincides with the production of interferon, has helped increase our knowledge of innate immunity against virus infection.

The direct demonstration of virus replication in lymphocytes is of substantial importance for understanding arenavirus pathogenesis, as these cells provide

a continuing source of virus that enters the circulation and play a key role in the temporal and quantitative control of the immune response (Murphy and Whit- ﬁeld, 1975). Viral antigen is present in the cells of the lymphatic system in mice persistently infected with LCM virus. Most of the virus in the blood of carrier mice is associated with approximately 2% of the total circulating lymphocyte population. Precursor or immature lymphocytes may support the replication in vitro of LCM virus when they are stimulated to proliferate by phytohaemagglutinin, in agreement with the general ﬁnding that arenaviruses grow best in actively dividing cells. Such clonal expansion may be triggered in vivo by viral antigen binding to appropriate lymphocyte receptors.

Arenaviruses can replicate in peritoneal and tissue macrophages. Virus can be recovered from mononuclear

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597

cells and macrophages of adult mice infected with LCM virus when these cells become activated as a result of the uptake of heterologous antigens. This does not occur in athymic mice, suggesting that infection of macrophages requires T cell activity.

Interferon Interferon is induced early in acute LCM virus

infection of mice, and its appearance correlates with the appearance of infectious virus in the blood. There have been few studies of the levels of a-interferon in acute arenavirus infection of man. Elevated levels can

be detected in the early stages of Argentinian haemorrhagic fever, and these coincide with the onset of fever and backache. Although there is no correlation between the titres of interferon and circulating virus, Levis and Saavedra (1984) have suggested that at least some of the clinical signs may be directly attributable to interferon, particularly the depression of platelet and lymphocyte numbers that result from Junı´n virus infection of leukocytes and macrophages.

The role of natural killer cells in controlling arenavirus infection is not clear, although many are found in the blood and spleen of LCM virus-infected mice as early as 1 day after infection. This response declines rapidly, however, until by the fourth day almost all the cytolytic immune activity is H-2- restricted.

Antibodies Antibodies against the nucleocapsid can be detected by

complement fixation (CF) and immunofluorescence early in the acute phase of most arenavirus infections. Infectious virus–antibody complexes can be detected 4 days after LCM virus infection of mice but there is no evidence that B cell responses play a role in the pathology of the acute infection. Immunity to arenaviruses appears in general to be type-specific; an infection with one member of the family does not necessarily confer protective humoral or cellular immunity against arenaviruses that can be distinguished by neutralisation tests in vitro. However, cross- reactive antibodies may confer some degree of protection in some instances, e.g. immunisation of guinea- pigs with Tacaribe virus protects against subsequent challenge with the normally virulent Junıń virus. These responses are clearly different from the anamnestic responses that may be induced as a result of antigenic

similarities between the nucleocapsid proteins of the two viruses concerned.

Cellular Immunity

The role of cellular immunity during acute LCM infection is manifested by a cytotoxic T cell response associated with the clearing of virus; e.g. CD8 + T cells cultured and cloned in vitro and injected intravenously reduce the amount of virus 100-fold in the spleens of acutely infected mice. Cytotoxic T cell responses are restricted by the need for activated T cells to recognise both viral antigen and host cell proteins encoded by the H-2 region, a concept developed in LCM-infected mice which as referred to above has radically altered our concept of the mechanisms by which the infected host clears virus from infected tissues. The generation of speciﬁc cellular toxicity is related to the replication of the virus in target organs; inoculation with live virus appears necessary as a primary cytotoxic T cell response is not seen if the virus is inactivated. This has implications for the development of inactivated arenavirus vaccines should the stimulation of cellular immunity prove essential for protection, as many workers believe. T cell clones from mice infected with the Armstrong strain of LCM virus lyse a wide range of LCM virus strains. This ﬁnding demonstrates that cytotoxic responses to arenaviruses are haplotype- restricted but show a broad cross-reactivity for conserved viral determinants. Some of these determinants have now been mapped to an immunodominant domain of GP2 (amino acids 278–286; Whitton et al., 1988). Such T cell clones can discriminate between cells infected with a given strain of virus containing only a single amino acid substitution in this region; this implies that mutations in this region of the genome may lead to selection of a virus variant with altered pathogenicity.

In contrast to LCM virus, the role of cellular immunity in Lassa virus infection seems to play only a minor role. The human host is clearly restricted in its ability to clear the virus and prevent virus replication in tissues, possibly because of an impaired cytotoxic T cell response. The poor neutralising antibody response and the high degree of viraemia contrast sharply with those in patients with South American haemorrhagic fevers, in whom there is little viraemia and neutralising antibodies develop rapidly during acute infection. The prospects of immunotherapy thus seem poor and greater emphasis has, therefore, been placed on the use of antiviral agents (see below).

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599 PERSISTENT INFECTION

ARENAVIRUSES

there is evidence for arenaviral RNA being transcribed into complementary DNA, presumably mediated by

Antibodies endogenous retroviral reverse transcriptase (Klener- man et al., 1997). This would imply that long-term

Mice persistently infected with LCM virus produce persistence of viral gene sequences as retroviral antibodies to all the major structural proteins. This

elements results in continual low-level expression of ﬁnding was contrary to the view previously held that

viral proteins. Thus immune responsiveness is main- viral persistence is established or maintained in vivo as

tained by the continued presentation of viral sequences

a result of an absence of speciﬁc B cell responses to as MHC–peptide complexes. some or all viral antigens. As viral proteins continue to

be produced in the tissues of such animals, circulating antigen–antibody complexes are formed which can be detected by binding Clq. It is worth noting that,

PATHOLOGY OF ARENAVIRUS INFECTIONS: despite the existence of antibody to all LCM virus

GENERAL FEATURES structural polypeptides, sera from persistently infected

mice are negative by CF tests; this was the original The mechanisms by which arenaviruses cause disease basis for the belief that carrier animals do not produce

in man are not fully understood. There is no evidence

a humoral response to this virus. Antibodies in the sera that either immunopathological or allergenic processes of such animals bind to the surface of virus infected

play any part in causing disease; it appears to be more cells, but are unable to mediate complement-dependent

likely that disease is caused by direct damage of cells cytolysis, suggesting that viral antigens at the plasma

by the virus. Post mortem studies on patients who died membrane may be either masked, thereby preventing

from Junı´n virus infection have shown generalised further immune reactions, or removed by antigenic

lymphadenopathy, endothelial swelling in the capil- modulation. This notion would imply that persistently

laries and arterioles of almost every organ, infected mice are deﬁcient in viral antibody of the

accompanied by a depletion of lymphocytes in the complement ﬁxing subclass of IgG, but this has not

spleen. The virus ﬁrst replicates in lymphoid tissue, been proven.

whence it invades the reticuloendothelial system and those cells concerned in the humoral and cellular immune responses; the host’s defence mechanisms are

Cell-mediated Immunity thus impaired. Fatal illness is invariably associated with capillary damage leading to capillary fragility,

Mice persistently infected with LCM virus should haemorrhages and irreversible shock (Johnson et al., mount a normal T cell response to unrelated immuno-

1973). Disseminated intravascular coagulation is not a gens, indicating a state of tolerance only to speciﬁc

typical feature. Although Lassa fever is often regarded antigens. However, it has been diﬃcult to distinguish

as being hepatotropic, the extent of hepatic damage is T cell suppression from an absence of virus-speciﬁc

insuﬃcient to account for the severity of the clinical T cell clones. Here it is pertinent to mention that

disease and serum transaminase values often remain persistence of LCM virus in mice infected at birth or in

within normal limits except in severe cases. Studies of utero was one of the important observations made by

Lassa virus-infected rhesus monkeys have shown that Burnet and Fenner to support the concept of tolerance

changes in vascular function may play a much greater to ‘self’ antigens. The time of infection is critical, as

role in pathogenesis, as a result either of viral LCM infection induced 24 h after birth results in a

replication in the vascular epithelium or secondary cytotoxic T cell response typical of acute disease. The

eﬀects of virus activity in diﬀerent organs. Platelet and failure of mice infected before this time to mount an

epithelial cell functions fail immediately before death adequate cytotoxic response is presumably related to

and are accompanied by a drop in the level of maturation of T cell function; it appears to be virus

prostacyclin; these functions rapidly return to normal speciﬁc because adult carrier mice challenged with

in animals surviving infection (Fisher-Hoch et al., other unrelated arenaviruses mount normal cytotoxic

1987). Impairment of the functions of vascular T cell responses. Thus, the block appears to be either in

epithelium in the absence of histological changes recognition of infected cells or in their expression of

appears to be a common feature of the ﬁnal stages of type-speciﬁc antigenic determinants. The relationship

viral haemorrhagic diseases in general and suggests between the virus and the host immune response may

that hypovolaemic shock may be amenable to treat-

be more complex than hitherto believed, however, as

ment with prostacyclin.

The pathogenesis of Argentinian haemorrhagic fever has been studied in guinea-pigs infected with Junıń virus, this being a suitable model of human disease. There is a pronounced thrombocytopenia and leukopenia characteristic of human infections, and animals die of severe haemorrhagic lesions. Bone marrow cells are destroyed with release of proteases and acid and alkaline phosphatases into the blood; this leads to consumption of the C4 complement component. These effects may lead in turn to progressive alterations in vascular permeability and platelet function (Rimoldi and de Bracco, 1980). The most extensive histopathological studies have been made on tissues from patients with Lassa fever (Walker and Murphy, 1987). How- ever, there are many similarities in the pathological lesions found in man following Junıń and Machupo virus infections. Focal non-zonal necrosis in the liver has been described in all three conditions, with hyperplasia of Kupffer cells, erythrophagocytosis and acidophilic necrosis of hepatocytes. Councilman-like bodies can be observed together with cytoplasmic vacuolations and nuclear pyknosis or lysis. As with other organs, there is little evidence of cellular inflammation. Lesions in other organs have been described, including interstitial pneumonitis, tubular necrosis in the kidney, lymphocytic infiltration of the spleen and minimal inflammation of the central nervous system and myocardium (Walker and Mur- phy, 1987). The hepatic changes may range from mild, focal necrosis to extensive zonal necrosis, involving up to 50% of hepatocytes. These changes are consistent with a direct cytolytic action of the virus; nevertheless, the simultaneous presence of Lassa virus and specific antibodies during the later stages of the acute disease suggests that antibody-dependent cellular immune reactions may also occur. Microscopic changes in the kidneys are minimal, although it is not clear whether the functional impairment is due to the deposition of antigen–antibody complexes.

Lymphocytic Choriomeningitis Clinical and Pathological Features

Infection is often inapparent but may present as an influenza-like febrile illness, as aseptic meningitis or as severe meningoencephalomyelitis. The great majority of LCM infections are, however, benign. The incubation period is 6–13 days. In the influenza-like illness there is fever, malaise, muscular pains and bronchitis. An early leukopenia followed by lymphocytosis is a constant finding. Generally, the mean value of mono-

nuclear cells is approximately 600 cells/mm 3 , although counts of up to 3000/mm 3 have been recorded. A coryza, together with retro-orbital pain, anorexia and nausea are common. During the acute phase a large number of mononuclear cells are present in the cerebrospinal ﬂuid as part of a pleocytosis, although the absolute number varies with time after onset.

As with all central nervous system diseases, the cerebrospinal ﬂuid (CSF) is at increased pressure, with

a slight rise in protein concentration, normal or slightly reduced sugar concentration, and a moderate number of cells, mainly lymphocytes (150–400/mm 3 ). It has been noted that the majority of such patients have a history of inﬂuenza-like illness immediately prior to the onset of meningitis. The meningeal form is more common; the same symptoms may remain mild and be of short duration and patients recover within a few days, but there can be a more pronounced illness with severe prostration lasting 2 weeks or more. Chronic sequelae have been reported on occasion, including parotitis and orchitis. Other symptoms include continuing headache, paralysis and personality changes. The few deaths reported have followed severe meningoencephalomyelitis. In this most severe form, patients may rapidly develop a bilateral papilloedema, confusion and paralysis of the extremities over a 1 week period. An erythematous rash followed by haemorrhage and death has also been reported. Virus can be isolated from blood, CSF and, in fatal cases, from brain tissue. However, the preferred method of diagnosis is antibody detection by immunoﬂuores- cence, although the test is not readily available in most clinical virology laboratories. Because of the possible diagnosis of related, more hazardous arenaviruses, samples should be referred to a national reference laboratory.

Epidemiology Man is usually infected through contact with rodents.

In the past, these have been acquired in laboratories, where LCM may be a contaminant in laboratory colonies of mice and hamsters. In particular, virus is shed from the urine of persistently infected animals, resulting in contamination of skin and working surfaces. Hamsters kept as pet animals have also played a role in human infection. The mechanism of transmission of the virus to man is not fully understood but is likely to involve dust contaminated by urine, the contamination of food and drink, or via skin abrasions.

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A variant of LCM virus has been isolated from captive New World primates. The histopathology in infected marmosets and tamarins is remarkably similar to that seen in Lassa virus infection in humans. It is suspected that these animals acquired the virus from infected Mus musculus rodents (Montali et al., 1995; Stephensen et al., 1991).

Diagnosis of Bolivian and Argentinian Haemorrhagic Fevers

Although the clinical features of Bolivian and Argen- tinian haemorrhagic fevers are similar, the laboratory diagnosis of these diseases is approached in a somewhat diﬀerent manner. In the case of Junı´n, virus can

be recovered consistently from the blood from the 3rd to the 8th day of illness; in contrast, direct recovery of Machupo virus from acutely ill patients is much more diﬃcult. In both instances, serological methods are more useful.

Complement-fixing antibodies may be detected sufficiently early in both cases, provided that suitable paired sera are available. Although this technique has now largely been superseded by the use of more sensitive immunofluorescence methods and ELISA, the appearance of CF antibodies may still provide useful information as to the course of the infection and signal the onset of convalescence. Early use of immunofluorescence techniques for the diagnosis of Argentinian haemorrhagic fever showed that specific antibodies could be detected by the indirect method approximately 30 days after onset of symptoms. Specific staining is generally seen as a bright, granular fluorescence evenly distributed over the cytoplasm of the fixed infected cell substrate. The titre of immunofluorescent antibodies increases from the 12th to the 20th day of illness and is a mixture of IgG and IgM antibodies.

Neutralising antibodies to both Machupo and Junıń viruses persist for many years at high titre, appearing simultaneously with CF antibodies. The sensitivity and specificity of neutralisation tests for detecting immunity to Junıń virus has proved to be of value retrospectively in the detection of subclinically infected individuals. The test may be carried out in Vero cell monolayers by varying virus dilution in the presence of

a ﬁxed concentration of serum. Antibody titres are then expressed as an index calculated by subtracting the logarithmic diﬀerences between the virus titre in control and experimental reactions. Inapparent infections have been shown in approximately 20% of

laboratory workers handling known or presumptively positive specimens by this method.

Argentinian Haemorrhagic Fever (Junı´n Virus) Clinical and Pathological Features

Argentinian haemorrhagic fever has been known since 1943 and Junıń virus, the causative agent, was first isolated in 1958. The virus causes annual outbreaks of severe illness, with between 100 and 3500 cases, in an area of intensive agriculture known as the wet pampas in Argentina. Mortality in some outbreaks has been in the range of 10–20%, although the overall mortality is generally 3–15%. After an incubation period of 7–16 days, the onset of illness is insidious, with chills, headache, malaise, myalgia, retro-orbital pain and nausea; these are followed by fever, conjunctival injection and suffusion, a pharyngeal enanthema and erythema and oedema of the face, neck and upper thorax. A few petechiae may be seen, mostly in the axilla. There is hypervascularity and occasional ulceration of the soft palate. Generalised lymphadenopathy is common. Tongue tremor is an early sign, and some patients present with pneumonitis. In the more severe cases the patient’s condition becomes appreci- ably worse after a few days, with the development of hypotension, oliguria, haemorrhages from the nose and gums (Figure 19.4), haematemesis, haematuria and melaena. Oliguria may progress to anuria and pronounced neurological manifestations may develop. Laboratory findings have included leukopenia with a decrease in the number of CD4 + cells, thromobocyto- penia and urinary casts containing viral antigen. Patients recover when the fever falls, followed by diuresis and rapid improvement. Death may result from hypovolaemic shock. Subclinical infections also occur. Man-to-man transmission has not been observed.

Epidemiology Argentinian haemorrhagic fever has a marked seasonal

incidence, coinciding with the maize harvest between April and July, when rodent populations reach their peak. Agricultural workers, particularly those harvest- ing maize, are, not surprisingly, the most commonly affected. The main reservoir hosts of Junıń virus are Calomys field voles that live and breed in burrows under the maize fields and in the surrounding grass banks (Figure 19.5). Other rodent species may also be

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PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

this therapy is directly related to the titre of neutralising antibody in the plasma; as a result a dose of no less than 3000 ‘therapeutic units’/kg body weight has been recommended (Enria et al., 1984). The late development of a neurological syndrome is seen in up to 10% of patients treated with immune plasma; it is often benign and self-limiting but points to the possible persistence of viral antigens on cells of the central nervous system well into convalescence. Treatment with immune plasma also restores the response of peripheral blood lymphocytes to antigenic stimuli, suggesting that administration of plasma also results in the modulation of cellular immunity.

Figure 19.4 Bleeding from the gum margin in a case of Argentinian haemorrhagic fever. Reproduced from Howard

Prophylaxis There have been attempts to produce a vaccine against

Argentinian haemorrhagic fever. The XJC1 3 strain of virus grown in the brains of suckling mice is relatively non-pathogenic and was administered to 636 volunteers between 1968 and 1970. Over a period of 3 years,

70 cases of Junı´n virus infection occurred among the population but there were no cases amongst those immunised. However, the vaccine often induced a mild febrile reaction or a subclinical infection and its use was discontinued, despite the fact that over 90% of vaccinees maintained neutralising antibody for up to

9 years. There have been renewed attempts during recent years to develop a new vaccine strain suﬃciently

Figure 19.5 The habitat for the rodent Calomys sp. in attenuated for human use and meeting modern day the wet pampas of Argentina. Reproduced from Howard

requirements as to derivation, manufacture and (1986)

potency. Several clones have been prepared from the original XJ isolate, one of which exhibits less

neurovirulence than the XJCl 3 strain, yet protected infected. Calomys spp. have a persistent viraemia and

rhesus monkeys against challenge with wild-type Junı´n viruria, and virus is also present in considerable

virus (McKee et al., 1993). This ‘Candidate 1’ vaccine quantities in the saliva. The mode of transmission of

has been tested in a double-blind study in volunteers. Junı´n virus to man has not been conclusively estab-

lished. The virus may be carried in the air from dust contaminated by rodent excreta or may enter by

Bolivian Haemorrhagic Fever (Machupo Virus) ingestion of contaminated foodstuﬀs.

Clinical Features Bolivian haemorrhagic fever was ﬁrst recognised in

Therapy 1959 in the Beni region in north-eastern Bolivia, with 470 reported cases in the years up to 1962. The disease

In contrast to Lassa fever, antibodies play a major role continued in that region more or less annually for a in recovery from Junı´n infection. Controlled trials of

number of years in the form of sharply localised immune plasma collected from patients at least 6

epidemics. Its incidence has decreased considerably months into convalescence have shown a dramatic

since the late 1960s and human infections are now reduction in mortality if plasma is given within the ﬁrst

rarely reported. It is worth noting that the discovery of

8 days of illness (Maiztegui et al., 1979). The eﬃcacy of

a common morphology and serological cross-reaction a common morphology and serological cross-reaction

The clinical disease is similar to Argentinian haemorrhagic fever. The incubation period ranges from 7 to 14 days and the onset is insidious, beginning with an influenza-like illness accompanied by malaise and fatigue. This is followed by abdominal pain, anorexia, tremors, prostration and severe limb pain. About one-third of patients show a tendency to bleed, with petechiae on the trunk and palate, and bleeding from the gastrointestinal tract, nose, gums and uterus. Almost half the patients develop a fine tremor of the tongue and hands, and some may have more pronounced neurological systems. The acute disease may last 2–3 weeks and convalescence may be protracted, generalised weakness being the most common complaint. Clinically inapparent infections are rare. Machupo virus, the responsible agent, is readily isolated from lymph nodes and spleen taken at necropsy. Isolation of the virus from acutely ill patients has proved difficult, however, the best results being obtained from specimens taken 7–12 days after the onset of illness.

Epidemiology The rodent reservoir of Machupo virus is the ﬁeld vole,

Calomys callosus ; over 60% of animals caught during the San Joaquin epidemic were found to be infected. The distribution of cases in the township was associated with certain houses and C. callosus was trapped in all households where cases occurred. Transmission to man is probably by contamination of food and water or by infection through skin abrasions. Transmission from man to man is unusual but a small episode took place in 1971, well outside the endemic zone. The index case, infected in Beni, carried the infection to Cochabamba and, by direct transmission, caused ﬁve secondary cases, of which four were fatal.

Abnormally low rainfall, combined with an increase in the use of insecticide, led to a rapid decline in the numbers of cats, with the result that the population of

Machupo-infected rodents increased dramatically, thus increasing the opportunity for human contact with contaminated soil and foodstuﬀs. This balance has since been restored, consistent with the decline in the number of reported cases over the past two decades.

Treatment As with AHF, treatment is largely supportive.

Although attempts have been made to use convalescent immune plasma from survivors of Machupo infection, a combination of a lack of facilities in Bolivia suitable for treating collected plasma and the absence of a controlled trial as to the eﬃcacy of its use means that the treatment of patients with immunoglobulin remains speculative. Ribavirin has been administered during the 1994 outbreak, but again there is no certain indication that ribavirin is eﬀective against Machupo infection.

Lassa Fever History . In 1969 Lassa virus made a dramatic

appearance in Nigeria as a lethal, highly transmissible disease. The ﬁrst victim was an American nurse, who was infected in a small mission station in the Lassa township in north-eastern Nigeria, whence the virus and the disease derive their names. The origin of the infection was never determined, although it is thought to have been acquired through direct contact with an infected patient in Lassa. When the nurse’s condition steadily deteriorated, she was ﬂown to the Evangel Hospital in Jos, where she died the following day. While she was in hospital she was cared for by two other American nurses, one of whom also became infected by direct contact, probably through skin abrasion. This nurse became unwell after an 8 day incubation period and died following an illness lasting

11 days. The head nurse of the hospital, who had assisted at the post mortem of the ﬁrst patient, fell ill

7 days after the death of the second patient for whom she had cared, and from whom she probably acquired the infection. This third case was evacuated to the USA by air. After a severe illness under intensive care she slowly recovered. A virus, subsequently named Lassa, was isolated from her blood by workers at the Yale Arbovirus Unit. One of these virologists became ill but improved after an immune plasma transfusion donated by the third case. Five months after this

infection, a laboratory technician in the Yale

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603

laboratories, who had not been working with Lassa virus, fell ill and died. The manner in which this infection was acquired has never been determined. This trail of events not unnaturally earned for Lassa virus a formidable notoriety, which was sharply enhanced by two more devastating hospital outbreaks—one in Nigeria, the other in Liberia.

The fourth outbreak was seen in Sierra Leone in October 1972. In sharp contrast to the previous outbreaks, this one was not conﬁned to hospitals, although hospital staﬀ were at considerable risk and several became infected. Most of the patients acquired their illness in the community and there were several intrafamilial transmissions. This led to a revision of the initial view, formed from experience of nosocomial infections, that Lassa fever has a high mortality.

Lassa fever has since continued to occur in west Africa, usually as sporadic cases (Monath, 1987). Between 1969 and 1978 there were 17 reported outbreaks affecting 386 patients, in whom the mortality was 27%. Eleven of the episodes were in hospitals, where the case fatality rate reached 44%; two were laboratory infections, two were community-acquired outbreaks, and two were prolonged community outbreaks. Eight patients were flown to Europe or North America. One of them was evacuated with full isolation precautions and the remainder, of whom five were infectious, travelled on scheduled commercial flights as fare-paying passengers. Fortunately, no contact cases resulted.

Clinical features . Lassa virus causes a spectrum of disease ranging from subclinical to fulminating fatal

infection. Studies in Sierra Leone show that most patients present with only a mild form of the disease and this is resolved by good primary health care. The incubation period ranges from 3 to 16 days and the illness usually begins insidiously. The disease is diﬃcult to distinguish in the early stages from other systematic febrile illnesses, most notably malaria, septicaemia and yellow fever. The most reliable clinical signs on presentation are a sore throat, myalgia, abdominal and lower back pains, accompanied by vomiting. Occasionally a faint maculopapular rash may be seen during the second week of illness on the face, neck, trunk and arms. Cough is a common symptom, and light-headedness, vertigo and tinnitus appear in a few patients. The fever generally lasts 7–17 days and is variable. Convalescence begins in the second to fourth weeks, when the temperature returns to normal and the symptoms improve. Most patients complain of extreme fatigue for several weeks. Loss of hair is

common and deafness aﬄicts one in four patients, and there may be brief bouts of fever.

In a significant number of cases, the symptoms suddenly worsen after the first week, with continuing high fever, severe prostration, chest and abdominal pains, conjunctival injection, diarrhoea, dysphagia and vomiting. One important physical finding is a distinct pharyngitis; yellow-white exudative spots may be seen on the tonsillar pillars together with small vesicles and ulcers. The patient appears toxic, lethargic and dehydrated; the blood pressure is low and there is sometimes a bradycardia relative to the body temperature. Patients in whom the disease is eventually fatal not uncommonly have a high sustained fever. There may be cervical lymphadenopathy, an encephalopathy, coated tongue, puffiness of the face and neck, and blurred vision. In approximately 25% of cases there is marked involvement of the CNS, manifested by disorientation, ataxia and seizures. Progression to severe haemorrhaging occurs in around one-fifth of patients and it is among such patients that mortality exceeds 50%. Death is due to shock, anoxia, respiratory insufficiency and cardiac arrest. Lassa fever is particularly severe in pregnant women. A study of 75 women in Sierra Leone showed that 11/14 deaths were the result of infection during the third trimester; a further 23 patients suffered abortion in the first and second trimesters.

Epidemiology . Lassa virus has been repeatedly isolated from the multimammate rat Mastomys nata-

lensis in Sierra Leone and Nigeria. This rodent is a common domestic and peridomestic species, and large populations are widely distributed in Africa south of the Sahara. During the rainy season it may desert the open fields and seek shelter indoors. Some genetic variation has been shown in Mastomys populations inhabiting different ecological niches; however, there appears to be no difference in the prevalence of antibody and virus in at least two of the karotypes found in West Africa. The animals are infected at birth or during the perinatal period. Like other arenaviruses, Lassa virus produces a persistent, tolerant infection in its rodent reservoir host with no ill effects and without any detectable immune response. The animals remain infectious during their lifetime, freely excreting Lassa virus in urine and other body fluids. The correlation between the prevalence of antibody in a community and the degree of infestation by infected rodents, however, is poor.

Studies of the ratio of clinical illness to infections have recently conﬁrmed that Lassa fever is endemic in several regions of West Africa. It has been estimated

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PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

605 that only 1–2% of infections are fatal—substantially

ARENAVIRUSES

Table 19.2 Diﬀerential diagnosis of arenavirus fevers less than the ﬁgures of 30–50% originally associated with the early nosocomial outbreaks. However, there

Yellow fever

may still be up to 300 000 infections per year, with as Malaria

Bacterial septicaemia

many as 5000 deaths (McCormick et al., 1986b). The Enteric fevers (typhoid, paratyphoid) seroconversion rates among villagers in Sierra Leone

Streptococcal pharyngitis vary from 4 to 22 per 100 susceptible individuals per

Typhus

year; up to 14% of febrile illness in such population

Trypanosomiasis

groups is due to Lassa virus infection. There is a

Leptospirosis

marked variation as to the severity of the disease Other viral haemorrhagic fevers according to diﬀerent geographical regions. This may

in part be due to genotypic variation of Lassa virus, or dose and route of infection, or a combination of these

laboratories equipped (Biosafety level P4) to provide factors (Fisher-Hoch, 1993). There is a relatively high

maximum containment to protect the investigator. rate of asymptomatic and mild infections in endemic

Suspected cases should be reported immediately to areas. One reason for this may be the frequency of

local and national public health authorities prior to reinfections; although about 6% of the population lose

any attempt to handle specimens. antibody annually, rises in antibody titre are also often

The diagnosis of Lassa fever is conﬁrmed by observed. It is not clear whether reinfection results in

isolation of the virus or demonstration of a speciﬁc clinical disease. A frequent ﬁnding of incomplete

serological response. Infection in the early stages can immunity after infection would have profound impli-

be confused clinically with a number of other cations for the use of a vaccine.

infectious diseases, particularly malignant malaria There may be secondary spread from person to

(Table 19.2) (Woodruﬀ, 1975). The two most reliable person in conditions of overcrowded housing and

prognostic markers of fatal infections are the titres of particularly in rural hospitals. There is a particularly

circulating virus and of aspartate aminotransferase high risk to staﬀ and patients on maternity wards, as

(AST). Patients in whom the titre of virus exceeds 10 4 Lassa fever is a major cause of spontaneous abortion.

TCID 50 /ml accompanied by AST levels above 150 IU Medical attendants or relatives who provide direct

have a poor prognosis, and fatality rates approach personal care are most likely to contract the infection;

80%. In contrast, patients with virus and enzyme levels as noted above, accidental inoculation with a sharp

below these values have a greater than 85% chance of instrument and contact with blood have caused

survival (Johnson et al., 1987). This demonstration of infection in a few cases. Airborne spread may take

an association between the degree of viraemia and place, as well as mechanical transmission. Although in

mortality is unique for virus infections and contrasts Sierra Leone there has been no evidence of airborne

with the diﬃculty in predicting the outcome in patients spread in hospital outbreaks, one of the 1970 out-

with Argentinian and Bolivian haemorrhagic fevers. breaks in Nigeria is believed to have been caused by

Although Lassa fever can be diagnosed accurately airborne transmission from a woman with severe

from the presence of IgM antibodies on admission, pulmonary infection.

there is no correlation between the time of appearance, Lassa fever is a major cause of spontaneous abortion

the titre of speciﬁc antibodies and clinical outcome. in West Africa. The virus is readily recovered from the

Lassa virus grows readily in Vero cell culture and blood and placenta of aborted fetuses. Women

virus can usually be isolated within 4 days. Virus can generally recover quickly after such abortions, showing

be cultured from serum, throat washings, pleural ﬂuid

a dramatic decline in viraemia, partially due to massive and urine; it is excreted from the pharynx for up to 14 bleeding at the time of abortion (A. Demby, personal

days after the onset of illness and in urine for up to 67 communication). Paediatric Lassa fever is known to

days after onset. Lassa infection can be diagnosed occur more commonly in male children, for unknown

early by detection of virus-speciﬁc antigens in con- reasons. Presenting as an acute febrile illness, the case

junctival cells using indirect immunoﬂuorescence. The fatality rate may approach 30% in children with

use of RT-PCR method is possible, although these widespread oedema, abdominal distension and bleed-

techniques are of limited practical use in endemic ing.

areas.

The most sensitive serological test for the detection Diagnosis . It is important to note that serodiagnosis

of Lassa antibodies is indirect immunoﬂuorescence; and virus isolation should be attempted only in

antibodies can be detected by this method in the antibodies can be detected by this method in the

Therapy . Although the passive administration of Lassa immune plasma may suppress viraemia and

favourably alter the clinical outcome, it does not always do so, particularly if the patient has a high virus burden (McCormick et al., 1986b). Failure may be due to the difficulty in assessing accurately the titre of viral neutralising antibodies in the plasma, the late and non- uniform nature of this response in convalescence, and antigenic variation. The widespread occurrence of human immunodeficiency virus (HIV) infections in West Africa precludes at present the use of immune plasma from convalescent individuals in this region. This is in marked contrast to the benefit of immune plasma in the treatment of Junıń infections. This may

be due either to the high titre of neutralising antibodies that develops soon after the acute phase or to the lesser importance of antibody in the resolution of Lassa virus.

Greater success has been achieved with antivirals. In one study of patients with a poor prognosis, treatment for 10 days with intravenous ribavirin (60–70 mg/kg/ day) within 6 days after the onset of fever showed a reduced case fatality rate of 5% (McCormick et al., 1986a). In contrast, patients treated 7 or more days after the onset of fever had a case fatality rate of 26%. In the Sierra Leone study, viraemia of greater than

10 3.6 TCID 50 /ml on admission was associated with a case fatality rate of 76%. Patients with this risk factor who were treated with intravenous ribavirin within 6 days of the onset of fever had a case fatality rate of 9% compared with 47% in those treated 7 days or more after the onset of illness. Oral ribavirin is less eﬀective.

A difficulty with its use, however, is that ribavirin can induce haemolytic anaemia in over 40% of patients. The introduction of vaccines against Junıń virus has stimulated the expectation that a vaccine could also be developed for the prevention of Lassa virus infections. However, the perceived necessity for a strong cell- mediated response would dictate the development of an attenuated vaccine; this raises concerns, however, as to a possible reversion to virulence of any attenuated Lassa virus vaccine. Given these technological difficul- ties and the limited numbers globally at risk of

infection, it is unlikely that such a vaccine will be developed in the near future.

Control . Containment of Lassa fever depends upon the strict isolation of cases, rigorous disinfection,

rodent control and effective surveillance. Nosocomial transmission presents a considerable risk and patient isolation—in isolators if available—is an absolute must. Strict procedures for dealing with body fluids and excreta need to be enforced. Disinfection with 0.5% sodium hyperchlorite or 0.5% phenol in detergent is recommended for instruments and surfaces. Given the higher virus burden in cases of Lassa fever compared to patients with Junıń or Machupo infections, surveillance of those having been in contact with Lassa fever patients is also a high public health priority. WHO recommends that those who have been in non-casual contact with cases should be observed for 3 months after their last contact with the patient. This follow-up should consist of taking body temperature measurements twice daily. Infection should be suspected if the body temperature exceeds 38.38C (1018F) and the contact hospitalised immediately.

Rodent control is frequently diﬃcult, although much can be done to minimise contact by isolating foodstuﬀs, preventing rodent entry into dwellings, and reducing the chance of inhabitants coming into contact with rodent excreta.

EMERGING ARENAVIRUS INFECTIONS

Brazilian Haemorrhagic Fever (Sabia´ Virus)

This arenavirus was isolated in 1990 from human cases at autopsy (Lisieux et al., 1994). The source of this infection was uncertain but is likely to have been acquired by exposure to infected rodents in an agricultural setting in an area immediately outside Saõ Paulo. As a continuing reminder of the potential severity of these infections, a laboratory worker became critically ill after having been accidentally exposed to an aerosol containing Sabia´ virus. The virus was first isolated from a fatal case of haemorrhagic fever. A laboratory-acquired infection was characterised by a febrile illness accompanied by leukopenia and thrombocytopenia. There is little information regarding the epidemiology of this virus, although the extensive liver necrosis seen in the first case is a warning that this and other haemophagic fevers may on first examination be mistaken for yellow fever.

606

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

607 Venezuela Haemorrhagic Fever (Guanarito Virus)

ARENAVIRUSES

became evident when in 1999 and 2000 three female patients residing in California presented with symp-

Between May 1990 and March 1991 an outbreak toms subsequently ascribed to infection with the occurred among residents of Guanarito municipality

same arenavirus. Although there was no obvious on the central plains of Venezuela. Originally mistaken

link between the three cases, each presented with as dengue fever, a total of 104 cases were recorded with

non-speciﬁc febrile symptoms and acute respiratory

a mortality rate of around 25%. The Guanarito virus distress. Two developed a lymphopenia and thrombo- was subsequently isolated from the spleens of such

cytopenia, and two also showed signs of liver failure cases at autopsy. The principal rodent hosts of this

and haemorrhage. All three died within 1–8 weeks of virus have been identiﬁed (Table 19.1) (Tesh et al.,

onset. Virus was recovered in one and all three gave 1994).

PCR products that were 87% identical with White- The disease has a clinical proﬁle similar to that of

water Arroyo virus.

Argentinian haemorrhagic fever, with patients mani- Yet further new isolations have been made recently. festing a thrombocytopenia, haemorrhaging and

A virus closely related, but distinct from, Whitewater neurological signs. Pharyngitis has been observed and

Arroyo virus has been isolated from the California deafness reported in convalescent patients. Although

mouse Peromyscus californicus. Infectious virus was initial reports suggest a high mortality for this infection,

recovered from 5/27 animals caught in the Santa Ana antibody prevalence rates of up to 3% have been found

mountains of southern California, close to the Bear among healthy individuals and up to 10% of household

Canyon trailhead. It cannot be ruled out that the contacts have anti-Guanarito virus antibodies.

tentatively dubbed Bear Canyon virus represents an additional arenavirus that has yet to be associated with human disease.

Oliveros Virus This new agent has been isolated from a small rodent,

SUMMARY Bolomys obscurus , within the endemic region of

Argentinian haemorrhagic fever (Bowen et al., 1997). The increasing numbers of human infections due to arenaviruses is beginning to require a greater vigilance

With a rodent host distinct from that of Junı´n virus, on the part of public health workers. Arenavirus approximately 25% of captured B. obscurus have been aetiology for febrile illnesses in individuals residing in found to contain antibodies to this virus. At present, endemic areas should be considered, particularly those there are no indications that this virus causes who are likely to have come into regular contact with signiﬁcant numbers of human infections (Mills et al., rodents by virtue of their lifestyle or occupation. 1996). Although until recently there has been little or no

evidence for human arenavirus infection in north America, Europe and Asia, this situation has changed

Whitewater Arroyo Virus and Other Isolates considerably since a greater awareness of the potential from the USA

for emerging infections has developed among clinicians and microbiologists, particularly in geographical areas

As a consequence of the 1993 hantavirus outbreak on where the last decades have seen clearance of wood- the Colorado Plateau, there has been intensive study of

land, forest and scrub in advance of extensive changes rodent populations in order to gauge the extent of Sin

in agricultural practices. This potential has been Nombre virus distribution and the risk that infected

augmented by changing or abnormal weather patterns, rodents present to rural populations in the USA.

these serving to promote behavioural, if not also During one such study, Kosoy et al. (1996) found an

numerical, changes in rodent populations. Particularly unexpectedly high level of arenavirus antibodies in

in the Americas, arenavirus investigations have pro- pack rats (Neotoma spp.) caught in the Whitewater

gressively become interleaved with studies on Arroyo of New Mexico. Members of the family to

hantavirus distribution, especially in endemic zones which Neotoma belongs are ubiquitous throughout the

where a particular species of rodent may be infected south-western part of the USA. Independently Ful-

with either a hantavirus or an arenavirus. The only horst and colleagues (1996) described the isolation of a

certainty is that the number of arenaviruses identiﬁed hitherto unknown arenavirus from trapped examples

hitherto will increase as more becomes known regard- of N. albigula. The importance of these ﬁndings

ing the natural history of these agents.

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

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20 Filoviruses

Susan P. Fisher-Hoch

University of Texas, Brownsville, TX, USA

INTRODUCTION epidemiological link with Africa could be found, and when laboratory-conﬁrmed human infections were

Human infections with ﬁloviruses are rare, but their documented during this epizootic, all were asympto- occurrence has invariably been dramatic and myster-

matic. The new virus was the closest relative of Ebola ious. Their ﬁrst appearance was in Marburg,

virus yet seen, serologically even more closely related Germany, in 1967 (Martini, 1971). Thirty-one people

than Marburg virus, and appeared to be Asian in were infected: laboratory technicians, medical person-

origin. Ideas about the epidemiology and clinical nel, animal care personnel and their relatives. Seven of

spectrum of ﬁloviruses had to be drastically revised. them died. Primary cases had been exposed to tissues

This was the position in 1994 when news came from and blood from African green monkeys imported into

Zaire (DRC) of a fresh outbreak of the original Zaire Germany and Yugoslavia from Uganda. A virus

strain of ﬁlovirus. Following this outbreak, relatively frequent epidemics of Ebola Zaire in Gabon and the

isolated from these patients was found by electron Congo, Ebola Sudan in Uganda and Marburg in microscopy to be unique among mammalian patho- eastern Congo have been reported, and several out- gens, having a strange, looped and branched breaks of Ebola Zaire are ongoing and apparently ﬁlamentous form, hence the name ‘ﬁlovirus’ (Figure closely linked to a major epizootic in great apes. This 20.1a) (Johnson et al., 1977). No more was heard of epizootic, combined with hunting and loss of habitat, these viruses until 1976 and 1979 when epidemics of a is now thought to threaten chimpanzees and gorillas in haemorrhagic disease with very high mortality in

the area with extinction.

northern Zaire (now the Democratic Republic of Congo; DRC) and in southern Sudan were found to

be due to two strains of a related, yet distinct ﬁlovirus (Bowen et al., 1980; Richman et al., 1983; Johnson et al. , 1977) This was named Ebola virus, after a river in

VIROLOGY Zaire (World Health Organization, 1978a, 1978b; Baron et al., 1983). Over the next 10 years rare,

Despite intensive eﬀorts, the natural history of sporadic cases of ﬁlovirus infections in Africa were the

ﬁloviruses remains one of the mysteries of virology. only continuing evidence of the existence of these

At ﬁrst they were thought to be related to rhabdoviruses. Their natural host and ecology remained

viruses, but it has become clear that they form a family elusive, but they were thought to be exclusively

of their own, designated Filoviridae on account of their African, occurring principally in Zaire, Sudan, Uganda

ﬁlamentous appearance (Kiley et al., 1982). Nucleotide and Kenya (Figure 20.2). The mystery deepened when

sequence analyses place the family in the order in 1989 a ﬁlovirus was isolated near Washington, DC,

Mononegavirales , which also includes the Para- from dying cynomolgus monkeys shipped to the USA

myxoviridae and Rhabdoviridae (Table 20.1) (Kiley from the Philippines (Jahrling et al., 1990). No

et al. , 1988; Pringle, 1991).

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

(a)

(b)

Figure 20.1 Electron micrographs of ﬁloviruses (a) Scanning electron micrograph of Ebola virus (6500). (b) Liver section from a cynomolgus monkey that died of Ebola (Reston) infection, showing extruding particles from cells in hepatic sinuses. Stars are in vacuoles also seen in Ebola-infected liver (68400). Both courtesy of C. Goldsmith

FILOVIRUSES

613

Figure 20.2 Map of Africa showing approximate locations and dates of Ebola and Marburg epidemics and origins of known index cases. Note the equatorial rain forest distribution of the outbreaks.

Table 20.1 Tree of the order Mononegavirales showing the Filoviruses undergo rapid, lytic replication in the suggested relationship of the Filoviridae to paramyxo- and

cytoplasm of a wide range of host cells. The rhabdoviruses, based on nucleic acid sequencing of the nucleic

protein gene nucleocapsids acquire the envelope with its surface

projections by a process of extrusion of cell membrane Family

Genus

Virus

rather than a discrete orderly budding, which may account for the striking pleomorphism of these viruses

Rhabdoviridae Vesiculovirus

Chandipura virus

(Johnson et al., 1977). Filoviruses are among the

largest known viruses, with highly variable length (up Lyssavirus

Vesicular stomatitits

virus

Rabies

to 14 000 nm), apparently due to concatamerisation (Regnery et al., 1980). The virions are of uniform

Paramyxoviridae Morbillivirus

80 nm diameter, with a helical nucleocapsid, consisting Paramyxovirus

Measles

Newcastle disease

of a central axis 20–30 nm in diameter, surrounded by

virus Mumps

a helical capsid 40–50 nm in diameter, with 5 nm cross-

striations. A host cell membrane-derived layer with Pneumovirus

Parainﬂuenza type 3

Respiratory syncytial

10 nm projections in regular array surrounds the

virus

nucleocapsid and the terminal windings of nucleocap-

Pneumonia virus of

sid found at one end of the particle. The virions

contain a single negative-strand RNA genome ranging Filoviridae

mice

Ebola

from 46106 (Marburg virus) to 4.56106 Da (Ebola

Marburg

virus) (Regnery et al., 1980). The RNA is a template for at least seven polypeptides, a nucleoprotein (NP), a

glycoprotein (GP), a polymerase (L) and four other undesignated proteins (VP40, VP35, VP30 and VP24), two of which are associated with the nucleocapsid (Elliott et al., 1985). The Ebola virus glycoprotein gene produces two molecular species, one a soluble truncated protein (sGP), which is secreted from infected cells, and the other the full-length glycoprotein consisting of two subunits, GP1 and GP2, the latter being anchored in the membrane. GP1 and GP2 are heavily glycosylated, and together provide the surface spikes seen by electron microscopy, and these proteins are responsible for viral entry. Marburg virus diﬀers in that it does not possess a sGP (Feldmann et al., 1999). An abundant but poorly glycosylated protein, VP40, and the nucleoprotein (N) are associated with the nucleocapsid. The VP40 has been shown to be able to bud from mammalian cells as lipid-bound virus-like particles, which are probably able to interact with cellular proteins, speciﬁcally a ligase (Licata et al., 2003). The importance of lipids is further illustrated by the observation that Ebola and Marburg viruses’ proteins compartmentalise within lipid rafts during viral assembly, and both entry and exit of the virus may be through these rafts (Bavari et al., 2002). The VP24 protein is believed to be a secondary matrix protein which appears to localise to the plasma membrane and the perinuclear region and may play a role in assembly and budding (Han et al., 2003).

Oligonucleotide restriction mapping of viral RNA has shown that there are about 60 diﬀerences between the Ebola (Zaire) and Ebola (Sudan) strains, whereas the 1976 and 1979 Ebola (Sudan) strains were almost identical (Cox et al., 1983). The Ebola viruses are genetically stable, with about 50–60% of their oligonucleotides distinct from those of Marburg virus (McCormick et al., 1983). Nucleotide and amino acid sequence analyses of Ebola (Zaire) and Marburg viruses show similar organisation and structure, with the nucleoprotein gene positioned at the extreme 3’ end of the genome (Sanchez et al., 1992). Homology in the N-terminal 400 residues of the nucleoprotein gene is strongest from positions 130–392 of the Marburg sequence, containing 34 identical amino acids, and having in turn a high degree of identity with paramyxovirus nucleoprotein sequences and, to a lesser extent, with corresponding rhabdovirus sequences. Computer-generated dendrogram analysis of this region among the Mononegavirales shows the closest relationship between Marburg and Ebola, suggesting common ancestral origin. The next nearest relative is respiratory syncytial virus, which in turn is related to the other paramyxo- and myxoviruses. Most distant are the rhabdoviruses.

Radioimmunoprecipitation studies of antibody responses in monkeys infected with different Ebola viruses show that though there was cross-reaction between Asian and African filovirus nucleoproteins. No cross-reacting antibody to the glycoprotein between Asian and African filoviruses is observed (Fisher-Hoch et al., 1992a). There is, however, apparently close identity at the glycoprotein level among Asian filoviruses, but not African filoviruses. The Asian viruses so far isolated are probably related very closely to each other, and may indeed be variants of the same strain.

EPIDEMIOLOGY Epidemics: Marburg Virus Disease

In 1967, a fulminating haemorrhagic fever struck a number of laboratory workers in Marburg and Frankfurt, Germany, and Belgrade, Yugoslavia (Mar- tini, 1971). The virus name was taken from the German city where most of the cases occurred. There were 31 human cases, 25 of which were primary infections with seven deaths (Table 20.2). None of six secondary cases died. Twenty primary cases were in Marburg, four in Frankfurt and one in Belgrade. All primary cases had handled blood or tissues from shipments of African green monkeys (Cercopithecus aethiops ) soon after being imported from Uganda, via London. Epidemiological investigations revealed that 20/29 persons with blood contact became infected, and 4/13 exposed to tissue culture. Among the primary human cases in Marburg, 10 had assisted in autopsies and three had trephined monkey skulls. One of the laboratory staﬀ had dissected kidneys, one had handled tissue culture from the monkeys, one broke

a test tube which had contained contaminated material, and ﬁve had merely cleaned contaminated glassware. None of the animal attendants not in contact with blood, or laboratory personnel who used precautions, such as protective gloves and clothing, were infected. There were no further primary cases upon institution of compulsory wearing of protective clothing with gloves and masks for work with monkeys and cleaning procedures, and use of disinfectants. Five of the secondary cases resulted from person-to-person contact at home or in hospital. The sixth was the wife of a veterinarian who became ill several weeks after recovery of her husband, from whose semen Marburg virus was later recovered. One physician inoculated herself accidentally with a needle through a rubber glove. In Belgrade, the single primary

614

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

615 Table 20.2 Table of the reported human cases and deaths from ﬁlovirus infections in Africa

FILOVIRUSES

Virus/year

Mortality (%) Marburg

Place of infection

Total cases

Marburg, Germany

Belgrade, Yugoslavia

Mount Elgon, Kenya

1999

76 52 68 Total

Durba, DRC (Zaire)

61 54 Ebola Zaire

Yambuku, Zaire

Tandala, Zaire

1980

Nzoia, Kenya

77 1994–1995

1994

Kikwit, Zaire

Republic of Congo

92*

80*

80 Ebola Sudan

Maridi, Sudan

53 Total

Uganda (Gula district)

425

224

54 Ebola Reston

Richmond, Virginia

1990

Manila, Philippines

Total cases

3120

2649

*Provisional figures, since these current outbreaks are ongoing, few cases have been laboratory confirmed and data are difficult to confirm. In these circumstances mortality may be artificially high, since only the most severe cases will be recorded.

case, a veterinarian, performed autopsies towards the however, better recorded. Three shipments of monkeys end of the monkeys’ 6 week quarantine. His wife fell ill

from the same source in Uganda were received, two of

10 days after nursing him at home. Despite the death which were in transit at Heathrow airport, London. of a total of 99 animals during quarantine in Belgrade,

The third arrived directly via Munich. Unusually high these were the only two human infections.

mortality during 6 weeks’ quarantine was noted in all About 400–600 animals originating from four

three shipments; 46/99 animals died from the ﬁrst, and shipments reached Europe from Uganda over a 3

20 and 30 from the second two. The Belgrade epizootic week period. Frankfurt received only 40–60 animals

was clearly characterised by ongoing transmission with from two shipments, and Belgrade about 300 animals

daily death of one or more animals (Figure 20.3) from three shipments. The remainder went to Mar-

(Martini, 1971).

burg. All spent 60–87 days in a holding facility in Epidemiological studies after the 1967 epidemics Uganda before being shipped to London, Heathrow,

concluded that two or three infected monkeys would where they spent 6–36 h in an animal hostel prior to

have been suﬃcient to initiate the epizootic and all being forwarded to Germany. In Marburg, the

three outbreaks of human disease. It was stated at the monkeys were housed in separate rooms with no

time that evidence clearly pointed to transmission recirculation of ‘air-conditioned’ ventilated air. Pub-

between monkeys in quarantine facilities by direct lished data are unclear as to whether ongoing enzootics

contact with equipment. Direct contact with blood and were observed inside Germany or the details of animal

tissues was documented for all human cases and there movements. Data on the Belgrade enzootics are,

was signiﬁcant evidence against transmission to

humans by air. No evidence was ever produced that supported the hypothesis that the monkeys were infected in transit in London from any number of a wide range of mammals and birds also temporarily lodging at the airport hostel. Furthermore, no evidence could be found of epizootics in Uganda, but later some indirect, controversial information emerged that there had at that time of the outbreak been excess deaths in monkey colonies in islands near Lake Kyoga, north of Lake Victoria, to the east of Mount Elgon in Kenya. In Uganda monkeys were captured in this area and placed in holding facilities, reportedly in single cages. They were then transported to Entebbe. Here they were held for at least 3 days before shipment. At the time of the outbreak the trade had expanded considerably and holding times had been reduced, and it may be imagined that some crowding may have ensued. During July and August 1967, 1772 C. aethiops were housed in Entebbe and 1290 exported, the majority to Germany and Yugoslavia (Henderson et al. , 1971).

For two decades only three further, isolated primary human Marburg infections and only two secondary cases were observed. These were adventurous tourists or expatriate residents: one a traveller in Zimbabwe and two others from the Mount Elgon region of western Kenya, not far from the shores of Lake Victoria (Gear et al., 1975; Teepe et al., 1983; Smith et al. , 1982). Extensive epidemiological investigations in Zimbabwe and on Mount Elgon revealed no clues to the origin of these infections. Medical facilities are limited in these areas, and isolated cases in local residents may go unnoticed unless dramatic epidemics supervene.

In 1999, Marburg re-emerged in the east of the Democratic Republic of the Congo, in the community of Durba. There were an estimated 76 cases and 52 deaths. A very diﬃcult investigation was conducted in what was essentially a war zone in a state of anarchy. Cases were found to be associated with illegal working of a partially ﬂooded gold mine infested with all manner of wildlife. There appeared to have been a series of primary cases among these illegal miners, rather than a point source epidemic, with secondary cases in their households. The investigating team had to leave for security reasons, and it is unclear what happened subsequently. It is possible that sporadic Marburg disease continues in the area, which is a region of continuing civil unrest.

Epidemics: Ebola Virus Disease

It was not until nearly a decade after the Marburg outbreak that simultaneous outbreaks of another lethal haemorrhagic fever struck suddenly in northern Zaire and Sudan in 1976 (Figure 20.2) (World Health Organization, 1978a, 1978b). Two more filoviruses were isolated: Ebola (Zaire) and Ebola (Sudan) (Johnson et al., 1977). In 1976 the largest outbreak recorded took place in equatorial rain-forest areas of northern Zaire. In retrospective case-searching exercises the case definition of probable Ebola haemorrhagic fever used was a person living in the epidemic area who died after 1 or more days with two or more symptoms and signs (headache, fever, abdominal pain, nausea and/or vomiting and bleeding) and who had received an injection or had had contact with a probable or proven case in the 3 preceding weeks. Virus isolation, visualisation of the virus by electron microscopy, or an Ebola virus-specific immunofluorescent antibody (IFA) titre of 64 or more was required in addition to confirm a case. Using these definitions, 280 deaths in 318 probable or confirmed cases were identified: case fatality 88%. The index case may have been a recent traveller in the northern Equateur region of Zaire, who attended the outpatient clinic of a mission hospital in Yambuku apparently for treatment of acute malaria, where he received an injection of chloroquine. It remains unclear whether this man was the source of the epidemic or whether he was infected by his injection, since another patient with

a similar illness was admitted to the hospital at about the same time. The subsequent nine cases, however, had all received treatment for other diseases at the hospital. Although all ages and sexes were aﬀected, the

616

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

Figure 20.3 A histogram of the epizootic of Marburg virus infections in monkeys imported into Belgrade in 1967.

Derived from Stojkovic et al. (1971)

highest incidence was in women aged 15–29 years, but these were frequently patients attending antenatal and outpatient clinics at the hospital, and the major risk factor was receiving an injection at this hospital. Needles were in short supply, and were not sterilised between use. Eleven of the 17 staff members of the hospital died, and the outbreak only terminated 4 weeks after it began, when the hospital was closed. Although transmission was focused in the outpatient clinics of the hospital, there was subsequent dissemination in surrounding villages to people caring for sick relatives or attending childbirths. The overall secondary attack rate was about 5%, but nearer 20% in close relatives of a case. Illness:infection ratios in one village exceeded 10:1. Ebola virus antibody in persons not ill and without contact with cases during the epidemic was only conclusively identified in four individuals. However, the following year, 1977, a single fatal case was identified in Tandala, also in northern Zaire (Table 20.2). There was no ensuing epidemic, but serological and epidemiological investigations uncovered two possible cases dating back to 1972, and 7% prevalence of IFA antibody in the local population (Heymann et al. , 1980). Unfortunately, non-specific reactions to the Ebola virus antigens used at that time make interpretation of much of these serological data uncertain.

At the same time as the 1976 Yambuku epidemic, an outbreak of a similar disease occurred in southern Sudan (Figure 20.2) (World Health Organization, 1978b). This outbreak was strongly associated with index cases in a single cotton weaving factory. There were 151 deaths in 284 cases identified (case fatality about 53% case definition not stated). The focus of the infection was in the town of Nzara, where the factory was located, and spread was to close relatives (67 cases). The epidemic was augmented by high levels of transmission at nearby Maridi hospital, following transfer of one of the Nzara patients, and further cases were transferred to Juba and Khartoum. There were 203 cases in Maridi, 93 of which were probably infected in the hospital, and 105 in the community. Forty-one staff members died, and at the height of the epidemic all wards contained patients with overt haemorrhage. The highest attack rates were associated with nursing a patient, but not with sleeping in the same room. In 1979 there was a similar outbreak, when 22/34 infections (65%) were fatal (Baron et al., 1983). Although closely related, the viruses from Zaire and Sudan were found to be distinct, confirming the conclusions of epidemiological investigations that the two 1976 epidemics were

independent. The two virus strains isolated in Sudan in 1976 and 1979, however, are identical (Cox et al., 1983).

Between 1979 and 1994 the disease fell silent, with only one case of human Ebola infection recorded (Teepe et al., 1983). In 1994 all this changed. A large, hospital-based outbreak in Kikwit resulted in the deaths of 242/315 cases. This outbreak resembled those that had gone before, in that a hospital was the amplifying focus of the outbreak, with many needle- borne transmissions, and the virus then spilled over into the community, with cases among close contacts of the sick (Reiter et al., 1999). The original source was traced to a single forest worker, but intensive studies still failed to reveal the primary host. Soon afterwards the ﬁrst of three epidemics appeared in Gabon. The index cases were apparently gold panners or hunters in the forest, one outbreak resulting from ﬁnding and handling the carcass of a dead chimpanzee in the forest. All the primary cases handled the uncooked meat at some stage. People who ate the cooked meat did not get sick.

Ebola Sudan did not reappear until October 2000, and when it did it was by way of a massive epidemic in Uganda. The origins of the outbreak are obscure, but it appeared to originate in the northern part of the country where rebel ﬁghting was ongoing. The epidemic was centred in the Gulu district and was not brought under control until mid-January 2001. There were a total of 425 cases and 224 deaths (case fatality 53%). Attack rates were calculated to be between 4.5 and 12.6/10 000 depending on the case deﬁnition (Okware et al., 2002).

Infections with Ebola Zaire re-emerged in Gabon and the neighbouring Republic of Congo in 2000 and continue to date. As of 30 December 2002, WHO reported 30 conﬁrmed cases (14 laboratory conﬁrmed and 16 epidemiologically linked), including

22 deaths, in Gabon and neighbouring villages across the Congolese border. Epidemics in the Congo are ongoing in 2003, and appear to be closely associated with the epizootics in great apes, described in the section on ecology. As of 27 February 2003, there were five confirmed and 92 probable cases of Ebola, with 80 deaths in the Cuvette Ouest Region of the Congo. These outbreaks are proving difficult to control because of the remoteness of the area, the association with the ongoing great ape epizootic and the fears and resistance of the communities which are affected. These fears were sufficient in one community for a schoolteacher to be attacked and killed for accusa- tions of being the cause of the epidemic.

FILOVIRUSES

617

Epizootics: Reston Virus In 1989 and early 1990, a ﬁlovirus closely related to

Ebola virus was isolated from Cynomolgus monkeys in quarantine facilities in Reston, Virginia, in Texas and in Pennsylvania (Jahrling et al., 1990; Centers for Disease Control, 1989). The monkeys had recently been imported into the USA from the Philippines. The first shipment arrived via Amsterdam, and exhaustive enquiries ruled out a link with African animals during transit. Early the following year more shipments of monkeys from the same source arrived, this time directly from the Philippines over the Pacific Ocean, and several more filovirus isolations were made from sick and dying monkeys (Centers for Disease Control, 1990a). No link with Africa or African animals could

be identified in the Philippines, and in the absence of such evidence this must be considered at present the first Asian filovirus. Pathogenicity for cynomolgus monkeys was uncertain because of a high rate of concurrent infection with simian haemorrhagic fever virus (SHFV), a DNA virus which is a known severe simian pathogen unrelated to the Filoviridae. This co- infection added enormously to the complexity of this episode (SHFV also produces haemorrhagic disease, although it does not apparently infect humans). In the first reported epizootic, 223/1050 exposed animals died, with increased handling a risk factor for disease and death. The natural host and geographical distribution are also unknown, although the infected monkeys (Macaca mulatta) apparently originated in India (Palmer et al., 1968).

In 1990, concern about the risk to humans led to a temporary ban on importation into the USA of cynomolgus, rhesus and African green monkeys. Evidence for ongoing epizootics and transmission was sought in the Philippine export facilities which had provided the monkeys (Hayes et al., 1992). Antigen detection enzyme-linked immunosorbent assays (ELISAs) on liver homogenates revealed that 85/161 (52.8%) of monkeys that died there over a period of less than 3 months were positive for ﬁlovirus antigen, as were 6.7% of monkeys tested in an initial serological survey. Incidence was calculated to be 24.4/ 100 animals, or 0.6/100 monkeys/day of follow-up. Documented case fatality at this institution was 82.4%, and survivors developed high-titre IFA antibody. Average duration of viraemia was 5.6–2.4 days. Diarrhoea and respiratory problems were the most frequently recorded manifestations. In the 73% of monkeys positive for ﬁlovirus by IFA at this facility the geometric mean antibody titre was 145. A protective factor was the presence of antibody to

filovirus at the time of entry to the facility (Hayes et al., 1992). As in the USA, many of the filovirus-infected monkeys were co-infected with SHFV, which means that all data from these epizootics are difficult to interpret and conclusions are open to question.

Epidemiology: Reston Virus

In the Philippines, 186 people were studied who lived in wildlife collection areas or worked in four primate export facilities in Manila (Miranda et al., 1991). Twelve (6%) were filovirus antibody-positive by IFA and in the facility experiencing the epizootic 22% were positive, significantly higher than the other export facilities [relative risk 5.6; 95% confidence interval (CI) 1.09–24.14] (Hayes et al., 1992). In that facility the workers in the animal hospital had the highest titres: 3/5 had titres of 4256. However, there was no illness in any of the positive individuals, and no association between seropositivity and other risk factors, such as bites, scratches or eating monkey meat. In the facility at Reston, Virginia, five animal handlers had a high level of daily exposure to infected and dying animals (Centers for Disease Control, 1990b, 1990c). Four of these had serological evidence of recent infection by IFA, and three were observed to seroconvert during the period of the epizootic. One cut his finger while performing a necropsy on an infected animal. Daily monitoring of this individual revealed transient viraemia and seroconversion, but neither he nor his colleagues had any illness attributable to filovirus infection.

Serological Surveys of Humans

Serosurveys of village populations in epidemic areas have shown the presence of low-titred Ebola and Marburg antibodies. If these reactions can be con- ﬁrmed by other methods and in prospective studies, then infection without a serious clinical disease is more common than realised at present. In serosurveys in Zaire, antibody prevalence to Ebola virus has been 37%. Serosurveys in individuals not in contact with patients in Sudan have had antibody prevalence of 1– 4% to an Ebola-related virus. Unfortunately, the IFA test used for these studies is unreliable at low titre or in the absence of a history of clinical disease, and serological reports need to be interpreted with considerable caution. Antibody, sometimes with high prevalence, has been reported in monkeys and humans from many geographical locations, including unlikely

618

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

populations such as Cona Indians from Central America (Heymann et al., 1980) and Alaskans. Specificity is not improved by the use of Western blot techniques, since non-specific antibody to some of the Ebola virus proteins can also be seen in this test. This problem appears to be unique to Ebola virus (Zaire, Sudan and Reston strains), and is not encountered with Marburg serology. Lack of correlation of serology with Ebola disease supports the conclusion that the titres are non-specific, although it remains possible that they may reflect past infection with unrecognised non-pathogenic filoviruses. ELISA and other techniques for antibody assays under development may allow some of these issues to be settled.

ECOLOGY Until the early 1990s, outbreaks of human disease may

have been unusual, because transmission from the natural reservoir to humans is rare. Searches for evidence of virus infection in many species of animals captured in central African countries have failed to provide any clues as to the possible reservoir. The original ecological setting of Marburg and Ebola appeared to centre around central and south central Africa (Figure 20.2), particularly the Mount Elgon area of western Kenya and Uganda. Surveys of wild monkeys from the Lake Kyoga area in northern Uganda in the 1970s, where the original Marburg- infected group originated, failed to yield virus but about 10% of sera reacted with crude Marburg antigen in a complement ﬁxation test, and in at least three animals neutralising antibody could be demonstrated. The 1987 case was a boy who had spent considerable time in Kitum Cave on Mount Elgon, collecting minerals and other items, near to the sugar cane factory where the 1980 case had worked. Extensive searches for the reservoir in the area around the sugar cane-processing factory and around Kitum Cave were unsuccessful. The cave contained large numbers of bats, and was visited by a wide range of mammals, birds, reptiles and insects. Sentinel animals and other means of searching for the virus failed to identify bats (or any of a number of wild mammals) as the ultimate source. Similarly, a thorough study of insects and mammals along the trail of the 1975 traveller in Zimbabwe did not produce evidence of the source of infection.

Bats remain highly suspect, since they were implicated directly in the Kitum Cave case, which contains enormous colonies of bats. In Sudan in 1976 and 1979 both index cases had been from a cotton factory, where

the roof was heavily infested with bats, concentrated over the store room where both index cases worked. Antibody could not be detected in bat sera, and no virus was isolated from bat tissues in either investigation, but limited sampling, particularly in the Sudan studies, could have missed infected animals (serological studies in bats and other exotic species present a serious challenge in terms of species-speciﬁc reagents). An animal study showed that Ebola virus can replicate in bats without causing disease (Swanepoel et al., 1996). This is strong evidence that bats can carry the silent infection, which is a prerequisite for maintaining the virus in a wild population over time. Other suggestions, such as rodents and even plants as primary hosts have never been substantiated. If this were a rodent virus, the ubiquity of most rodents would mean that primary infections in humans would

be much more frequent, as is the case with Lassa fever, so rodents are unlikely as hosts. There is no precedent for a plant virus infecting humans.

Given the insensitivity and lack of specificity of the original tests, early data on animal surveys, including bat studies, probably do not reflect the real situation. What we do know is that things changed in the 1990s, and that large apes, specifically chimpanzees and gorillas, have become increasingly implicated in transmission to humans. The first indication of the significance of chimpanzees in the chain of transmission came with the report of an outbreak in Gabon in 1996, resulting from the handling and butchering of a dead chimpanzee found in the forest by some villagers (Georges et al., 1999). Since 1994, four human epidemics have occurred in north-east Gabon, and ape carcasses have been found near the sites of three of these. That the great apes are not the natural host is made clear by the fact that mortality in both species is as high as in humans. From 2000 on, repeated epidemics in Gabon and in bordering DRC and the Republic of Congo have been linked with the epizootic in great apes. This epizootic has contributed in large part to a catastrophic decline in great ape populations, threatening them with extinction. The populations have been reduced by more than a half between 1983 and 2000. A great ape nest group survey in Gabon near Minke´be´, an area where there had been a human epidemic, recorded a 99% fall in nest groups over the study period (Walsh et al., 2003). In Mekambo in neighbouring Congo, many ape carcasses have been found, and in Lossi, a population of 143 individually identified gorillas has been reduced to seven. The epizootic is gradually making its way south and west. Logging and a booming commercial bush meat trade also contributes to the declines, but Ebola is thought to

FILOVIRUSES

619

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

have been responsible at least equally. Of further occupant of a gang cage (six-fold increase of risk; concern is that handling of dead carcasses, with the

p5 0.00l; OR 5.96; 95% CI 2.87–12.38) (Hayes et al., potential of commerce in infected bushmeat, increases

1992). Although one infected monkey was identiﬁed at the probability of human epidemics, which is exactly

a second exporting facility, no transmission could be what is being seen in the same areas. Extermination of

documented. Signiﬁcantly, this second facility routi- the great apes would certainly reduce the possibility of

nely housed their animals in single cages. Ebola human Ebola epidemics, but that seems too high a

(Reston) has been identiﬁed at high titre in respiratory price to pay. Furthermore, the natural host, be it bat or

secretions in monkeys, and respiratory transmission at other occult species, will remain as the primary source.

close quarters may be a factor in epizootics with this Eﬀorts to control the epizootic are called for,

virus. This last hypothesis is supported in part by the particularly as it threatens the great wildlife refuges,

genetic relationship between the ﬁloviruses and known but given the remoteness of the area and the lack of a

respiratory pathogens. However, there was strong vaccine and a means of delivery, it is diﬃcult to know

evidence for monkey-to-monkey transmission by re- what measures might be eﬀective.

use of needles for routine procedures, such as tuberculin testing or antibiotic administration.

TRANSMISSION AND RISK FACTORS Laboratory Infections

Person-to-person spread has been the major mode of transmission in epidemics. Contact with patients ill

The outbreak of Marburg virus in 1967 was caused by with Ebola is the most important factor in determining

infection of individuals handling fresh monkey tissues risk of illness. Other risk factors associated with

or contaminated equipment without gloves or other human-to-human transmission are infection from

protective clothing. Otherwise there has only been one contaminated materials such as needles, contact with

reported laboratory-acquired infection (needlestick) blood or secretions, preparation of a body for burial

with Ebola virus, in 1976 (Emond et al., 1977). or, occasionally, sexual contact. Close contact with

Because of its lethal potential, Ebola has been a blood or tissues of infected monkeys is also important.

candidate for biological warfare. Little information is The virus enters through mucous membranes or skin

available, but it has been handled extensively in lesions, and outbreaks have been abruptly terminated

biological research, and further accidental infections when blood and needle transmission were interrupted

may have occurred, speciﬁcally in the former Soviet (Fisher-Hoch, 1993). In later sporadic outbreaks, one

bloc. The key to safe laboratory handling of this virus secondary Marburg case nursed the index patient with

is extreme care in avoiding accidental inoculation. no protective clothing, and later assisted with resusci-

tation procedures. She also handled wet tissues from the companion of the index case, who was also infected

DISEASE (Gear et al., 1975). In 1980, a patient died 5 h after

being admitted to a Nairobi hospital and the attending doctor subsequently became ill with, but survived,

Clinical Spectrum Marburg infection. Furthermore, epidemiological stu-

The incubation period for Marburg virus disease is 3–9 dies in Zaire and Sudan do not suggest spread through

days (Martini, 1971) and for Ebola virus about 10 casual contact or by aerosol transmission. A formal

days; 5–7 days for needle transmission and 6–12 days study of risk factors for virus transmission in the

for person-to-person spread (World Health Organiza- Sudan epidemic in 1979 showed that caring for an ill

tion, 1978a, 1978b; Baron et al., 1983). The incubation patient carried a relative risk ﬁve times greater than

period for the Ebola-related virus from Sudan may be persons with a lesser degree of physical contact, and no

slightly longer than for the more lethal Ebola (Zaire) cases occurred in persons who entered the room of an

strain, and appears to be dose-dependent. The illness- ill patient but had no physical contact. These data

to-infection ratio for Marburg and Ebola viruses conﬁrm that Ebola is not an airborne disease and

approaches unity, since few if any asymptomatic depends on close contact, probably with infected blood

infections have ever been observed. In contrast, or secretions, for its propagation. The mode of

Ebola (Reston) virus, in all individuals documented acquisition of primary infection is totally unknown.

to have been infected, was uniformly asymptomatic The most signiﬁcant risk factor for the monkeys

(Table 20.2) (Centers for Disease Control, 1990b, infected in the epizootic in the Philippines was being an

1990c; Miranda et al., 1991).

621 The human disease caused by the African viruses is

FILOVIRUSES

Mortality dramatic (Figure 20.4). The onset is abrupt, with fever,

severe headache (usually periorbital and frontal), The death-to-infection ratio is related to virus strain myalgia, arthralgia, conjunctivitis and extreme

(Table 20.2). Of the 29 known primary Marburg malaise. Sore throat is a common symptom, often

infections, 10 died (35%). No fatalities occurred associated with severe swelling and dysphagia, but no

among the 10 secondary cases (overall mortality exudative pharyngitis. A papular, eventually desqua-

25.6%). The mortality ratios during the two epidemics mating rash may occur in some patients, especially on

of Ebola disease in Sudan were 55% and 65%, while the trunk and back, and morbilliform rash has been

that during the Zaire epidemic in 1976 was 88%. observed on white skins. In non-human primates

Patients in these epidemics received little or no medical petechiae are striking. Gastrointestinal symptoms

care, and mortality might be substantially lowered develop in most patients on the second or third

with modern intensive care. Reduction in case fatality day of illness, with abdominal pain, and cramping

with human transmission has also been observed in followed by diarrhoea and vomiting. Jaundice is not a

Ebola infections, where human-to-human transmission feature of Marburg or Ebola disease. The bleeding

was followed-up to six generations (Figure 20.5). This begins on about the ﬁfth day of illness and is most

may represent some degree of viral attenuation with commonly from the mucous membranes: gastrointest-

human passage.

inal tract, gingiva, nasopharynx and vagina. Death occurs in a large proportion of patients, and is associated with hypovolaemic shock and severe bleeding. Infection in pregnancy results in high maternal

ANIMAL MODELS fatality and virtually 100% fetal death. The persistence

of vomiting and the onset of any signs of mucosal African green monkeys that had been recently bleeding carry a high risk of fatal outcome. Central

imported from Uganda were the source of the nervous system involvement has led to hemiplegia and

Marburg outbreak (Henderson et al., 1971), and disorientation, and sometimes frank psychosis. Even in

cynomolgus monkeys were the source of the newly convalescence patients show prolonged weakness,

described Asian ﬁlovirus (Jahrling et al., 1990; Centers severe weight loss, and in a few survivors serious but

for Disease Control, 1989). Both are important species reversible personality changes are recorded, namely

for importation and medical research, and the monkey confusion, anxiety and aggressive behaviour.

has been the most successful animal used for the study of the pathogenesis of ﬁloviruses (Bowen et al., 1980; Fisher-Hoch et al., 1983; Baskerville et al., 1978). The ability of any of the viruses to kill guinea-pigs is variable. Ebola (Zaire) kills guinea-pigs consistently

Figure 20.5 Mortality of cases of Ebola haemorrhagic fever Figure 20.4 Frequency of symptoms and signs in Ebola

in Sudan in 1979 showing the eﬀect of successive human-to- haemorrhagic fever in Zaire in 1976

human transmission

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

tion, but progression to death was slowed if the inoculum titre was reduced.

Ebola (Reston) was first identified in an epizootic in an animal-handling facility, where transmission and a high death rate were observed in cynomolgus monkeys (Centers for Disease Control, 1989). However, the disease is characterised by slower development of viraemia, delayed onset of enzyme and immune disturbances, and lower mortality than in the African viruses (Figure 20.6). However, since these animals were co-infected with SHFV, the observations on filovirus pathogenesis are inconclusive (Jahrling et al.,

Figure 20.6 Comparison of the outcome and viraemia in 31 1990). Viraemia rises and peaks later than in Sudan monkeys challenged with ﬁloviruses. Dotted lines denote

infection. There was also histologically less evidence of mean viraemia in monkeys infected with African ﬁloviruses

and solid lines mean viraemia in monkeys infected with Asian extracellular distribution of ﬁlovirus antigen. When

ﬁloviruses. Numbers of deaths are shown by bars, dark bars disease is severe or fatal, peak liver enzyme levels may are from African virus infections, and light bars from Asia

be very high. It is clear that the host genetics also virus infections

contribute, in that African green monkeys are less susceptible to severe or fatal disease due to Ebola (Sudan) or Ebola (Reston) than cynomolgus monkeys. Ebola (Zaire) infection, however, seems uniformly

after several adaptive passages; the Sudan strain and fatal in all species so far challenged. Marburg virus do not. Only the Zaire virus was found to be lethal for suckling mice (McCormick et al., 1983).

Rhesus monkeys inoculated intraperitoneally with PATHOGENESIS AND IMMUNOLOGY 103–104 guinea-pig infectious units of Ebola (Zaire)

virus become febrile 3–5 days after inoculation, and Pathogenesis and immunology need to be discussed develop a petechial rash on the forehead, face, limbs

together, since they are inextricably linked, and some and chest on day 4–5. Severe prostration with

of the major manifestations of disease may be diarrhoea and bleeding leads to rapid death in almost

immunopathological in origin. The processes are all animals. Although similar in onset, the disease

clearly complex, reﬂecting a host–parasite relationship caused by the ﬁlovirus from Sudan is characterised by

which is in no way adapted, and which may result from lower incidence of viraemia, enzyme and immune

chance similarities in viral and host proteins favouring disturbances and some survivors, in keeping with the

a wide range of events, including viral entry, replica- 53% case fatality reported from the 1976 human

tion and induction of host proteins, which damage Sudan outbreak (Figure 20.6), and lack of pathogeni-

rather than protect. Other factors, such as infecting city in suckling mice (Baron et al., 1983; World Health

dose, route of infection and host genetics, also Organization, 1978b; McCormick et al., 1983). Pete-

undoubtedly play major roles. Indeed, the more we chiae are rarely seen, and then only in dying monkeys.

learn, the more we understand the complexity of the Viraemias and liver enzymes do not reach the levels

processes involved and how little we yet understand seen in Ebola (Zaire) ﬁlovirus infections, but as the

them.

illness progresses severe thrombocytopenia, neutrophi- Among the long-established facts are that high titres lia and lymphopenia developed, with very high

of virus are found in serum and tissues taken at aspartate transaminase (AST) and lactate dehydrogen-

autopsy, and particles may be seen in large numbers, ase (LDH) levels. The monkeys are obviously very

with some obvious tropism for reticuloendothelial cells sick, including the survivors, but haematological and

(Figure 20.1b) (Baskerville et al., 1985; Fisher-Hoch et biochemical parameters return essentially to normal by

al. , 1992a). The most profound physiological altera- day 20, and recovery is rapid and complete. Although

tion, and that which is invariably associated with systematic comparisons have not been made, Marburg

death, is shock, manifested by hypotension, eﬀusions virus infection in monkeys apparently resembles Ebola

and facial oedema. Severe, acute ﬂuid loss, often with (Sudan) infection. However, African green monkeys

frank bleeding into the tissue and into the gut, is inoculated experimentally with the virus soon after the

characteristic and results in dehydration and electro- 1967 outbreak all died, regardless of route of inocula-

lyte and acid–base imbalance. Several years before the

623 discovery of cytokines and other cellular messengers, it

FILOVIRUSES

liver, lymphatic organs, kidney, testes and ovaries, but was observed that aortic endothelial cells from

usually not suﬃcient to produce organ failure, and experimentally infected primates, although intact by

there is little inﬁltration of inﬂammatory cells in areas histology and electron microscopy, had markedly

of tissue damage.

reduced in vitro ability to produce prostacyclin That the Asian ﬁloviruses have a lesser pathogenic compared with endothelium from normal uninfected

potential in primates is consistent with observations in control animals (Fisher-Hoch et al., 1985). Thus,

accidental human infection. Other biological proper- profound disruption of biochemical integrity of the

ties, such as the speed of replication in tissue culture, endothelial cell was proposed, and subsequent data

support the contention that there are clear diﬀerences show that this is probably the case. Since there is no

in virulence between African and Asian ﬁlovirus. In a evidence for extensive destruction of endothelial cells

Reston-infected monkey, virus particles were observed by virus by histology or electron microscopy, it must

embedded in the basement membrane of lung alveoli,

be assumed that the ﬂuid losses are due to these and replication in the lung may occur with this functional changes rather than lytic destruction of the

ﬁlovirus, with which respiratory manifestations have endothelium by replicating virus. Nevertheless, profuse

been a feature in epizootics. The possibility of extracellular antigen suggests that overwhelming viral

pulmonary involvement needs further study. replication may be the underlying process causing the rapid multi-system collapse seen in Ebola (Zaire) infections (Baskerville et al., 1985).

Past Infection and Persistence Bleeding is prominent, manifested as petechiae, uncontrolled bleeding from venepuncture sites and

Humans and non-human primates with documented haemorrhagic eﬀusions. Thrombocytopenia is invari-

acute infection with Ebola (Zaire), Sudan or Reston able, but bleeding is not usually of suﬃcient volume to

ﬁloviruses seroconvert promptly to high-titre IFA to account for the shock, neither is it associated with solid

antigens prepared from all three viruses. Low-titre evidence of signiﬁcant disseminated intravascular

antibody may be associated with ﬁlovirus infection in coagulation (DIC) in the small number of animals or

the past, and also with immunity, but this remains to humans studied. As in Lassa fever, platelet dysfunction

be demonstrated. Although Marburg virus has been has been described in experimentally infected primates,

isolated from semen 7 months after acute infection in which there is a decline in in vitro platelet

(Martini, 1971) and from the anterior chamber of the aggregation, beginning 1–3 days prior to the onset of

eye 2 months after acute infection (Gear et al., 1975), bleeding and shock and progressing to virtually no

human infections have been few, survivors scarce, and aggregation at death. This leads to an increase in

most experimentally infected non-human primates platelet factor 4 (PF4) levels in plasma, and concomi-

have not survived (Heymann et al., 1980; World tant decrease in platelet-derived PF4, suggesting that

Health Organization, 1978a, 1978b; Fisher-Hoch et the platelets are being stimulated but are unable to

al. , 1985). A systematic study of the potential of the aggregate (Fisher-Hoch et al., 1985). In primates,

Ebola ﬁlovirus strains to persist in non-human profound lymphopenia early in disease is followed by

primates failed to detect persistent ﬁlovirus. This was marked neutrophilia. Laboratory evidence of only

despite intensive eﬀorts, including laparotomy to moderate DIC appears late in disease and is probably a

obtain serial specimens, co-cultivation and use of consequence of the process, rather than the underlying

polymerase chain reaction (PCR). It cannot at present primary pathology. Liver enzymes, aspartate (AST)

be excluded that passage in animals, speciﬁcally into and alanine transaminases (ALT) are raised, but the

additional non-human primates, or use of a more rise in AST is disproportionately higher than ALT, as

sensitive, or more broadly reactive PCR primer system, was described in the early Marburg cases (Martini,

might not identify a few virions in tissues of recovered 1971; Fisher-Hoch et al., 1985).

animals, perhaps in immunologically privileged sites At autopsy both Marburg- and Ebola-infected

such as the eye. However, these are likely to be at such humans and primates show widespread haemorrhagic

low titre as to present an insigniﬁcant risk to people. diathesis into skin, membranes and soft tissue. There is

Immunosuppression of such animals might also lead to focal necrosis in liver, lymph nodes, ovaries and testis.

reactivation, but ﬁlovirus disease in monkeys after Most prominent are eosinophilic inclusion bodies in

release from quarantine has never been reported, and hepatocytes (Councilman-like), without signiﬁcant

since many have been experimentally immunosup- inﬂammatory response (Baskerville et al., 1985).

pressed for many years, reactivation of disease should Focal necrosis is observed in many organs, including

have been observed by now, were it occurring.

Very little is understood about the immunology of Ebola virus infections except for the observation, made many years ago, that neutralising antibodies are difficult to demonstrate in both humans and primates, and that like many zoonotic viruses, notably Lassa virus and now the SARS HCoV, appear to be able to circulate in humans in the presence of detectable antibody and to show varying ability to persist at least for short periods following acute infection. Recent studies using specimens from patients in Gabon have shown that the innate immune system plays a very important role in the disease (Baize et al., 1999). Evidence from these studies and from animal studies suggest that the proinflammatory response is a central figure in both pathogenesis of severe disease and protection from disease, in that an early, orderly innate immune response was observed in infected individuals who never developed disease (Leroy et al., 2000). Conversely, primate studies have shown that infection of monoculear phagocytes is critical, that this triggers

a cascade of cytokines/chemokines and oxygen free radicals, and that it is this process, not direct viral replication destroying critical cells, which leads to the manifestations of disease. These manifestations are associated with massive intravascular apoptosis (Baize et al. , 1999; Hensley et al., 2002).

LABORATORY OBSERVATIONS General Laboratory Findings

Clinical laboratory observations are limited to the Marburg outbreak. No acute-phase investigations were undertaken during the care of the one laboratory infection. There is invariably biochemical evidence of hepatic disease, with elevated AST levels maximal by day 7 of illness. Bilirubin is not elevated, and ALT is disproportionately low. These findings are reproducible in non-human primates experimentally infected with Ebola virus (AST:ALT ratio, 7:1) and the prothrombin time is relatively normal until the final stages of disease. Marked wasting in these diseases is dramatic and due to actual loss of muscle mass. The pathophysiology of this is not understood, although the raised AST may reflect some associated extrahepatic process. Lymphopenia and thrombocytopenia are marked. As soon as 24 h after infection a marked lymphopenia is observed in experimentally infected animals. The lymphocytes disappear rapidly at the same time as a significant neutrophilia develops, with

absolute counts as high as 20610 9 /1, in the absence of

concurrent bacterial infection.

Laboratory Diagnosis

Care should be taken in both drawing and handling blood specimens, since virus titre may be extremely high and the virus is stable for long periods even at room temperature (Elliott et al., 1982). Gloves should

be worn at all times, and discarded into disinfectant. All sharp instruments, needles, syringes, etc. should be discarded to a puncture-resistant container with a lid, as is recommended for HIV-infected specimens (Cen- ters for Disease Control and National Institutes of Health, 1988; Centers for Disease Control, 1988a). A blood specimen should be taken without anticoagulant and serum separated. All procedures should be carried out by the most experienced staff under the most stringent safety facilities available at the admitting hospital or clinic (Biological Safety Level 3 if possible) (Centers for Disease Control, 1988b). Serum should be transferred to a leak-proof plastic container and double wrapped in further leak-proof containers in which it may be transported to a suitable reference laboratory. Sera may be safely handled for immunological tests by inactivating with g-irradiation, or, if this is unavailable, heating to 608C for 30 min. High or rising titre filovirus- specific IgG is diagnostic, as is the presence of IgM by IFA. Virus may be isolated and identified within 2–3 days if suitable facilities are available.

Although morphologically similar, Marburg and Ebola viruses are immunologically distinct. The indirect immunofluorescent assay for Marburg and Ebola viruses was the original basic diagnostic serological test for these viruses evaluated for the diagnosis of human Ebola virus disease (Wulff and Johnson, 1979). In acute infections, a rising filovirus-specific IFA titre (four-fold) in paired serum, or a high IgG titre (464) and presence of IgM antibody with a clinical illness compatible with haemorrhagic fever are consistent with the diagnosis (Ksiazek et al., 1992). Marburg virus antibody is usually specific, but Ebola virus serology in the absence of a history of recent disease was consistently plagued by low-titre, non-specific reactions using the IFA. This problem appears to be related to the use of native antigens. Newer systems using ELISA technology based on recombinant antigens have superior specificity and have replaced the older assays. ELISAs using recombinant nucleoprotein (NP) are the most broadly reactive and the most sensitive (Groen et al., 2003). Use of other proteins as antigens, e.g. recombinant VP35, results in a strain-specific assay but is less sensitive (Ksiazek et al., 1992). False positives are also seen in Western blot and radioimmunoprecipitation assays using native proteins.

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PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

Most filoviruses may easily be isolated from serum, blood or tissue specimens stored at minus 708C in Vero E-6 cells, but biological safety level 4 containment facilities are recommended for isolation. Blood specimens unrefrigerated for up to 10 days have yielded virus strains. Marburg and the Zaire variant of Ebola virus are readily isolated from blood specimens taken in the first week of illness, by inoculation of Vero or other mammalian cell cultures and identified by IFA within 2–5 days after inoculation of cells. The Sudan variant of Ebola virus, however, may be more difficult to recover from blood or tissues of patients and success may depend on blind passage of cultured cells in guinea-pigs monitored for febrile response. Specimens, including throat washings, urine, various soft tissue exudates, semen and anterior eye fluid, may also contain virus. Virus identification is made generally by direct immunofluorescence of the tissue culture using monoclonal antibodies. Impression preparations made from a postmortem liver biopsy may be probed with monoclonal antibodies by IFA for presence of virus antigen which is abundant in liver and spleen. Using this system, sensitivity for the Reston virus in liver is

0.96 and speciﬁcity 0.98 compared with viral culture. An antigen-detection ELISA system in serum has been used extensively, employing monoclonal antibodies (Ksiazek et al., 1992; Hayes et al., 1992). This assay has been used extensively in human and epizootic outbreaks. One study showed that a positive antigen test was obtained in symptomatic patients, but once symptoms had resolved in survivors, the test became negative (Baize et al., 1999). More sensitive is PCR, which may still be positive after symptoms have resolved.

PATIENT MANAGEMENT General Patient Management

Fluid, electrolyte, respiratory and osmotic imbalances should be managed carefully. Patients may require full intensive care support, including mechanical ventilation, along with blood, plasma or platelet replacement. The maintenance of intravascular volume is a particular challenge but every eﬀort is justiﬁed, since the crisis is short-lived and complete recovery can be expected in survivors. Pregnant patients may present with absent fetal movements, and maternal survival may depend on aggressive obstetric intervention.

Although much has been written about disseminated intravascular coagulation (DIC), particularly the use of heparin, there is little evidence for the role of DIC in

pathogenesis, and this drug remains controversial and potentially dangerous. The only experience has been the therapy of two individuals with Marburg disease in South Africa. However, both of these patients were secondary Marburg infections, and none of 10 known secondary patients with Marburg infection have died.

Specific Therapy

No antiviral therapy, including convalescent plasma, ribavirin or related compounds have been shown to be effective against either Marburg or Ebola virus infection in patients or in experimentally infected non-human primates, although some compounds are currently under investigation. An undefined ‘rapid response’, recorded after administration of passive antibodies and interferon to the laboratory worker who suffered accidental inoculation of Ebola virus, led to early optimism that convalescent plasma might be effective, and this approach was used in the Kikwit outbreak, with uncertain outcome since all those treated were infected later in the outbreak, and it may be that subsequent generations of infections show some attenuation of the virus. Certainly blood or plasma transfusion might be effective in symptomatic support of a patient with Ebola infection. However, its value as specific therapy is doubtful, since it does not protect animals from simultaneous challenge with homologous virus. Suitably screened and stored human plasma is, in any event, unavailable. Human interferon is ineffective in vitro.

Efforts continue to seek antiviral agents effective against filoviruses. A product isolated from a cyano- bacterium, Nostoc elliposporum, named cyanovirin-N (CV-N), has shown some potential to prevent transmission of human immunodeficiency virus (HIV) by binding to surface glycoproteins specifically through oligosaccharides. Since Ebola glycoproteins are heavily glycosylated, CV-N was studied for its ability to do the same with Ebola virus and it was found to bind with considerably affinity to carbohydrates on GP1 and GP2 (Barrientos et al., 2003). Studies of this kind give hope that eventually we will be able to develop effective antiviral therapy.

CONTROL Containment

Since the reservoir(s) of the viruses are not known, no speciﬁc precautions can be identiﬁed which would

FILOVIRUSES

625

avoid infections from the natural source of the viruses. The obvious exception to this generality is containment of monkeys which might be infected with one of these viruses. Prompt identiﬁcation of active cases is critical, and is in great part dependent on an accurate and detailed history (Centers for Disease Control, 1988b). Interruption of person-to-person spread of the virus is essential to control. The most important issue is that of awareness in the medical community that these diseases exist, and they may result in extensive nosocomial spread if not recognised early and if appropriate isolation of the patient is not achieved. Thus, early institution of safe and orderly care of the ill can be set up, with eﬀective surveillance of high-risk contacts and prompt isolation of further cases. This has been shown to ensure rapid control of an epidemic.

The experience in Uganda provided the only unique data showing the value of a national approach to control combined with strong community participation (Okware et al., 2002). This extensive outbreak presented major logistic problems, threatening to spread throughout the country. The Ugandans set up

a series of Task Forces at different administrative levels, including a District Talk Force in each district of the country. Programmes included public education, mobilisation of communities, training for health care workers and village volunteers, and timely dissemination of information using radio, mobile phones and foot and motor patrols. Scouts were recruited for active case finding. Isolation wards and a temporary field laboratory were set up. One of the major obstacles was lack of laboratory facilities. Burial teams were established for safe burial, since it soon became clear that the preponderance of female cases was associated with ritual cleansing of the dead.

Contacts High-risk contact is associated with direct contact with

blood or body ﬂuids from acutely infected humans or animals, or sexual contact with a convalescent case. Laboratory accidents must be treated seriously, with careful review of level of risk. If this is thought to be high, isolation for the incubation period (17 days is adequate) and tracing and surveillance of any further potential contacts, such as family members, are necessary. If the risk is low, simple surveillance of the individual by daily telephone contact for fever during the incubation period suﬃces. It must be remembered that biocontainment facilities do not protect against injury with needles or other sharps. There is no evidence for or against the use of passive

antibody in prophylaxis. Suitably screened and stored material is in any event unavailable.

Hospital Containment

The key to prevention of transmission in both endemic and non-endemic areas has consistently been good hospital and laboratory practice, with strict isolation of febrile patients and rigorous use of gloves and disinfection. Intensive care, surgery and air transport should not be denied. Patient isolators are not recommended, since the hazard to contacts is not via aerosols, but by direct inoculation of virus in blood or other material. Isolators induce loss of manual dexterity and fatigue, inhibit intensive care procedures and communication, do not protect against sharp instrument injury and have no provision for resuscita- tion. The 1988 CDC Guidelines for the Management of Patients with VHFs recommends routine patient isolation in a single room, preferably but not necessarily with negative air pressure gradient from the hallway, through an anteroom to the patient room (Centers for Disease Control, 1988b). Staﬀ education and strict supervision, use of gloves, gowns and masks, and rigorous disinfection with fresh liquids are mandatory. The recommendations issued for management of AIDS patients are also adequate for containment of ﬁloviruses (Centers for Disease Con- trol, 1987).

Vaccine

There is no candidate vaccine for filoviruses. Fears that this agent might be used in biological warfare have stimulated studies to develop suitable candidates, primarily by genetic engineering of constructs in vaccinia virus or other vectors. The potential target population for a vaccine, however, military interests apart, is vanishingly small, and it is therefore unlikely that resources for vaccine development will be available in the near future. Simple challenge studies with Ebola (Zaire) in a very small number of animals surviving less pathogenic filovirus infections—Ebola (Sudan or Reston strains)—showed variable protection (Figure 20.7) (Fisher-Hoch et al., 1992a). Filoviruses do not induce classical neutralising antibodies, and reliable protection may be difficult to achieve, particularly with a killed vaccine. The accept- ability of an attenuated live virus is difficult to conceive, but the apparently benign course of Ebola (Reston) infection in humans is an interesting possibility. Presumably, safety testing (phases I and II) would

626

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

FILOVIRUSES

Publisher's Note: Permission to reproduce this image online was not granted by the copyright holder. Readers are kindly requested to refer to the printed v ersion of this chapter.

Figure 20.7 Outcome, antibody proﬁle and viraemia in two of four cynomolgus monkeys that survived Ebola (Reston) challenge and that were subsequently challenged with Ebola (Zaire). In this experiment, one of the monkeys died on rechallenge. A further monkey that survived Ebola (Sudan) survived rechallenge with Ebola (Zaire). Reproduced from Fisher-Hoch et al. (1992a) by permission of The University of Chicago Press

be difficult if not impossible. Nevertheless, a live for efficacy in rodents (guinea-pigs mostly) consistently homologous vaccine may be effective, since an associa-

failed to protect primates (Geisbert et al., 2002). tion was observed between protection and possession

Development of an eﬀective vaccine requires a better of pre-existing antibody to ﬁlovirus at the facility in

understanding of the pathophysiological and immu- Manila during the epizootic of Ebola (Reston), and

nological diﬀerences between rodents and primates protection correlated with antibody titre. Even then, a

infected with ﬁloviruses, and this presents a signiﬁcant small number of animals (six) with pre-existing anti-

scientiﬁc challenge.

body were infected (Hayes et al., 1992). Eﬀorts to produce newer generation vaccines such as Venezuelan equine encephalitis virus (VEE) replicons, liposomes containing inactivated vaccine and DNA vaccines have had mixed results. A highly acclaimed

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21 Rabies and Other Lyssavirus Infections

Mary J. Warrell

John Radcliﬀe Hospital, Oxford, UK

INTRODUCTION disease was spread by saliva. In the sixteenth century, Fracastoro strengthened the concept of rabies as a

Rabies is a zoonosis infecting a variety of mammal contagious disease. A scientiﬁc or experimental species in diﬀerent areas of the world. The domestic

approach to rabies was delayed until 1793, when dog is the most important vector, mainly in Asia and

John Hunter suggested that the transmission of rabies Africa, and is responsible for >90% of human

should be studied by inoculating saliva from rabid infections. Sylvatic or wildlife rabies is endemic in

animals and humans into dogs and that attempts Asia, Africa, the Americas, Australia and most of

should be made to inactivate the ‘poison’ in the saliva. Europe. Almost all infections are due to classical

These ideas may have inspired the animal experiments rabies, genotype I, viruses. The remainder are due to

on transmission by Zinke and Magendie and Breschet the six genotypes of rabies-related viruses. In humans

(The´odoride`s, 1986).

the disease is invariably fatal in unvaccinated people; Galtier found that rabbits could be infected with during the last 25 years, the very few previously

rabies and were more convenient experimental animals vaccinated survivors have had profound neurological

than dogs. In 1881, he first demonstrated specific deficits. Prevention with pre- or post-exposure rabies

immunisation against the disease (The´odoride`s, 1986). immunisation is therefore crucial to prevent fatal

Pasteur adopted the use of rabbits in his studies of infection.

rabies beginning in 1880. He was the ﬁrst to recognise In English, rabies is also known as ‘hydrophobia’,

that the major site of infection was the central nervous ‘lyssa’ and ‘mad dog bite’: in French, la rage or

system. Pasteur was able to protect dogs from l’hydrophobie ; in Italian, la rabbia; in Spanish la rabia;

challenge by immunising them with a virus attenuated in Portuguese, a raiva or hidrofobia, and in German,

in desiccated rabbit spinal cord and in 1885 he used die Tollwut .

this as a vaccine successfully for the ﬁrst time in Joseph Meister and Jean-Baptiste Jupille, boys who had been severely bitten by rabid dogs. The reputation of

HISTORY modified forms of Pasteur’s vaccine increased during the first half of the twentieth century, but its efficacy

Rabies in dogs and the importance of saliva in its remained uncertain. The bite of a mad dog then caused transmission may have been recognised in pharaonic

rabies in only about 30% of untreated patients, times and in China in the seventh century BC although with wolf bites the risk was higher. In Iran,

treatment of bites by rabid wolves with classical rabies in animals but seemed to deny that humans

(The´odoride`s, 1986). Aristotle (322 BC ) described

Semple brain tissue vaccine only reduced the case could be infected or could die from the disease. Celsus,

fatality about 10% compared with unvaccinated

patients. A dramatic natural experiment in 1954, phobia in aﬄicted humans and recognised that the

in De medicina (ﬁrst century AD ), described hydro-

when a rabid wolf invaded a village and bit 29 people,

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

demonstrated the protective value of passive immuni- inﬂuenza-like symptoms occur occasionally, and two sation in patients with severe bites (Baltazard and

cases of encephalitis have been reported (Quiroz et al., Bahmanyar, 1955). This led to a general recommenda-

1988). The genus Lyssavirus (lyssa=rage, frenzy; tion that, in addition to the vaccine, this should be

Greek) comprises seven genotypes: genotype 1, classi- included in rabies post-exposure prophylaxis.

cal rabies; genotype 2, Lagos bat virus; genotype 3, Negri described his diagnostic inclusion body which

Mokola virus in shrews and cats; genotype 4, Duvenh- allowed the laboratory diagnosis of rabies in 1903.

age virus in bats; genotypes 5 and 6, European bat The introduction of the more speciﬁc and sensitive

lyssavirus , found in insectivorous bats; and genotype 7, immunoﬂuorescence method in 1958 has now replaced

Australian bat lyssavirus in ﬂying foxes (fruit bats). All the Seller’s stain for Negri bodies. Remlinger, in 1903,

these rabies-related viruses have been associated with showed that rabies was caused by a ﬁlterable agent. It

human disease except for Lagos Bat virus (see Table was not until 1936 that the size of the virus was

21.1, and section on Human Infections with Rabies- established and it was ﬁrst seen by electron microscopy

related Viruses, below). The recently identiﬁed bat in 1962 (The´odoride`s, 1986).

lyssaviruses from Eastern Europe: Aravan, Khujand, Improvements in Pasteur’s vaccine were achieved by

Irkut and West Caucasian bat virus have yet to be Semple and Fermi, who killed the virus rather than

classiﬁed.

attenuated it, and by Fuenzalida and Palacios, who The genus Lyssavirus has been divided into two developed a suckling mouse brain vaccine which

phylogroups as a result of serological and genetic carried a lower risk of neuroparalytic complications.

analyses (Badrane et al., 2001). Phylogroup I com- Growth of rabies virus in tissue culture was achieved

prises all genotypes except Mokola virus and Lagos bat by the 1930s, leading to the development, by Wiktor

virus , which form phylogroup II. All phylogroup I and his colleagues, of the ﬁrst tissue culture vaccine for

genotypes have caused fatal rabies-like encephalitis in human use.

man, whereas Mokola virus probably caused three known human infections, one of which was a fatal encephalitis without typical features of rabies. Experi-

CLASSIFICATION mentally, phylogroup II viruses are less pathogenic and there is little if any cross-neutralisation with the

The family Rhabdoviridae (rhabdos=rod; Greek)

phylogroup I lyssaviruses.

includes several genera of viruses found in plants, arthropods, ﬁsh, birds, reptiles and mammals. Almost all of those known to infect man belong to two

VIRUS STRUCTURE (Wunner, 2002) morphologically similar genera, Lyssavirus and Vesi- culovirus . Members of the genus Vesiculovirus cause

The bullet-shaped rabies virions measure 180675 nm vesiculostomatitis of cattle and horses. In man,

(Figure 21.1). The genome is a single non-segmented

Table 21.1 The genus Lyssavirus Genotype

Source

Known distribution

Phylogroup I 1 Rabies virus

Dog, fox, raccoon, skunk, bat, etc.

Widespread

4 Duvenhage

South Africa, Zimbabwe (very rarely identiﬁed) 5 European bat lyssavirus

Insectivorous bat (e.g. Nycteris thebaica)

1a. Bats, e.g. Eptesicus serotinus

Northern and Eastern Europe

1b. Bats, e.g. Eptesicus serotinus

Western Europe

6 European bat lyssavirus

2a. Myotis dasycneme bat

The Netherlands (rare)

Myotis daubentonii bat

UK (and Ukraine in other bat spp.)

Switzerland (very rare) 7 Australian bat lyssavirus

2b. Myotis daubentonii bat

Flying foxes (Pteropus spp.)

Australia

Insectivorous bats

Phylogroup II 3 Mokola

South Africa, Nigeria, Cameroon, Ethiopia (rare) 2 Lagos bat virus

Shrews (Crocidura spp.), cats

Bats, cats

Africa (rare)

Has not been detected in man

RABIES AND OTHER LYSSAVIRUS INFECTIONS

Figure 21.1 Negatively stained electron micrograph of Ho¨gyes strain of ﬁxed rabies virus in mouse cerebral cortex. Courtesy of G. Gosztonyi

strand of negative-sense non-infectious RNA of of viral P protein, often in large accumulations of

11.9 kb which bears coding sequences for ﬁve poly- ﬁlamentous matrix, forming the diagnostic Negri bodies peptides. This RNA is associated with a nucleoprotein

(Figure 21.2), which may also include other proteins. (N) a phosphoprotein (P) and an RNA-dependent

The phosphoprotein has 297 amino acids (*40 kDa), RNA polymerase (L) to form a tight helical coil, the

and exists in at least two forms. It has previously been ribonucleoprotein (RNP) complex core of the virion. A

known as membrane-associated 1 (M1) protein and layer of matrix (M) protein covers this cylindrical

non-structural (NS) protein. Its functions include structure. The outermost lipoprotein envelope is

binding to nascent N, so preventing its polymerisation, composed of a host-derived lipid bilayer and the

and non-speciﬁc binding to cellular RNA. P also forms virion-encoded glycoprotein (G) bearing spikes pro-

a complex with L protein and so has roles in genome jecting 8–10 nm above the virion surface. Each

transcription and replication. P has been shown to projection is a trimer of G molecules with a distal

attach to dyenin light chain (LC8) molecules, which knob. The envelope covers all except the ﬂat end of the

suggests an involvement in the transport of rabies virus virion, resulting in the characteristic bullet shape.

components intracellularly, including by the dyenin The viral nucleoprotein has 450 amino acids,

microtubular system which eﬀects retrograde axonal (*57 kDa) and is an integral component of the RNP

transport (see section on Pathogenesis, page 638). complex. It is the most conserved protein throughout

The viral RNA polymerase is the largest protein the genus Lyssavirus, and so its antigens are employed

(hence the designation L), containing approx 2142 both for genus-speciﬁc simple immunoﬂuorescent

amino acids (*190 kDa).The action of this RNA- antibody techniques and genotype-speciﬁc monoclonal

dependent RNA polymerase is essential early in antibody and PCR assays for diagnosis. N protein

infection of a cell, to transcribe the primary genomic encapsidates viral RNA, protecting it from ribonu-

RNA. The many functions of L include transcription, cleases, and has a role in regulating RNA transcription.

replication, polyadenylation and protein kinase activities. The lyssavirus N is phosphorylated, unlike all other

The P protein is an essential enzymic co-factor. rhabdovirus nucleoproteins. Nascent N molecules

The matrix protein has 202 amino acids (*25 kDa) rapidly aggregate together or combine with molecules

and covers the RNP complex. It has several functions,

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

Figure 21.2 A Negri body (arrow) in a cerebellar Purkinje cell. Courtesy of F. A. Murphy including the compacting of the loose helical core

as they are the only inducers of rabies neutralising structure, regulation of viral RNA transcription and

antibodies, which are protective. They can also induce interaction with the lipid bilayer and the G protein to

helper and minimal cytotoxic T cell responses. enable budding of virus from the cell membrane. The surface glycoprotein is N-glycosylated with branched chain oligosaccharides, which show micro-

REPLICATION (Wunner, 2002) heterogeneity within the virion. It has 505 amino acids

(*65 kDa). The external domain of the G molecule is Entry of virus into a cell occurs by cell receptor-

a trimeric spike connecting via a transmembrane mediated adsorptive endocytosis into an endosome anchor to a cytoplasmic domain, which is closely

(see section on Pathogenesis, below). If this is acid associated with the M protein. The distal knob on the

containing, the surface viral glycoprotein enables low surface projection is important in pathogenesis, as it

pH-dependent fusion with the endosomal membrane, bears binding sites for cellular receptors and eﬀects

uncoating and liberating the nucleocapsid (or RNP pH-dependent fusion with endosomal membranes. A

complex) into the cytoplasm. Catalysed by the viral L reversible change in conformation of the protein into

protein, the primary genomic negative-strand RNA is an activated hydrophobic state initiates membrane

transcribed, starting with a short leader RNA and fusion. A single amino acid replacement at arginine

followed by 5’ monocistronic positive-strand mRNAs, 333, at antigenic site III on the ectodomain, can

and later the full-length antigenome replicative RNA. radically reduce the virulence of the virus (Dietzschold

The amount of mRNA produced is greatest from et al. , 1983). The G also promotes the passage of virus

the 5’ end, in the order of N, P, M, G and L. from cell to cell, is involved in axonal transport and it

Protein synthesis proceeds in the cytoplasm on host is essential for trans-synaptic spread (see section on

cell ribosomes. The unstable nascent nucleoprotein Pathogenesis, below). G epitopes are most important

and phosphoprotein encapsidate and stabilise the and phosphoprotein encapsidate and stabilise the

INACTIVATION OF VIRUS AND STABILITY OF VACCINE ANTIGEN

Rabies virus in a PBS buffer solution is rapidly inactivated by heat: at 568C the half-life is 51 min; at 378C the half-life is prolonged to about 3 h in moist conditions and at 48C to 3–4 days experimentally. The stabilising presence of serum or other buffer solutions enhances survival. The lipid coat of the virion renders it vulnerable to disruption by detergents and simple 1% soap solution. Ethanol (45%), iodine solutions (with 1/10 000 available iodine), 3% sodium hydrox- ide, 1% benzalkonium chloride, 3% formalin, neat chloroform and acetone all inactivate the virus (Kaplan et al., 1966). Hypochlorite and glutaraldehyde solutions are suitable for laboratory use with the normal precautions, but phenol is not an effective virucidal compound.

Although benzylkonium chloride and other qua- ternary ammonium

compounds

are virucidal

experimentally, they are not recommended for cleaning rabies-infected wounds at the concentrations in normal clinical use because they are inactivated by the presence of soap (Kaplan et al., 1966), which is recommended as ﬁrst aid treatment of animal bites.

The potency and immunogenicity of lyophilised cell culture vaccine is retained after tropical ambient temperatures for 11 weeks (Nicholson et al., 1983). In reconstituted liquid form, the potency may fall sig- niﬁcantly after a week or two, and it is unwise to keep an open vial for several days because of the risk of microbial contamination causing denaturing of antigen, loss of potency and possible infection of the recipient.

EPIZOOTIOLOGY AND EPIDEMIOLOGY

Rabies is transmitted within populations of relatively few species of mammals, principally within the domestic dog population, causing urban rabies. The vectors of sylvatic, or wildlife, rabies vary in diﬀerent parts of the world and the virus circulates within the species group, maintaining infection with a constant virus type. As a result of the geographical compart- mentalisation of rabies within a mammalian species, there has been a tendency for adaptation and divergence of virus strains, which can be identiﬁed by monoclonal antibody tests and genetic analysis (Smith, 2002). There is occasional overspill between these intraspecies cycles, e.g. when a rabid raccoon bites a fox in North America. Transmission occurs more commonly to non-vector mammalian species, e.g. when a cow or sheep is bitten by a rabid red fox in Europe.

Rabies is enzootic in terrestrial mammal species in most of the world, but rabies-related lyssaviruses alone are found in bats in a few countries (Figure 21.3). Areas of the world which have been reported to be free of rabies include: Ireland, Iceland, Finland, Sweden, mainland Norway, Portugal, Italy, Greece, the Mediterranean islands, New Zealand, Papua New Guinea, Japan, Taiwan, Hong Kong Islands (but not the New Territories), Singapore, Sabah, Sarawak, some islands of Indonesia (e.g. Bali), many islands of the Indian Ocean and Oceania (e.g. Solomon Islands, Fiji, Samoa and Cook Islands), Uruguay, some Caribbean islands (e.g. Barbados, Bahamas, Jamaica, St Lucia, Antigua and others), Antarctica and, until recently, the UK. Some countries are generally free but infected animals occasionally cross land borders. The epizootiology is constantly changing, so local advice should be sought for detailed information.

Rabies is spread between mammals by bites; by contamination of intact and abraded mucosal membranes by virus-laden saliva; by ingestion of infected prey; transplacentally and possibly by inhalation of aerosols (in heavily populated bat caves). The principal reservoir or vector species in diﬀerent geographical areas are as follows:

Europe (World Health Organization; www.who-rabies-bulletin.org)

. Red fox (Vulpes vulpes)—from Poland to Slovenia and then eastwards to include the Russian Federation.

. Arctic fox (Alopex lagopus)—northern USSR.

RABIES AND OTHER LYSSAVIRUS INFECTIONS

635

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

. Raccoon dog (Nyctereutes procyonoides)—The Africa (King et al., 1994) Baltic States, Poland, Belarus, Ukraine and the

Russian Federation. . Domestic dog (Canis familiaris)—rabies is pre- . Wolf (Lupus lupus)—Lithuania, Belarus, Ukraine,

dominant throughout the continent and in Turkey and the Russian Federation.

Madagascar.

. Domestic dog (Canis familiaris)—Dominant vector . Black-backed jackals (Canis mesomelas)—South- in Turkey. Cases also found from the Baltic States

ern Africa.

south to Croatia and eastwards to the Russian . Yellow mongoose (Cynictis penicillata)—Southern Federation, which are likely to be due to infection

Africa.

from foxes or other wildlife. . Bat-eared fox (Otocyon megalotis)—Southern and . Insectivorous bats harbour the rabies-related

East Africa.

European bat lyssaviruses (Amengual et al., . Frugivorous and insectivorous bats—Duvenhage 1997). Transmission to terrestrial mammals has

virus very rarely found (see section on Human very rarely been found, to date only in four sheep

Infections with Rabies-related Viruses, p 647). in Denmark and a stone marten in Germany.

. Genotype 5, EBL 1a is found mainly in Eptesicus serotinus (serotine bats), in The Netherlands, Denmark, Germany, Poland, Hungary and the

North America (Centers for Disease Control: Russian Federation (isolates also from the

http://www.cdc.gov/ncidod/dvrd/rabies/ Slovak Republic and Czech Republic, but

Epidemiology/Epidemiology.htm) typing not available).

. EBL 1b is also found in Eptesicus serotinus in . Arctic fox (Alopex lagopus)—Alaska, north-west The Netherlands and in France and in several

Canada.

species in Spain. . Red fox (Vulpes fulva)—Ontario, Quebec and . Genotype 6, EBL 2a is rarely identiﬁed in M.

north-east USA, but many are infected here by dasycneme (pond bat), in The Netherlands but

raccoons.

three isolations were made in the UK: two from . Grey fox (Urocyon cinereoargenteus)—Arizona, Myotis daubentonii (Daubenton’s bat) in New-

Texas.

. haven in 1996, in Lancashire in 2002 and from Striped skunk (Mephitis mephitis) and other species—Saskatchewan, Manitoba, (central Canada)

the Scottish patient in 2002. Single infected bats California, Central USA and probably infected by were found in the Ukraine and Germany raccoons in north-eastern coastal states. (Johnson et al., 2003). . Raccoon (Procyon lotor)—Eastern coastal states of . EBL 2b was isolated from a M. daubentonii in USA from Florida to the Canadian border, and Switzerland and from the Swiss or Finnish adjacent regions of Canada. human case (see section on Human Infections . Insectivorous bats—widespread throughout conti- with Rabies-related Viruses, page 647). nental USA, e.g. Mexican free-tailed bat (Tadarida

braziliensis mexicana ), red bat (Lasiurus borealis), Middle East

big brown bat (Eptesicus fuscus), hoary bat (Lasirus cinereus), silver-haired bat (Lasionycteris

. Dominant vectors are the fox and the wolf. Dogs noctivagans ) and eastern pipistrelle (Pipistrellus may be infected from wildlife.

subﬂavus ).

. Other vectors include jackals, e.g. in Israel, and . Coyote (Canis latrans)—a few cases persist in hyenas, e.g. in Jordan.

Southern USA.

Asia Caribbean . Domestic dog (Canis familiaris) is the dominant vector.

. Vampire bat (Desmodus rotundus )—Trinidad, . Frugivorous and insectivorous bats in the Philip-

Tobago.

pines are seropositive for lyssavirus (see section on . Mongoose (Herpestes spp.)—Granada, Puerto Recovery from Infection in Animals, p 641).

Rico, Cuba, Dominican Republic.

637 Central and South America

RABIES AND OTHER LYSSAVIRUS INFECTIONS

Most indigenous human rabies in the USA is associated with insectivorous bat rabies virus, usually . Domestic dog rabies in Mexico and some areas of

the silver-haired bat or the eastern pipistrelle. In the Central and South America.

last 10 years, a total of 28 deaths from rabies were . Vampire bat (Desmodus spp.) causing bovine

reported, of which six (21%) were imported during the paralytic rabies from Mexico south to the northern

incubation period in patients infected by dogs abroad. parts of Argentina and Chile (McColl et al., 2000).

All 22 indigenous patients were infected with bat virus but only one had recognised a bat bite. It has been suggested that rabies is under-diagnosed in the USA (Messenger et al., 2002).

Australia In the UK nine people have died of rabies since 1980, eight of whom were infected abroad. Four were

. Flying foxes or fruit bats (Pteropus spp.) and infected in the Indian subcontinent, three in Africa and occasionally insectivorous bats in Eastern coastal

one in the Philippines. After a century of freedom from regions harbour Australian bat lyssavirus (McColl

indigenous rabies, a man died of EBL 2a infection in et al. , 2000) (see section on Human Infections with

Scotland in 2002 (see p 648). In continental Europe Rabies-related Viruses, p 648; Table 21.1).

about 10 rabies deaths are now reported annually in Vampire bat rabies has serious economic eﬀects on

the Russian Federation or in Turkey. farmers. The bats feed on the blood of large mammals,

particularly cattle, transmitting, in the process, a form of paralytic bovine rabies. This disease causes an

PATHOGENESIS estimated loss of 50 000 head of cattle each year in Brazil. Recent outbreaks have been reported from

Entry of Virus into the Nervous System Ecuador. Bat rabies in the Americas is all due to

genotype 1 virus, whereas bats in the rest of the world Bites by rabid animals usually inoculate virus-laden harbour only the rabies-related lyssaviruses: European

saliva through the skin into muscle. Local replication bat lyssavirus , Australian bat lyssavirus or Duvenhage

of virus in striated muscle may occur and account for virus (McColl et al., 2000).

the long incubation periods often observed (Charlton Cyclical epizootics of rabies, such as the fox

et al. , 1997); then virus becomes detectable experimen- epizootic in Europe , result from an uncontrolled

tally at local motor or sensory nerve endings (Murphy, increase in the population of the key reservoir species.

1977). Direct invasion of neurons has also been shown This epizootic started in Poland at the end of the

after peripheral inoculation. Second World War and spread to France, but has now

The rabies virus infects a great variety of cell types in been controlled by vaccination in Western Europe (see

vitro and in vivo, and viral attachment has been control).

demonstrated to several types of cell surface receptors, Many other species of domestic and wild mammals

including carbohydrates, phospholipids and sialylated have been found to be infected with rabies. These are

gangliosides. This binding is not speciﬁc and there is thought to be infected by local known vector species.

no evidence yet that these receptors are important in vivo , but it might explain the diversity of tissue culture cell lines which will support rabies infection.

Speciﬁc binding occurs at neuromuscular junctions, INCIDENCE OF HUMAN RABIES

where it co-localises with the nicotinic acetylcholine receptor (Lentz et al., 1982; Jackson, 2002b). Binding

The true global incidence of human rabies has been at this postsynaptic site is competitive with cholinergic obscured by under-reporting and is not reﬂected in

ligands, including the snake venom neurotoxin, a- oﬃcial ﬁgures. The areas of highest known incidence

bungarotoxin, which shows sequence homology with are where dog rabies is endemic, as dogs are the cause

rabies virus glycoprotein. This may explain the varied of more than 95% of human infections, e.g. in India,

susceptibility of diﬀerent species to rabies infection. Bangladesh and Pakistan an estimated total of 40 000

The muscle cells of the susceptible fox bear more deaths occur annually, according to the World Health

nicotinic acetylcholine receptors than those of the Organization (2001). There are very few data from

resistant opossum, and rabies virus binds well to fox Africa. In Latin America, the highest mortalities are

muscle membranes in vitro, but not to opossum reported from Brazil, Mexico, Bolivia and Peru.

membranes (Baer et al., 1990). Furthermore, there is membranes (Baer et al., 1990). Furthermore, there is

Entry of virus into a cell can occur by endocytosis into a vesicle and pH-dependent fusion of the viral glycoprotein with the endosomal membrane, releasing the ribonucleoprotein complex into the cytosol.

Transport of Virus to the Brain Rabies antigen can be found along the nervous

pathways from a peripheral site of infection towards the brain during the incubation period. The migration of virus from a wound in a peripheral nerve to the CNS occurs within motor axons via the retrograde fast axonal transport system. Its progress can be blocked by sectioning nerves or by metabolic inhibition with locally applied colchicine, which disrupts the axon microtubular system. The rate varies experimentally but in human neurons it is estimated at 50–100 mm/ day. Viral movement is not aﬀected by the presence of extracellular neutralising antibody (Tsiang, 1993). Since rabies has strictly retrograde axonal movement, it has been used as a tracer to identify neural pathways. The rabies P protein associates with LC8, a component of a cytoplasmic dynein light chain, suggesting a means of attachment of the viral RNP complex to the retrograde dyenin axonal motor (Raux et al., 2000; Jacob et al., 2000). Further studies show that binding of viral proteins to the LC8 molecule is not essential for pathogenesis (Mebatsion, 2001), and that the viral

G protein alone can take advantage of the retrograde microtubular transport (Mazarakis et al., 2001). Viral replication is intraneuronal, but the mechanism of interneuronal spread is unknown. The fact that budding of virus is very rarely seen at synapses by EM suggests that infectious naked nucleocapsids may

be transferred trans-synaptically (Gosztonyi, 1994). However, interneuronal infection has been shown to be dependent on the presence of viral G protein (Etessami et al. , 2000). Although rabies infection downregulates host cell gene expression overall, a few genes are upregulated, including ones associated with synaptic vesicle function (Prosniak et al., 2001).

Effect of Infection on the Brain Rabies progresses rapidly through the spinal cord and

brain, with massive intraneuronal viral replication and

accumulation of viral proteins, resulting in inclusion formation and eventually Negri bodies (see section on Virus Structure, above). Virus has been observed emerging from post-synaptic membranes with viral pinocytosis at axon terminals in animals. Contact with the extracellular environment is thereby avoided. Infected glial cells have rarely been observed in man, and they are not a signiﬁcant route of dissemination of virus. Infection of the limbic system aﬀects the behaviour of the host which, in a vector species, increases the chance of transmitting the disease.

The areas of maximum inﬂammatory change in the human brain do not correlate with the distribution of Negri bodies. EEG evidence of pathology is greater in infections with attenuated virus than virulent street strains (Tsiang, 1993) and neuronal death is not a prominent feature at post mortem. Pathogenic strains of virus cause less neuronal apoptosis than avirulent ones; indeed, rabies infection may have surprisingly little visible pathological eﬀect on the brain. The level of viral glycoprotein expression on the neuron surface correlates with the degree of apoptosis and there is downregulation of glycoprotein expression in virulent strains, and so apoptosis only appears at a terminal stage of infection (Dietzschold et al., 2001) The cause of the gross neuronal dysfunction of living cells remains elusive but it may be associated with altered activity at a variety of neurotransmitter binding sites (Tsiang, 1993; Jackson, 2002b). Rabies infection of neurons alters cellular gene expression, not only in infected cells but probably also in uninfected neurons (Prosniak, 2001). The depletion of metabolic pools may limit cell survival (Dietzschold et al., 2001). Changes in the functional expression of some channels, and attenuation of inhibition of others, have been shown in vitro (Iwata et al., 2000). The suggestion that nitric oxide might cause pathogenic neurotoxicity in rabies remains unproven (Jackson, 2002b).

Centrifugal Spread of Virus from the Brain

Finally, there is a phase of diﬀuse centrifugal spread by axonal transport of virus from the brain by many nerve pathways, including the autonomic nervous system. Virus has been isolated from human tissues such as peripheral nerve, skeletal and cardiac muscle, kidney, lung, skin, salivary, lacrimal and adrenal glands (Helmick et al., 1987). Rabies antigen has been detected in nerves and ganglia adjacent to these organs and the gastrointestinal tract, and extraneurally in tongue tissues, often without an inﬂammatory reaction (Jackson et al., 1999). Viraemia is not thought

638

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

639 to occur (Helmick et al., 1987). Virus is shed from

RABIES AND OTHER LYSSAVIRUS INFECTIONS

are crucial. Clearance of rabies virus infection from the human salivary and lacrimal glands, the respiratory

central nervous system has only been demonstrated in tract, rarely in urine (Helmick et al., 1987) and possibly

rodents by treatment with a single rabies virus- in milk.

neutralising monoclonal antibody before the onset of The predominant site of replication changes as the

clinical signs (Dietzschold et al., 2001). Although virus reaches the salivary gland. In contrast to neural

rabies G and N proteins bear epitopes which can cells, there is profuse production of extracellular virus

induce activation of CD8 + T cells, virulent strains do from acinar cells. Viral budding occurs into the gland

not do so. The role of cytotoxicity in rabies encepha- lumen or intercellular canniculae. Virus is secreted into

litis is not clear. Infected human brains show the saliva, from which it is available to infect a new

remarkably little pathology, so apoptosis is unlikely host.

to be a signiﬁcant cause of death, which is in keeping with experimental ﬁndings.

IMMUNOLOGY Response in Human Encephalitis

Immune Responses to Rabies Virus In unvaccinated humans with encephalitis, neutralising

Following a rabid bite, no immune response can be and other antibodies appear in serum about 7 days detected until after the development of symptomatic

after the ﬁrst symptom, later in the cerebrospinal ﬂuid rabies encephalitis, suggesting that the virus can evade

(CSF) (Anderson et al., 1984). In patients whose lives or suppress the immune system while in transit into

are prolonged by intensive care, antibody titres may and out of the brain, during the entire incubation

rise to very high levels. A low level of rabies-speciﬁc period. An immunosuppressive eﬀect of rabies infec-

IgM is sometimes detectable in serum and CSF, but tion has been shown experimentally to rabies antigens

there is no evidence to date that it precedes the IgG and also unrelated viral antigens (Camelo et al., 2001).

response (Warrell et al., 1988). Rabies neutralising antibody, which is only induced by

A minimal lymphocyte response is detected histolo- antigens on the glycoprotein envelope (Wunner, 2002),

gically in brain, blood and CSF (Warrell et al., 1976) is protective against rabies following challenge in

and there is no pleocytosis in 40% of patients in the animals. In vitro, this antibody impairs viral attach-

ﬁrst week of illness, or in 13% in the second week ment and penetration of cells, and reduces cell to cell

(Anderson et al., 1984). Evidence of cell-mediated spread of virus. Although the presence of neutralising

immunity, by speciﬁc lymphocyte transformation tests, antibody is the best available indicator of protective

was found in 6/9 furious encephalitis patients, but not immunity, the level does not correlate with protection

in seven with paralytic disease (Hemachudha et al., in all experiments, showing that other immune

mechanisms are also eﬀective (Lodmell et al., 2001). It has long been recognised that the rabies incuba- RNP antigens can confer protection from death

tion period is shorter than average in patients who experimentally, in the absence of neutralising anti-

were inadequately vaccinated and develop rabies. body. T and B cell speciﬁc epitopes have been

Although high levels of neutralising antibody are identiﬁed on the N molecule, and N can prime the

protective, low levels can accelerate the terminal immune system to enhance the neutralising antibody

phase of the illness, resulting in the ‘early death’ response to G protein. This RNP-induced immunity is

phenomenon (Prabhakar and Nathanson, 1981). The more cross-reactive with other strains, including

eﬀect can be produced in mice by adoptive transfer of rabies-related viruses, than that stimulated by G

antibody or immune B lymphocytes during the protein. There is evidence that N acts as a weak

incubation period.

superantigen by directly inducing proliferation of Interferon-a (IFN-a) prevents rabies viral replica- human CD4 Th2 cells bearing the Vb8 TCR (Lafon,

tion in vitro, but only very low levels have been found 1997) but the eﬀect of this unexpected ﬁnding on

in the serum and CSF of one-third of patients with disease or the host immune response remains elusive.

encephalitis (Merigan et al., 1984). Mice die of rabies Survival of infection in animals is associated with a

despite high levels of IFN-a and IFN-b in the brain, strong immune response, including neutralising anti-

and high doses of intravenous and intrathecal IFN-a body, and upregulation of MHC class II mRNA

after the onset of symptoms of human rabies expression in the CNS early in infection (Irwin et al.,

encephalitis have not proved eﬀective therapy in man 1999). The activity of CD4 + T lymphocytes and B cells

(Merigan et al., 1984; Warrell et al., 1989). In contrast,

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

the early appearance of IFN-g in infected rodent brain population of vaccinees 10% had poor, relatively was associated with survival or delayed mortality

delayed antibody induction (Kuwert et al., 1981). (Koprowski and Dietzschold, 1997).

Immunosuppression due to drugs, HIV, cirrhosis or other disease can also impair the response to vaccine.

Immune Response to Rabies Vaccine Despite the anergy seen in patients with encephalitis,

ROUTES OF INFECTION rabies virus proteins are highly immunogenic when

Human infections usually result from inoculation of given as vaccine. Neutralising antibody usually virus-laden saliva through the skin by the bite of a becomes detectable in serum 7–14 days after starting rabid dog or other mammal. Broken skin and intact

a primary course of vaccine. Antibody production is mucosae can admit the virus, and scratches, abrasions accelerated if the dose of tissue culture vaccine is given and previous open skin lesions can be contaminated in multiple sites, either by increasing the dose of with infected saliva. Intact skin is an adequate barrier intramuscular treatment or by dividing a single dose to the infection. The following are very unusual routes between several intradermal sites (Suntharasamai et al.,

of human infection:

1987; Turner et al., 1976). The early IgM component of the antibody response does not protect against

1. Human-to-human transmission has only been docu- rabies experimentally, has low aﬃnity for viral

mented in recipients of corneal transplant grafts. antigens and is conﬁned to the vascular system, so it

Six virologically proven cases have been reported in is unlikely to be important in immediate post-exposure

which infected corneae were transplanted from prophylaxis (Turner, 1985).

donors who had died of unsuspected rabies. The No antibody or T lymphocyte function test predicts

one patient who survived received high-dose post- protection against disease, but the level of neutralising

exposure treatment and IFN (Sureau et al., 1981). antibody, induced by rabies G protein, gives the best

Two further cases resulted in clinically diagnosed correlation available, and should be used to evaluate

rabies in India. Rare reports of infection by human vaccines. The standard mouse serum neutralisation test

bite or kissing (Fekadu et al., 1996) have been is cumbersome and takes 2–3 weeks, but is now

persuasive, but infection from another source could replaced by tissue culture methods: the rapid immuno-

not be excluded. Despite this, thousands of people ﬂuorescent focus inhibition test (RIFFIT; Smith et al.,

dying of rabies have been nursed in poverty- 1996), which takes 24 h, or the similar ﬂuorescent

stricken homes, yet their relatives in intimate antibody virus neutralisation (FAVN) test (Cliquet

contact with saliva and tears have not developed et al. , 1998), which is easier to read and automate, but

rabies. This indicates that the infection is not easily takes 40 h. The results are expressed in International

transmitted from man to man. Units (IU/ml), compared with an International

2. Inhalation has been reported from the USA, where Standard serum. An arbitrary minimum neutralising

there were two laboratory accidents involving the antibody level of 0.5 IU/ml indicates unequivocal

inhalation of ﬁxed virus during vaccine preparation seroconversion and is taken to be the minimum

(Centers for Disease Control, 1977; Winkler et al., adequate response after a pre- or post-exposure course

1973). Airborne rabies virus was assumed to be the of vaccine.

cause of death of two people 50 years ago who had Other antibody tests employing killed virus antigen

recently visited caves in Texas that were very are more convenient but detect a variety of non-

densely populated with insectivorous Mexican neutralising antibodies, whose titres show less correla-

free-tailed bats. Aerosol transmission of rabies tion with protection from disease. Reﬁnements in the

virus was demonstrated in caves experimentally, ELISA methodology have led to increasing speciﬁcity.

presumably from infected bats’ nasal secretions and Following vaccination, human peripheral blood lym-

possibly urine (Winkler, 1975). However, no further phocytes were transformed in response to a variety of

human cases have been reported. Infection could rabies and rabies-related virus antigens. The neutralis-

also be due to unnoticed physical contact with a bat ing antibody level did not correlate with the

(Messenger et al., 2002; Gibbons, 2002). proliferative indices.

3. Vaccine-induced rabies (Rage de laboratoire). The amount of antibody induced by vaccine is partly

Incomplete inactivation of virus in human vaccine determined by the host. Lower titres were found in

should no longer be a problem, but in the worst people over 50 years old. Kuwert observed that in a

incident 18 people developed paralytic rabies in

641 Fortaleza, Brazil, in 1960. The incubation period

RABIES AND OTHER LYSSAVIRUS INFECTIONS

Recovery from Infection and Chronic Rabies in was 4–13 days after receiving the vaccine (Para´,

Dogs, Vampire Bats and other Mammals 1965).

4. Transplancental infection has occurred in animals, Rabies is not a universally fatal disease in mammals. but only once documented virologically by antigen

The pathogenicity of rabies viruses can vary greatly detection in the brain tissue of a mother and infant

when tested in another mammal species. Dogs, cats, in Turkey. Several women with rabies encephalitis

bats and, most often, mice have recovered from have been delivered of healthy babies.

experimental infection with street rabies virus. It is

5. Oral infection has been shown in animals, although conceivable that a low dose of virus inoculated in the high titres are needed. The transmission of rabies

wild could immunise without causing disease. from mother to suckling infant via the breast milk

Although the fox is one of the most susceptible species has been suspected in at least one human case and is

to rabies, about 3% of animals survive the infection said to occur in animals. However, there have been

and become immune. Animals of several species have no documented cases of transmission of rabies by

been found to be seropositive and so are assumed to ingestion of milk from an infected animal. Boiling

have recovered from rabies. These include the natural and pasteurisation inactivate rabies virus. Trans-

rabies vectors: mongooses in Granada; foxes and mission of virus is theoretically possible from

raccoons in Alabama; hyenas in Tanzania; bats in ingestion of raw milk, but it is not a criterion for

Europe, the Philippines (Arguin et al., 2002) and post-exposure immunisation. Nevertheless, in the

Australia; and, very rarely, stray or unvaccinated dogs US people who had drunk milk from rabid cows

in Ethiopia and Nigeria (Se´rie´ and Andral, 1962; were given rabies prophylaxis (Centers for Disease

Ogunkoya et al., 1990).

Control, 1999b). Vampire bats have long been considered chronic carriers of rabies virus, and some bats were reported to have carried rabies in their saliva for more than 2 months before death, but there is now doubt about the

CLINICAL FEATURES OF RABIES IN ANIMALS validity of these reports (Jackson, 2002b). Some bats recover from infection but there is no evidence of

In domestic dogs, the incubation period ranges from 5 excretion of virus following recovery, and experimental days to 14 months, but is usually 3–12 weeks. It is

inoculation has failed to induce a carrier state. under 4 months in 80% of cases. Prodromal symptoms

The repeated shedding of rabies virus in the saliva of include change in temperament, fever and, as in many

apparently healthy dogs is a cause for great concern. humans, intense irritation at the site of the infecting

This chronic infection is exceptionally rare (Jackson, bite. The familiar picture of a ‘mad dog’ with furious

2002b) but has been reported in an Indian dog, and rabies is seen in only 25% of infected animals. The

in Ethiopian and Nigerian dogs. Thorough search- commoner paralytic or dumb presentation is less

ing in other areas has not revealed any chronically dramatic and more dangerous, as it may not be

infected animals. The incidence of chronic rabies is recognised. The clinical features of furious canine

presumed to be so low that it has not inﬂuenced the rabies include irritability, convulsions, dysphagia,

recommendations for post-exposure treatment. laryngeal paralysis causing an altered bark, hyper-

In Tanzania recently 37% of hyenas were found to salivation,

be seropositive and rabies was detected by PCR restlessness, causing the animal to wander for miles.

techniques in 13% of saliva samples from seropositive Dogs with furious rabies attack and swallow inanimate

animals, although all attempts at virus culture failed. objects, often breaking their teeth and injuring their

Infection was not associated with disease or decreased mouths in the process. Dogs with paralytic rabies may

survival. The virus strain was distinct from those of

be reclusive and exhibit paralysis of the jaw, neck and other local species and may be of low virulence. It is hind limbs, dysphagia and drooling of saliva. Virus

assumed that prolonged and perhaps repeated infec- may be excreted in the saliva 7 days before the

tion occurred with a strain of low virulence (East et al., appearance of symptoms and the animal usually dies

within 7–10 days of onset. Among other species, signs Infection of apparently healthy bats by European bat are usually furious in horses, cats, mustelids and

lyssavirus (genotype 5, EBL 1), has been found in wild viverrids, and usually paralytic in foxes and bovines.

Myotis and other species of Spanish insectivorous bats Although rabid animals are commonly unable to

and bats in a Dutch zoo, with an antibody prevalence swallow, they do not exhibit hydrophobia.

in up to 20% of some populations (Serra-Cobo et al.,

2002). Wellenberg et al. (2002) found rabies RNA in brain and other tissues of healthy-looking bats (Wellenberg et al., 2002). Sublethal EBL 1 infection of bats in Seville similarly was revealed by PCR in oropharyngeal swabs of live bats (Echevarria et al., 2001). To date there has been no isolation of live virus from these asymptomatic persistently infected bats in Europe.

CLINICAL FEATURES IN HUMANS Incubation Period

The extreme range is from 4 days to 19 years or more.

A Thai patient was savagely bitten on the shoulder by

a rabid dog, but received no active or passive immunisation against rabies. This unusually deep and damaging bite may have inoculated virus directly into the brachial plexus, explaining the shortest reported incubation period of 4 days after naturally occurring infection with street virus; it is 20–90 days in more than 60% of cases. The incubation period is reported to be more than 1 year in 1–7% of cases. It tends to be shortest with severe bites on the face, head and neck, especially in children, and in experimental animals injected with larger doses of virus. Average incubation periods for bites on the head and neck are 25–48 days; for the extremities 66–69 days; for the upper limb 46 and for the lower 78 days. Some of the very long incubation periods mentioned in the literature may have been explained by a second more recent, but forgotten, exposure. Alternatively, the virus may remain quiescent until activated by some kind of stress, which has been shown experimentally and suspected in immigrants to the USA with incubation periods up to 6 years (Smith et al., 1991). The incubation appears to be shorter in patients who have received (unsuccessful) post-exposure treatment than in those who have not (Hattwick, 1974).

Prodromal Symptoms

A vague feverish illness associated with a change in mood may precede, by up to about a week, the appearance of deﬁnite signs of rabies encephalomyelitis. The patient becomes anxious, agitated, apprehensive, restless, irritable and tense and may suﬀer from nightmares, insomnia, loss of concentration and depression. Physical symptoms include vague malaise, anorexia, headache, other aches and pains, weakness, tiredness, fever, chills, sore throat and other

symptoms suggestive of upper respiratory tract infection, inﬂuenza or gastroenteritis. This wide range of misleading symptoms has encouraged inappropriate referral to psychiatrists, otolaryngologists, gastro- enterologists and other specialists. Itching, tingling, burning, pain, numbness or some other paraesthesia at the site of the healed bite wound is experienced by about half the patients and may be associated with trembling, fasciculations, muscle contractures or weakness of the bitten limb. Most of the patients infected by corneal transplants noticed pain behind the grafted eye. Although local paraesthesia is suggestive of imminent rabies encephalomyelitis, this symptom is also surprisingly common in healthy victims of animal bites who fear that they are developing rabies. It might

be the result of trauma by the bite or by vigorous local inﬁltration of rabies immunoglobulin. Pruritis is, in our experience, by far the commonest local prodromal symptom; neuritic pain is also reported. Itching may be so intense that the patient excoriates large areas of skin by scratching.

Clinical Presentations of Rabies Encephalomyelitis

Rabies can take two clinical forms. In the more familiar type, furious or agitated rabies, the brainstem, cranial nerves, limbic system and higher centres bear the brunt of the infection, while in dumb or paralytic rabies the medulla, spinal cord and spinal nerves are principally involved. The predominantly but not exclusively paralytic picture seen in human victims of vampire bat transmitted rabies and other evidence from experimental rabies infection in animals suggests that the virus strain or size of infecting inoculum may contribute to the pattern of central nervous system infection. Host factors may also modify the pattern of the disease; the apparent frequency of the dumb form of rabies varies from species to species (it is common in bovines but rare in cats) and there is a widely quoted impression that dumb rabies is more likely to develop in those who have received antirabies vaccine (Hattwick, 1974). Many diﬀerent clinical features and patterns of presentation of human rabies have been described over the past 100 years and, since the recognition of strains of classical rabies Lyssavirus genotype I and rabies- related viruses, clinicians have been interested in attempting to associate distinctive clinical patterns with these various viruses. This is premature. Even the use of the term ‘classical rabies’ for a clinical presentation has become misleading, as it has been applied to the furious form of the disease (Jackson,

642

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

2002a) and also to both furious and paralytic forms, to distinguish them from hypothetical atypical patterns (Hemachudha et al., 2002).

Furious (Agitated) Rabies This is the more familiar and probably the commoner

presentation in humans, except in those infected by bats. Without intensive care, most patients with furious rabies die within a week of their first prodromal symptom and within a few days of developing hydrophobia. This short and hectic clinical course is characterised initially by hydrophobia, aerophobia and periods of extreme excitement, alternating with lucid intervals, features of autonomic system dysfunction and finally by unconsciousness and complete paralysis. Hydrophobia (from the Greek, ‘dread of water’) is a reflex series of forceful jerky inspiratory muscle spasms provoked by attempts to drink water and associated with an inexplicable terror (Figure 21.4). Driven by thirst to confront this terror, the patients attempt to drink but, even before the liquid has reached their lips, a rapid succession of violent inspiratory gasps is provoked. Forcible contractions of the diaphragm depress the xiphisternum and contractions of the accessory muscles of

inspiration, particularly the sternocleidomastoids, are visible during these spasms. There is usually no evidence of laryngospasm and most patients deny pain in the neck or retrosternal region. There is no difference between provoked ‘phobic’ spasms and inspiratory spasms. A draught of air on the skin produces a similar reflex response, ‘aerophobia’. Initially, the spasms affect mainly the inspiratory muscles, but a generalised extension response may be produced, not infrequently ending in opisthotonos and generalised convulsions with cardiac or respiratory arrest. The hydrophobic response may be reinforced by unpleasant consequences of the inspiratory spasms, such as aspiration of water up the nose or into the trachea. However, the first attack may occur without preceding difficulty in swallowing and with no opportunity for the establishment of a conditioned reflex. The hydrophobic response may eventually be produced merely by the sight, sound, mention or thought of water, touching the palatal mucosa, splashing water on the skin, loud noises, bright lights and by the patient’s attempting to speak. There may be associated excitement, agitation and aggression and many patients develop generalised convulsions and die during a hydrophobic spasm.

Cardiac tachyarrhythmias and ECG evidence of myocarditis may be observed during this phase of the

RABIES AND OTHER LYSSAVIRUS INFECTIONS

643

Figure 21.4 The terror of furious rabies in an 18 year-old Nigerian, bitten on the temple by a dog. After 13 days he developed classical hydrophobic spasms. Periods of lucidity were interspersed with episodes of extreme agitiation, violent struggling, shouting and terrifying hallucinations. Courtesy of D. A. Warrell

disease (Warrell et al., 1976; Jackson et al., 1999). There is transient hypertension during the hydrophobic spasms and the violent inspiratory spasms may result in pneumothorax (Warrell et al., 1989). Pneumo- mediastinum may result from alveolar rupture or tears in the lower oesophagus. If the patient survives this phase of the disease and sinks into coma, hydrophobia is replaced by a variety of respiratory arrhythmias (cluster or Biot’s breathing and Cheyne–Stokes respiration), with long apnoeic periods.

Pathophysiology of Hydrophobia (Warrell et al., 1976)

There is some clinical evidence that the inspiratory spasms of hydrophobia may be an exaggeration of reflexes which normally protect the respiratory tract from foreign bodies. These include sneezing, coughing, the neonate’s aspiration reflex and perhaps the immersion reflex, all of which have a powerful inspiratory contraction as their main motor effect. In furious rabies there is a selective brainstem encephalitis which damages cells in the region of the nucleus para- ambigualis (where the inspiratory motor neurons are situated) and in the limbic system. These lesions could disinhibit the respiratory tract protective reflexes and explain the associated terror of hydrophobia. The responses could be reinforced by conditioning, resulting in the heightened reaction to a variety of sensory stimuli which provoke spasms.

Other Features of Furious Rabies Alternating phases of extreme arousal and calm, lucid

intervals are typical of furious rabies. Physical ﬁndings include meningism, cranial nerve lesions (especially III,

VII, VIII), upper motor neuron lesions, muscle fasciculations and involuntary movements. However, conventional neurological examination may fail to disclose any abnormality unless a hydrophobic spasm is observed. Evidence of autonomic stimulation includes lacrimation, hypersalivation and excessive sweating, piloerection, high ﬂuctuating body temperature and blood pressure, pupillary abnormalities and Horner’s syndrome. Involvement of the amygdaloid nuclei and limbic system may explain the increased libido, priapism and spontaneous orgasms/ejaculations in a small minority of patients. These symptoms are reminiscent of Klu¨ver–Bucy syndrome, which can be induced experimentally in rhesus monkeys and by surgical treatment for temporal lobe epilepsy in

humans following bilateral temporal lobectomy. Evidence of an extensive axonal neuropathy was found in two patients with furious rabies in the second week of intensive care. The normal progression of furious rabies is to coma and death within a week of the ﬁrst symptom, but some patients have been kept alive for several months in intensive care units. In these cases a variety of other complications can develop (see Table 21.2).

Paralytic Rabies

This form is less likely to be recognised as rabies than furious disease, which has contributed to its being apparently less common in humans. The patients may become literally dumb (‘rage muette’), because their laryngeal muscles are paralysed, but the term ‘dumb rabies’ also describes the quieter symptoms and more insidious and protracted clinical course exempliﬁed by its other French name, ‘rage tranquille’. There was a famous outbreak of paralytic rabies in Trinidad, involving 20 people, mostly children, between 1929 and 1931. After prodromal fever and headache, burning, tingling, numbness, cramp or weakness developed in one foot, followed by a gradually ascending paralysis and hypoaesthesia, eventually aﬀecting the respiratory and deglutitive muscles. Constipation, urinary retention, high fever and profuse sweating were common. Some patients survived for 3 weeks and only one showed terminal hydrophobic spasms. Vampire bats were eventually implicated. Paralytic rabies was also seen in patients with post- vaccinal rabies (due to defective inactivation of vaccine) and in two patients who inhaled a vaccine

644

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

Table 21.2 Complications of rabies encephalitis Cardiovascular

Arrhythmias, hypotension, cardiac failure Respiratory

Asphyxiation (hydrophobic spasm) Pneumonia Respiratory failure Pneumothorax, pneumomediastinum Adult respiratory distress syndrome

Neurological Convulsions Diabetes insipidus Syndrome of inappropriate anti-diuretic hormone Hypo- or hyperpyrexia Cerebral oedema

Gastrointestinal Stress ulcers Mallory–Weiss syndrome Haemorrhage

645 strain of virus. Neurological signs include quadripar-

RABIES AND OTHER LYSSAVIRUS INFECTIONS

Table 21.3 Diﬀerential diagnosis of rabies esis with predominant involvement of proximal muscles, loss of deep tendon reﬂexes and progressive

Furious rabies

paralysis. In most patients sensation is intact, but in Hysterical pseudo-hydrophobia

(Cephalic) tetanus

some there is evidence of ascending myelitis with loss Other brainstem encephalitides (e.g. complicating serum of pinprick and joint position sense and fasciculations.

sickness)

Myxoedema has been claimed to be a distinguishing Other causes of muscle spasms (e.g. phenothiazine feature of paralytic rabies (Hemachudha et al., 2002).

dystonia, tetany, strychnine poisoning) However, this transient local mounding of muscle in

Delirium tremens

response to percussion, which was used historically to Porphyria Cerebrovascular accident, epilepsy lateralise pulmonary tuberculosis, is unlikely to be a

helpful sign as it is associated with a variety of

Paralytic rabies

pathological conditions (wasting, myxoedema, hypo- Post-vaccinal encephalomyelitis natraemia) and may also be found in healthy people

Paralytic poliomyelitis

(Jackson, 2002a). Fasciculation in the muscles becom- Other causes of acute ascending paralysis (e.g. acute ing involved in the ascending paralysis was mentioned idiopathic inﬂammatory polyneuropathy—

Guillain–Barre´ syndrome)

in the original description of paralytic rabies, by Herpes simiae (B virus) encephalomyelitis (after monkey Gamaleia in 1887. Cranial nerve involvement may

bites)

result in ptosis, external ophthalmoplegia, paralysis of the tongue and deafness. Most patients with paralytic rabies do not experience hydrophobia, but in a few, it may be represented by some terminal respiratory spasms. The course of the disease is less acute and

In those countries where nervous tissue vaccines are stormy than furious rabies. Even without intensive

still used, post-vaccinal encephalomyelitis (PVE) is the care, patients may survive for up to 30 days before they

commonest diﬀerential diagnosis of paralytic rabies. In succumb to bulbar and respiratory paralysis.

PVE, symptoms usually develop within 2 weeks of the ﬁrst dose of vaccine, but no clinical or laboratory features reliably distinguish the two conditions while the patient is still alive, save for the absence of

Differential Diagnosis of Rabies and Infections demonstrable rabies virus in skin biopsies, saliva or by Rabies-related Viruses

CSF. Herpes simiae (B virus) encephalomyelitis, transmitted by monkey bites, has a much shorter

In a rabies endemic area, the diagnosis of furious incubation period than rabies (3–4 days). Vesicles may rabies may be obvious in a patient with hydrophobic

be found in the monkey’s mouth and at the site of the spasms who has a history of being bitten by a dog

bite. The diagnosis can be conﬁrmed virologically and within the previous few weeks or months. However,

the patients treated with aciclovir. exposure to a mammal bite or scratch may not be

remembered, as in the majority of cases of Genotype 1 insectivorous bat-transmitted rabies in the USA (Noah et al. , 1998). Although hydrophobia is a pathogno-

Clinical Diagnostic Methods nomic sign of furious rabies, it may be misdiagnosed as

in Human Rabies

a laryngopharyngeal or even a psychiatric problem. Some of the reported misdiagnoses and diﬀerential diagnoses of furious and paralytic rabies are listed in

Haematological and Biochemical Tests Table 21.3. Rabies phobia, an hysterical response to

Routine haematological and biochemical tests are the fear of rabies, diﬀers from true rabies in its shorter

initially normal, apart from neutrophil leukocytosis. incubation period, which may be only a few hours after

Later, hypernatraemia or hyponatraemia may reﬂect the bite, exaggerated aggressive and dramatic symp-

diabetes insipidus or the syndrome of inappropriate toms and its excellent prognosis. Severe (cephalic)

ADH secretion, respectively. In a majority of tetanus, involving the cranial nerves, is distinguished

patients, there is a lymphocytic pleocytosis and some- by its shorter incubation period, the presence of

times an elevated protein concentration in the CSF. trismus, the persistence of muscular rigidity between

Hypoglycorrhachia has been reported (Roine et al., spasms and the absence of pleocytosis.

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

Neurological Investigations neutralising antibody levels were very high. The clinical features typical of rabies were the apparantly

The electroencephalogram (EEG) may be normal or subjective parasthesiae of the bitten limb, the cardiac may show the expected wide range of non-speciﬁc

conduction defect (if it was a new ﬁnding) and the changes, including evidence of seizure activity. Elec-

high antibody level. Cerebellar signs have rarely tromyography has revealed evidence of primary axonal

been reported in rabies. The rabies antibody neuropathy.

response to nervous tissue vaccines are higher than Most reported computed tomographic (CT) scans in

usual in patients with severe post-vaccinal encephalitis rabies have been normal, especially in the early stages.

(Hemachudha et al., 1989).

Hypodensities of the basal ganglia and of cortical The second patient was a 9 year-old boy in Ohio, lesions have been described.

USA in 1970, who was bitten by a proven rabid big Reports of magnetic resonance imaging (MRI) have

brown bat (Eptesicus fuscus) on the thumb, and began mainly been in patients with paralytic rabies, although,

treatment with duck embryo vaccine the next day surprisingly, both paralytic and encephalitic rabies

(Hattwick et al., 1972). After 20 days he developed a patients are said to show a similar distribution of ill-

meningitic illness progressing to encephalitis, with deﬁned, mildly T2-weighted hyperintensity of the

unilateral weakness maximal in the bitten arm. Focal brainstem, hippocampus, hypothalamus, deep and

seizures, paralysis, cerebral oedema and coma ensued, subcortical white matter and deep and cortical grey

lasting more than a week. He also had an atrial matter (Haemachudha et al., 2002). Minimal gado-

arrhythmia. Prolonged intensive care resulted in linium enhancement, indicating mild inﬂammation,

complete recovery in 6 months. The CSF and serum may be seen in the later stages of the clinical course.

rabies antibody levels were high. Features suggesting Similar changes in the spinal cord, suggestive of

rabies were the dominant signs in the bitten limb, a myelitis, might indicate a diagnosis of paralytic rabies.

cardiac arrhythmia and the high antibody level. Now, The non-speciﬁc changes reported to date would not

30 years later, the patient lives a normal life.

be diagnostic of rabies infection. The third case was a 32 year-old laboratory worker in New York in 1977 who was thought to have inhaled an aerosol of a ﬁxed strain of rabies (SAD) virus (Centers for Disease Control, 1977). He had had pre-

Recovery from Rabies Encephalitis exposure prophylaxis with duck embryo rabies vaccine with a neutralising antibody titre of 1:32 6 months

Animals of several species have recovered from rabies before exposure. Fever, encephalitic symptoms, spastic (see above) and humans with paralytic rabies can

hemiparesis, myoclonus, impaired conciousness and survive for several weeks, especially with intensive

respiratory arrest developed over 2 weeks, and the care, but the illness progresses relentlessly. Five

rabies neutralising antibody titres were high in the patients over the last 30 years have been claimed as

serum and CSF. A gradual improvement was followed survivors of encephalitis. All had received some rabies

by onset of a personality disorder and dementia. Two vaccine before the onset of symptoms. No virus or

and a half years later he still had profound neuro- viral antigen was detected in any patient and so the

logical deﬁcits.

diagnoses were based on ﬁnding high rabies neutralising

A Mexican boy severely bitten on the head by a antibody levels in the CSF.

proven rabid dog was given a course of vero cell Two of the patients were treated post-exposure with

vaccine (see section on Prophylaxis, below), starting rabies vaccines of nervous tissue origin. In Argentina

the following day, but no rabies immune globulin in 1972, a 45 year-old woman was bitten by her

(RIG) treatment (Alvarez et al., 1994). Nineteen days clinically rabid dog, began a course of suckling mouse

later he developed encephalitis with fever and convul- brain vaccine 10 days later (Porras et al., 1976). Three

sions. Intracranial hypertension and coma ensued. He weeks after the bite she had parasthesiae of the bitten

improved over 3 weeks, and reacted to painful stimuli, arm, with tremors, myoclonic spasms, ataxia and other

but quadriplegia persisted, he became blind and deaf signs of cerebellar dysfunction. She recovered but

and eventually died after 2 years and 10 months. High relapsed twice following booster doses of rabies

titres of rabies antibody appeared in the CSF and the vaccine. Clinical features included hypertonia, tetra-

serum. A second boy with similar clinical features paresis, dysphonia, dysphagia, varying levels of

survived at least 9 months.

consciousness and a cardiac conduction defect, with The most recent case of survival was reported from slow resolution over a year. The CSF and serum

India (Madhusudana et al., 2002). A 6 year-old girl

647 was severely bitten by a stray dog, which died 4 days

RABIES AND OTHER LYSSAVIRUS INFECTIONS

Initially she had a cough and vomited but later became later. The wound was not cleaned but she was given

drowsy, confused and weak. She died 3 days after puriﬁed chick embryo cell rabies vaccine (see section

admission. Mokola virus was isolated from brain tissue, on Prophylaxis, below) on days 0, 3 and 7 after the

and a Coxsackie A virus isolated from a rectal swab bite. Sixteen days later she would not drink and

(Familusi et al., 1972). A laboratory worker recovered developed fever, hallucinations and impaired con-

from a mild Mokola virus infection (Crick, 1981). sciousness, which progressed to coma with excessive salivation and focal seizures. Rabies antibody titres rose to very high levels in serum and CSF. After 3

Duvenhage (Lyssavirus Genotype 4) months of coma, improvement began very slowly but after 18 months she had spasticity, tremors and

This virus was named after a 31 year-old white South involuntary limb movements.

African man who had been bitten on the lip by a bat of Neurological illnesses following human diploid cell

unknown species (Meredith et al., 1971). One month rabies vaccine has been reported rarely (see below; Side

later he noticed headache, dizziness and aching in the Eﬀects of Tissue Culture Vaccine) but have been

neck and back. He began to sweat profusely and relatively mild.

suﬀered involuntary spasms of the face and limbs. Rabies virus was not isolated, nor was antigen

During the next 3 days he became progressively more identiﬁed, in these patients thought to have recovered

confused, irritable and aggressive, had nightmares and from rabies, but false negative results may have been

noticed diﬃculty in swallowing. He showed typical obtained because samples were taken late, when there

hydrophobia. He was agitated, and had involuntary was already a high titre of antibody neutralising the

spasms of the neck and back. Touch provoked spasms virus or covering the epitopes of the antigens. The

of the face, arms and trunk and aggressive episodes. diagnosis of rabies in such cases might more readily be

These features became more marked before he died conﬁrmed in future, with the development of sensitive

24 h after admission.

RT-PCR techniques for detecting viral RNA. The diagnosis may remain in some doubt in those patients given vaccines of nervous tissue origin, as post-vaccinal

European Bat Lyssaviruses

encephalitis can produce similar signs and symptoms (Label and Batts, 1982).

Genotype 5, EBL 1 . Two human deaths from rabies The term ‘survival’ is more appropriate than ‘recov-

have been reported from Russia in 1977 and 1985, ery’ in the three patients given tissue culture vaccines, as

following bat bites (Selimov et al., 1989) One month all had profound residual neurological deﬁcits. Severe

after being bitten on the hand, a 15 year-old girl impairment of nervous function was irreversible before

became unwell with fever, anxiety and paraesthesia of the infection was controlled, presumably by the

the bitten hand. She developed an acute ascending immune response.

paralysis, encephalitis and myocarditis and died 5 days later. An 11 year-old girl was bitten on the lip by a bat in Belgorod. Three weeks later she became unwell with

Human Infections with Rabies-related Viruses pain in the bitten cheek, weakness and drowsiness. She (see section on Classification, above)

developed typical furious rabies and died 6 days later. No rabies antigen was detected in brain impression smears by the immunoﬂuorescent test, but virus was

Mokola (Lyssavirus Genotype 3) isolated in suckling mice and proved to be an EBL 1. Two cases of Mokola virus infection occurred in girls admitted to University College Hospital, Ibadan, in

Genotype 6, EBL 2 . Two human infections due to 1968 and 1971. The ﬁrst was a 3½ year-old child who

EBL 2 virus have been documented. An unvaccinated presented with a febrile convulsion, and had a

30 year-old Swiss zoologist in Finland who studied temperature of 1058F and a sore throat. She recovered.

bats had been bitten several times by bats in Malaysia Virus was isolated from her cerebrospinal ﬂuid, but no

and Switzerland during the previous 5 years. Most speciﬁc complement-ﬁxing antibodies were detected. A

recently, in southern Finland he had been bitten by a similar strain of Mokola virus, isolated from a shrew,

sick Daubenton’s bat (Myotis daubentonii) 51 days was being handled in the laboratory at the same time

before the ﬁrst symptoms, which were numbness of the (Familusi and Moore, 1972). The second case, was a 6

palm of his right hand and pain in the neck radiating year-old, admitted after 6 days of feverish illness.

to his right cheek. Next day, he became unable to walk, to his right cheek. Next day, he became unable to walk,

The second patient, a 55 year-old unvaccinated bat conservationist, had been bitten by bats on several occasions in Scotland, most recently in Angus in 2002 on the left hand about 4 months before his illness. He developed pain, paraesthesiae and diminished sensation in the left arm. He was prescribed non-steroidal antiinﬂammatory drugs and, 5 days later, presented to

a hospital in Dundee, Scotland with acute haematemesis attributable to these drugs. He was feverish, disinhibited, dysarthric and had gaze-evoked nystagmus. His upper limbs were areflexic but the lower limb tendon reflexes were brisk. He showed truncal, upper limb and gait ataxia. Sensation to touch was decreased over the left arm. There was no meningism and CSF was normal, apart from a mildly raised protein concentration. Five days after admission, he became suddenly confused, aggressive and agitated and, the next day, deteriorated with a collapsed lung and respiratory failure, decreased consciousness and hypersalivation. Neither aerophobia or hydrophobia was observed. He was mechanically ventilated in the intensive care unit but he became comatose and generally flaccid with extensor plantar responses. There was electrophysiological evidence of a peripheral, predominantly motor axonal neuropathy. He died

19 days after the ﬁrst prodromal symptom (Nathwani et al. , 2003). EBL RNA was identiﬁed ante mortem in saliva by RT-PCR. Post mortem IF and RT-PCR tests were positive on brain samples and EBL 2a was isolated.

Australian Bat Lyssavirus (Lyssavirus Genotype 7)

There have been two reported human fatalities from Australian bat lyssavirus infection in Queensland, Australia. A 39 year-old woman, who cared for bats, had been scratched by bats during the previous 30 days. She was admitted to hospital with a history of a few days of left shoulder pain, dizziness, vomiting,

headache, fevers and chills. Her left arm became progressively weaker, she became ataxic with cerebellar signs, slurred speech, diplopia and dysphagia. Over the next few days she developed bilateral facial palsies, progressive quadriplegia with asymmetrical reduction of deep tendon reﬂexes and ﬂuctuating level of consciousness. She continued to deteriorate and died

20 days after the start of her illness. Rabies antibody was detected and viral RNA was identiﬁed by PCR in the CSF (Samaratunga et al., 1998). The virus isolated from post mortem brain was the insectivorous bat (Saccolaimus sp.) variant of Australian bat lyssavirus.

The second case was a 37 year-old woman who had been bitten on her left hand by a flying fox or fruit bat (Pteropus sp.) (Hanna et al., 2000). 27 months later she presented to hospital with a 5 day history of fever, vomiting, anorexia, pain around the left shoulder girdle, paraesthesiae of the dorsum of the left hand, sore throat and difficulty in swallowing. She was feverish, unable fully to open her mouth, was drooling saliva and had difficulty speak- ing. There was increased muscle tone, painful spasms provoked by examination and spasmodic attempts to swallow provoked by examination of the throat. She had a neutrophil leukocytosis. She deteriorated over the next 12 h, becoming agitated with dysphagia, dysphonia and increasingly severe and frequent muscle spasms, which were uncontrollable by med- ication. She was mechanically ventilated but died 19 days after the first symptom. Australian bat lyssavirus was detected by PCR in saliva. A post mortem revealed diffuse pancarditis, and virus was isolated from the brain.

DIAGNOSIS Intravitam Diagnosis

of Human Rabies Encephalitis (Table 21.4)

The laboratory diagnosis of rabies is rarely attempted in developing countries, but confirmation of infection will be useful to guide the appropriate management of the patient, relatives and staff, prevent unnecessary investigations and allow characterisation of the virus. Routine haematological and biochemical tests are likely to be normal initially, but a plasma neutrophil leukocytosis may be present. A mild pleocytosis is only seen in 60% of patients in the first week (Anderson et al. , 1984). The diagnosis can be made by virus isolation, identification of antigen or, in unvaccinated

648

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

649 Table 21.4 Human rabies diagnosis

RABIES AND OTHER LYSSAVIRUS INFECTIONS

Intravitam REPEAT until a diagnosis is made: Skin punch biopsy*

Antigen detection

IFA test on frozen section

RNA detection

PCR

Saliva, tears, CSF

Virus isolation

Tissue culture Mouse inoculation test

RNA detection

Antibody test Unvaccinated, test immediately Vaccinated, save for comparison later

Test immediately with serum Post mortem

CSF

Antibody detection

Brain.* Needle biopsy (see text) or

IFA test on impression smear brainstem and cerebellum

Antigen detection

RNA detection

PCR

Virus isolation

Tissue culture Mouse inoculation test

Serum

Serology

Antibody test

*Collect in small dry container, well sealed, keep cool during transport. Do not freeze.

people, antibody detection, and taking serial samples for all tests will enable rapid detection of the infection.

Isolation of Rabies Virus Culture of the virus from saliva, throat, tracheal or eye

swabs, brain biopsy samples and, rarely, the CSF is most successful during the ﬁrst week of illness (Anderson et al., 1984) and in seronegative patients. The isolation of virus is best attempted in murine neuroblastoma cells, which can be performed in microtitre plates in 4 days (Webster and Casey, 1996), whereas the inoculation of suckling mice yields results in 1–3 weeks.

Antigen Detection The most rapid diagnosis of rabies during life can be

made by direct immunoﬂuorescent antibody (IFA) identiﬁcation of antigen in a skin biopsy (Noah et al., 1998). A full-thickness biopsy, preferably taken with a disposable biopsy punch, must include the bases of hair follicles. It is taken from a hairy area, usually the nape of the neck, and in addition near the original bite wound, if there is an adjacent proximal hairy area. Tissue is collected in a small dry container and kept

Figure 21.5 Vertical section through a skin biopsy, showing a hair (small arrow) surrounded by its follicle. The bright

cool but not frozen. Vertical frozen sections through ﬂuorescence (large arrow) indicates rabies antigen in neurones hair follicles indicate rabies antigen in the nerve

surrounding the follicle, and is a rapid means of diagnosing twiglets around the base of the follicles, in a

rabies during life. (Courtesy of M. J. Warrell) rabies during life. (Courtesy of M. J. Warrell)

PCR techniques now seem to be a reliable means of identifying and genotyping rabies strains (Noah et al., 1998). The RT-PCR test has given positive results using RNA extracted from saliva samples, and also from CSF, skin biopsy tissue and urine. Nested PCR techniques enhance the sensitivity still further (Tri- marchi and Smith, 2002).

Antibody Detection In unvaccinated patients, the diagnostic appearance of

rabies antibody often occurs during the second week of illness, but it may not happen until after the third week. Antibody may be detectable in the CSF a few days later (see section on Immunology, above). A low level of rabies-speciﬁc IgM has been detected in the serum and occasionally in the CSF of rabies patients, but it did not appear earlier than IgG, and IgM has also been found in post-vaccinal encephalitis patients (Warrell et al., 1988). In vaccinated people, very high levels of antibody in the serum, and especially in the CSF, have been considered diagnostic (Hattwick et al., 1972).

Post Mortem Diagnosis in Humans Post mortem diagnosis can be made by any of the

above tests, but rabies virus is most readily detected in the brainstem and cerebellum. Brain tissue can, however, be obtained without a full post mortem examination. Needle necropsies are taken with a Vim– Silverman or other long biopsy needle via the medial canthus of the eye, through the superior orbital ﬁssure; via the nose through the ethmoid bone; by an occipital approach through the foramen magnum; or through burr holes or open fontanelles in children. Tissue is collected in small dry containers and kept cool but not frozen. The IFA test for antigen takes about 3 h to

perform on brain impression smears (Trimarchi and Smith, 2002). Viral isolation from brain by inoculation of murine neuroblastoma cells or of suckling mice might be successful even if the IFA staining is negative.

A retrospective diagnosis using formalin-ﬁxed brain specimens is possible by protein digestion and antigen detection by IFA or enzymatic techniques. Genomic and mRNAs can also be analysed by in situ hybridisation (Warner et al., 1997; Trimarchi and Smith, 2002). Rabies-related viruses may give a weak or negative reaction in the IFA test.

Diagnosis in the Biting Mammal (Trimarchi and Smith, 2002)

Suspect rabid animals should be euthanised immediately and their brains tested for rabies infection (World Health Organization, 1997). In practice this is often impossible if the animal escapes, or if laboratory facilities are not available. Observation in captivity is potentially dangerous and uncertain. Animal brain samples should include brainstem and cerebellum, but tissue can be obtained without craniotomy, via the occipital foramen. Tissue is collected in small dry containers and kept cool but not frozen. The diagnosis can be confirmed by a direct IFA test on acetone-fixed brain impression smears. A rapid enzyme immunodiagnosis kit will detect rabies antigen in a suspension of brain tissue. This is useful for laboratories without a fluorescence microscope, although the method is up to 3% less sensitive than the IFA test. No single laboratory method is sufficiently accurate for this crucial diagnosis. The IFA test has been found to be about 2% less sensitive than virus isolation in tropical dog rabies endemic areas, while false positive results are rare. Culture of virus should be attempted in antigen- negative samples. The sensitivity of detection, especially on decomposing samples, can be further increased using PCR methods, which can also identify genetic sequences in the viral G or N proteins characteristic of the vector species and geographical origin of the rabies or rabies- related virus. The diagnosis can also be made on formalin-fixed brain specimens (see section on Diag- nosis in Humans, above).

MANAGEMENT OF HUMAN RABIES ENCEPHALOMYELITIS

The undisputed mortality from human rabies is 100% in unvaccinated patients. All ﬁve of the patients reported to have ‘survived’ had received some rabies

650

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

651 vaccine before the onset of symptoms. Despite many

RABIES AND OTHER LYSSAVIRUS INFECTIONS

inﬁltrates (Babes’ nodules) are less frequent. All attempts at intensive care treatment over 30 years, no

changes may be widespread and are most pronounced patient has recovered mentally or physically (see

in the grey matter of the brainstem and spinal cord, section on Recovery, above). Life can be prolonged

and in paralytic rabies spinal cord pathology may by this treatment, but many complications arise (see

predominate. Children often show meningeal inﬂam- Table 21.2). Heavy sedation and analgesia should be

mation (Perl, 1975). The degree of histopathological given to relieve the agonising symptoms. Ketamine, an

change in rabies encephalitis varies from absence of anaesthetic agent and a non-competitive antagonist of

any inﬂammation (Tangchai et al., 1970; Iwasaki et al., the N-methyl- D -aspartate (NMDA) receptor, has

1985) to complete disruption of neuron structure in a speciﬁc anti-rabies activity (Lockhart et al., 1992),

patient treated with intensive care for several weeks. but the human therapeutic dosage is unlikely to give an

Peripheral nerve changes include axonal degeneration adequate concentration for an antiviral eﬀect in the

of myelinated and unmyelinated nerve ﬁbres with brain (Jackson et al., 2003). Immunosuppressive drugs,

leukocyte inﬁltration and degeneration in dorsal root including corticosteroids, rabies hyperimmune serum

ganglia, especially in the region of the site of the bite. antiviral agents, such as vidarabine, cytosine arabino-

Extra-neural changes include focal degeneration of side, ribavirin (Centres for Disease Control, 1984;

salivary and lacrimal glands, liver, pancreas, adrenal Warrell et al., 1989) and IFN-a (Merigan et al., 1984;

medulla, lymph nodes and ocular tissues. An inter- Warrell et al., 1989) have not proved useful.

stitial myocarditis with round cell inﬁltration has been Until a new speciﬁc therapy is available, palliative

described (Warrell et al., 1976; Metze and Feiden, care is recommended. Patients and their relatives

1991) and tissue in the myocardium is also aﬀected, should be advised that although intensive care therapy

which may account for associated cardiac arrhythmias. may prolong life, there can be no expectation of survival without severe permanent neurological disabilities (Jackson et al., 2003).

HUMAN RABIES PROPHYLAXIS Rabies Vaccines

PATHOLOGY Two rabies vaccines are now licensed for use in the UK

Rabies causes an acute non-suppurative meningo- and USA: human diploid cell vaccine (HDCV) (Aventis encephalomyelitis, usually accompanied by diagnostic

Pasteur) and puriﬁed chick embryo cell (PCEC) vaccine intracellular inclusions known as Negri bodies (Figure

(Rabipur TM , Chiron Behring). Both are in 1 ml dose vials. 21.2). These are masses of dense eosinophilic material

PCEC is cheaper to produce. Elsewhere, puriﬁed vero cell in neuronal cytoplasm. Electron microscopy shows

vaccine (PVRV) (Verorab TM , Aventis Pasteur) is widely they are composed of disorganised ﬁlaments in an

available, but the single dose vial contains 0.5 ml. amorphous matrix, largely consisting of rabies ribo-

nucleoprotein. The inclusions described by Negri contained a basophilic inner body, which may include

Pre-exposure Prophylaxis fragments of cellular organelles and occasional virions,

(World Health Organization, 1997; probably mechanically trapped by the fusion of

Centers for Disease Control, 1999a) smaller inclusions. Negri bodies are found in about 75% of patients and are most numerous in the

No rabies deaths have been reported in those given pre- hippocampus, Purkinje cells, medulla and ganglia

exposure prophylaxis with post-exposure boosting. Pre- (Perl, 1975). Inclusions with no internal structure

exposure immunisation is indicated for residents of, or were called ‘lyssa bodies’ by Goodpasture. These

visitors to, areas where dog rabies is endemic and all contain the nucleoprotein alone and are usually

those at occupational risk of contact with a rabid animal smaller than Negri bodies.

or rabies virus in quarantine facilities, customs depart- By the time the patient dies, cerebral congestion with

ments, zoos, laboratories or hospitals. Those staying in some petechial haemorrhages is usual, but without

rural areas of foreign countries where rabies is enzootic gross oedema (Tangchai et al., 1970). Inﬂammation

in other mammals (foxes, jackals, wolves, coyotes, with perivascular mononuclear cell inﬁltrate is com-

mongooses, bats, etc.) should seek advice about the risk. mon, with neutrophils seen only at an early stage.

Subsequent post-exposure treatment will be simpli- Neuronophagia, microglial reaction, ganglion cell

ﬁed and much cheaper after a prophylactic course. degeneration, foci of demyelination and perineural

Immunisation is especially important for children. The

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

high cost of vaccine is the only constraint to

be lower following i.d. vaccine, but the quality of the widespread pre-exposure

secondary immune response to a booster dose is recommendations on the need for pre-exposure rabies

immunisation. Oﬃcial

similar for i.d. and i.m. injections. Chloroquine vaccine use a risk assessment analysis to deﬁne a length

antimalarial chemoprophylaxis may inhibit the induc- of stay in the endemic area, but as rabies is a fatal

tion of rabies antibody after i.d. vaccination, so the disease, and a primary vaccine course is only required

larger dose must be given i.m. once, treatment should be encouraged for all visiting endemic areas.

Booster Doses of Vaccine

Pre-exposure Vaccine Regimens (Figure 21.6)

A booster dose i.m. or i.d. 1 year later enhances and prolongs the immune response, which lasts more than One dose of HDCV, PCEC or PVRV is given i.m. into

10 years in 96% of people (Strady et al., 1998). The the deltoid on days 0, 7 and 28 (or 21). An economical

neutralising antibody level after 3 years has been used but eﬀective alternative is to give the vaccine intra-

to decide whether to give a booster dose every 3 years dermally (i.d.) if more than one person is to be

or every 10 years (Strady et al., 2000). If no serology is immunised. The dose of 0.1 ml of any of the these

available or aﬀordable, doses may be given every 5–10 vaccines is injected i.d. over the deltoid to raise a

years to those at continued risk of infection. In the papule. If the injection is too deep, the needle

USA, frequent serology and boosters are recom- should be withdrawn and the procedure repeated.

mended only for those at high risk, but no boosters Opened ampoules should be stored in the fridge

are given after the primary course for travellers and used the same day. The ampoules do not

(Centers for Disease Control, 1999a). An antibody contain preservatives and so cannot be sanctioned

test should be performed 6 monthly for rabies pharmaceutically as multi-dose vials, hence the need to

laboratory staﬀ and all those at continued high risk use or discard after 1 day. The level of antibody may

and a booster dose given if the titre is <0.5 IU/ml.

Figure 21.6 Intramuscular rabies vaccine regimens. Each arrow represents one dose (whole ampoule) of vaccine. RIG=rabies immune globulin

653 Measurement of neutralising antibody levels after

RABIES AND OTHER LYSSAVIRUS INFECTIONS

If rabies exposure is suspected or proven, post- treatment is necessary only if immunosuppression is

exposure prophylaxis must be started as soon as suspected, e.g. in patients with AIDS. Those with low

possible. The greater the delay in starting treatment, CD4 + counts may fail to mount any antibody response

the greater the risk of virus entering a peripheral nerve, to repeated doses of vaccine.

where it becomes inaccessible to immune attack. If in doubt, vaccinate; it is worth giving prophylaxis even if several weeks have elapsed. The recommended criteria

Post-exposure Treatment for treatment are shown in Table 21.5. Post-exposure treatment is in great demand, especially

where dog rabies is endemic. According to WHO data, Primary Post-exposure Treatment about 5 million people in China, 1 million in India and 500 000 in Vietnam are treated with vaccine annually.

For those who have not had a previous course of Any bite or close contact with an animal in a rabies

vaccine, the treatment consists of three parts: wound endemic area is a potential exposure to rabies virus.

treatment, active immunisation with vaccine, and Evaluation of the risk of infection depends on the

passive immunisation with rabies immune globulin. history and clinical features of both the patient and the biting animal, and also knowledge of the local vectors

Treatment of wounds . Immediate vigorous wash- and epidemiology of rabies.

ing, scrubbing and ﬂushing with soap and water, detergent or water alone are recommended for all animal bite wounds, including those without risk of

The Site of Infection? rabies. Kaplan showed that this ﬁrst aid treatment can

be 50% eﬀective in preventing rabies experimentally The virus gains access through any bite, scratch or

(Kaplan and Cohen, 1962). He also suggested local contamination of broken skin or mucous membrane

inﬁltration, proximal to the wound, with procaine by animal’s saliva, but intact skin is an adequate

hydrochloride 1% in saline for pain, as this may also barrier against infection. The risk of infection is

have some antiviral action, followed by application of greatest from bites on the head, neck and hands and

either 70% ethanol or povidone iodine. Suturing the multiple bites carry a higher risk than single bites.

wound should be avoided or postponed. Tetanus Other routes of infection are very rare (see section on

prophylaxis may be required. Giving a prophylactic Route of Infection, above).

antimicrobial agent should be considered for serious bites or those on the hands (co-amoxyclav, doxycycline or erythromycin for dog or cat bites).

The Animal Species and Behaviour? Vaccine regimens (Figure 21.6) .

A course of ﬁve An unprovoked attack by a known local rabies vector

i.m. injections of HDCV or PCEC vaccine are given (see section on Epidemiology, above), or an animal

into the deltoid on days 0, 3, 7, 14 and 28 (World which may have been in contact with a vector species,

Health Organization, 1997; Centers for Disease suggests a high risk of exposure. Abnormal behaviour,

Control, 1999a). Economical intradermal (i.d.) post- either excitable or with partial paralysis, is typical of

exposure treatment has been used for 15 years in Asia rabies and wild animals can appear unusually tame.

(see below). Although an i.d. regimen may have some Vaccination of domestic animals is not always

advantages, it is not oﬃcially recommended in Europe protective; 20% of proven rabid dogs in Bangkok

and North America, where full i.m. treatment is freely had been vaccinated (Mitmoonpitak and Tepsumetha-

available. If immunosuppression is suspected, due to non, 2002).

diseases such as AIDS or drugs, the antigenic stimulus can be increased by doubling the initial dose of i.m. vaccine (one i.m. dose into each deltoid) or by dividing

To Confirm Exposure

a whole single dose between multiple sites intrader- mally (see below). A shortened course of i.m. treat- Every eﬀort should be made to obtain a virological

ment, requiring four instead of ﬁve doses of tissue diagnosis, by examining the animal’s brain for rabies

culture vaccine, is used occasionally outside the UK. antigen and other tests if appropriate (see section on

Two doses are injected on the ﬁrst occasion, one into Diagnosis of Biting Animal, above).

each deltoid muscle. A single dose is given on days 7

and 21 (Vodopija et al., 1988). Multisite injections might accelerate the antibody response (Anderson et al., 1981; Suntharasamai et al., 1987) but the antibody titre wanes more rapidly than with the conventional regimen. Pregnancy is not a contraindication to rabies vaccination.

Rabies immune globulin (RIG) . RIG should be given with every primary post-exposure treatment. It is

most important for severe exposure to infection—bites on the head, neck or hands or multiple bites. Passive immunisation provides some protection for the 7–10 days before vaccine-induced immunity appears. RIG apparently neutralises virus in the wound and enhances the T lymphocyte response to rabies (Celis et al., 1985). The dose of 20 units/kg body weight of human RIG (or 40 units/kg equine RIG, outside Europe and North America) should be infiltrated deep under and around the wound. If this is anatomically impossible, e.g. in a bitten finger, give the rest by i.m. injection at a site remote from the vaccine, but not into the gluteal region. For multiple bites the RIG can be diluted two- or three-fold in saline to ensure infiltration of all wounds. The recommended dose of RIG must not be exceeded, as this will impair the immune response to the vaccine. Serum sickness has not been reported with human RIG. As there are problems of production and supply of RIG worldwide, efforts to find alternative products continue, including the use of a collection of monoclonal antibodies and novel strategies to manufacture immune globulins in vitro.

Post-exposure Treatment in Previously Vaccinated Patients

Treatment of animal bites, including immediate wound cleaning, is always urgent. Provided that a complete pre- or post-exposure course of a tissue culture vaccine has been given previously, or if a serum rabies neutralising antibody level of >0.5 IU/ml has been recorded, an abbreviated course of only two doses of vaccine may be used. It is injected i.m. into the deltoid on days 0 and 3. RIG treatment is not necessary (World Health Organization, 1997; Centers for Disease Control, 1999a). If there is any uncertainty about past treatment, the full post-exposure regimen and RIG must be used.

How Effective is Post-exposure Treatment? The untreated mortality from proven rabid dog bites

in India was 35% and 57% in separate studies more than 35 years ago. Optimal modern post-exposure treatment, started on the day of the bite, in healthy recipients is practically 100% eﬀective. ‘Failures of treatment’ to date are due to failure to deliver the three components correctly and promptly, or failure of the patients’ immune response, e.g. if there is delay in starting treatment; failure to clean the wound; failure to complete the course of vaccine; injections of vaccine or RIG into the buttock; failure to inﬁltrate the wound with RIG; or immunosuppression by drugs, HIV,

654

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

Table 21.5 The decision to give post-exposure treatment Category

Type of contact with a suspect or confirmed rabid domestic or wild* animal, or animal unavailable for observation

Recommended treatment

I Touching or feeding of animals Licks on intact skin

None, if reliable case history is available

II Nibbling of uncovered skin Minor scratches or abrasions without bleeding Licks on broken skin

Administer vaccine immediately. Stop treatment if animal remains healthy throughout an observation period { of 10 days or if animal is killed humanely and found to be negative for rabies by appropriate laboratory tests

III Single or multiple transdermal bites or scratches Contamination of mucous membrane by animals’ saliva

Administer rabies immmunoglobulin and vaccine immediately. Stop treatment if animal remains healthy throughout an observation period { of 10 days or if animal is killed humanely and found to be negative for rabies by appropriate laboratory tests

This table is a simpliﬁcation of the WHO recommendations (1997). *Exposure to rodents, rabbits and hares seldom, if ever, requires speciﬁc anti-rabies treatment. {

This observation period applies only to dogs and cats. Other domestic and wild animals suspected as rabid should be killed humanely and their tissues examined using appropriate laboratory tests. An exception may be made for animals of threatened or endangered species.

cirrhosis or other illness. There has been no suggestion that treatment has failed because the potency of the recommended vaccines was low, or that the vaccine virus strain did not protect against the infecting virus. Only two people are known to have died despite complete prompt treatment with modern products (Hemachudha et al., 1999). Nevertheless, if wounds are extensive and severe, especially from wolf bites, primary post-exposure treatment cannot ensure survival.

Efficacy Against Rabies-related Viruses (see pp 632, 636)

Tissue culture vaccines do aﬀord some protection against European bat lyssavirus (genotypes 5 and 6) but they are probably less eﬀective than they are against the genotype 1 rabies strains and Australian bat lyssavirus (genotype 7). Vaccine is less protective against Duvenhage virus (lyssavirus genotype 4) and gives little or no protection against Mokola virus (genotype 3) (King and Turner, 1993; Badrane et al., 2001).

Side-effects of Tissue Culture Vaccines There is very wide variation in the incidence of side-

eﬀects in diﬀerent groups of recipients. Mild erythema and pain at injection sites are reported in 7–64% of HDCV recipients, and local irritation is more common (13–92%) after i.d. injections. Generalised symptoms of headache, malaise and fever occur in 3–14% (World Health Organization, 1997) and up to 3% reported a ‘rash’ distant from the injection site. There are similar data for PCEC and PVRV vaccine.

Rare case reports of neurological illness temporally associated with tissue culture vaccine treatment have described a Guillian–Barre´-like syndrome (Bøe and Nyland, 1980; Bernard et al., 1982; Knittel et al., 1989; Chackravarty, 2001), a relapsing mild hemiplegia (Tornatore and Richert, 1990) or symptoms restricted to an arm in two patients (Gardner, 1983). The incidence of neurological disease after these rabies vaccines is no more than after other commonly used vaccines.

Late booster doses of HDCV were followed, 3–13 days later, by a systemic allergic reaction in 6% of vaccinees in the USA (however, reaction rates were not evenly distributed between the groups of volunteers) (Dreesen et al., 1986). The urticarial rash, angioedema and arthralgia respond to symptomatic therapy. It is possibly caused by an IgE- mediated reaction to b-propiolactone-modiﬁed vaccine

components (Warrington et al., 1987). Repeated booster doses of vaccine for people at continued high risk of infection can be avoided by serological testing to conﬁrm the need for further treatment.

What to Do if Bitten in a Rabies Endemic Area

If bitten by a mammal in a rabies endemic country (or if any direct contact with a bat in the Americas), immediately wash the wound thoroughly (see above). Seek advice on the local epidemiology from a doctor. If there is a risk of rabies infection, start post-exposure treatment without delay. Tissue culture vaccines are too expensive for worldwide use, and so nervous tissue vaccines are still produced in many developing countries. The potency of Semple (sheep brain) or suckling mouse brain vaccines is variable and they are associated with neurological reactions; the incidence is estimated at 1/200 for Semple vaccine (Bahri et al., 1996). Suckling mouse brain vaccine carries a lower risk; estimates vary between 1/8000 and 1/27 000 courses. Nevertheless, if only locally produced animal brain vaccine is available it might be reasonable to begin treatment immediately, despite the risk of allergic encephalomyelitis, and change to one of the European vaccines as soon as possible. If RIG is not available initially, it should be given up to 7 days after starting vaccine. Equine RIG is widely used in Asia and Africa, and there is a 1–6% incidence of serum sickness, but anaphylaxis is rare. If the risk of rabies exposure seems high, it is worth curtailing a holiday to seek a recommended vaccine and RIG.

Two other post-exposure treatment regimens are recommended by the World Health Organization (1997) (Figure 21.7). They are economical multisite intradermal regimens which use only 40% of the amount of vaccine needed for the standard i.m. course. The eight-site regimen gives an accelerated antibody response: on day 0 use a whole 1 ml vial to inject about

0.1 ml PCECV or HDCV i.d. at eight sites (deltoids, thighs, suprascapular, lower anterior abdominal wall); on day 7 give 0.1 ml i.d. at four sites (deltoids, thighs); and on days 28 and 91, 0.1 ml i.d. at 1 site (Warrell et al. , 1985). There is no report of the use of PVRV, which contains 0.5 ml/ampoule, with this schedule, but the equivalent dose would be 0.05 ml/site.

The two-site intradermal post-exposure regimen has been used widely in Asia accompanied by RIG. It was designed for use with PVRV (Chutivongse et al., 1990), with an i.d. dose of 0.1 ml/site. If other (1.0 ml) vaccines are used, each i.d. dose must be 0.2 ml. On

RABIES AND OTHER LYSSAVIRUS INFECTIONS

655

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

Figure 21.7 Intradermal post-exposure rabies vaccine regimens. The height of the arrows indicates the amount of vaccine used. 1 ml here represents one ampoule of vaccine=one i.m. dose=usually 1 ml, but 0.5 ml for PVRV

days 0, 3 and 7, give one i.d. dose at two sites transmission of infection; the suitability of animal (deltoids); on days 28 and 91, give one i.d. dose at one

rabies vaccines; the social and cultural attitudes of the site (deltoid).

population to the vector species; and the ﬁnancial The two i.d. regimens use a similar total amount of

resources to implement a control programme. vaccine, and aseptic techniques are essential when sharing ampoules. Opened ampoules should be used within 24 h. A comparative study showed that the

Urban Rabies eight-site method induces neutralising antibody more rapidly and to higher levels than the two-site regimen,

This is an epizootic of infection in domestic and stray which is important when RIG is not available

dogs. Muzzling, vaccinating and restricting the move- (Madhusudana et al., 2001).

ment of owned dogs and reducing the number of strays was eﬀective in eradicating rabies from some islands and peninsulas. In several large cities in South

CONTROL OF ANIMAL RABIES America, intense mass vaccination programmes were dramatically eﬀective in reducing the incidence of

The ability to control animal rabies depends on: the canine rabies and eliminating human disease. The size prevalence and host range of rabies in wild and

of urban dog populations, as with wild species, is domestic mammals in the region; the eﬀectiveness of

determined by availability of food, water and shelter. the local rabies surveillance network; the mode of

Attempts to eliminate stray dogs by shooting and

657 poisoning are diﬃcult, unpopular and ineﬃcient as

RABIES AND OTHER LYSSAVIRUS INFECTIONS

which bats, but not cattle, are highly sensitive. fertility increases, restoring the numbers. Oral rabies

Vaccination of bats is being investigated. vaccines for stray dogs have not proved very eﬀective

No attempts have been made to control rabies in or easy to distribute; but methods of clearing rubbish

some vector species despite their potential to infect to reduce the food supply, with parenteral vaccination

man. Insectivorous bats in North America and Europe and fertility control of strays, can be successful in

are examples, due to their inaccessibility. Practical localised areas. The involvement of the residents is

measures to avoid contact with bats and a low essential. The role and inﬂuence of traditional methods

threshold for giving post-exposure treatment are the of rabies prophylaxis must be taken into account and

only means of preventing human infection (Centers for overcome tactfully by discussion and education.

Disease Control, 1999a). In rabies endemic areas, Vaccination of owned dogs and cats should be

people should be educated to avoid unnecessary mandatory in these areas. Facilities for viral diagnosis,

contacts with wild and domestic carnivores and to disease surveillance and pre- and post-exposure

seek medical advice immediately if they are bitten or vaccination of humans are also necessary. Countries

scratched.

free of rabies should prevent reinvasion by controlling the importation of mammals and by enforcing strict quarantine regulations from areas with urban rabies.

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22 Papillomaviruses

Dennis McCance

University of Rochester, Rochester, NY, USA

INTRODUCTION (animal and human cutaneous PVs). There are 11 groups under super-group A, two under B and C and

Papillomavirus is one of the two genera of the family one group under each of D and E. The classiﬁcation Papovaviridae . However, as will be seen, the viruses

results from the deduced evolutionary relationships belonging to this group are quite diﬀerent from the

amongst the papillomaviruses as judged by the sequence other genera, both in genome size and organisation as

similarity of their genomes. For example, A9 contains well as pathogenesis.

the commonly isolated virus HPV-16 and the related The papillomaviruses (Latin: papilla=nipple; oma=

viruses HPV-31, -33, -35, -52 and -58. Table 22.1 has a tumour) produce in their host benign skin tumours

breakdown on the classiﬁcation. Note that two of the (papillomas), containing variable amounts of infec-

animal viruses, rhesus monkey and pygmy chimpanzee tious virus, and have been a recognised lesion since the

papillomaviruses, are grouped with the human genital ﬁfth century BC . Common hand and plantar warts are

isolates in groups A9 and 10, respectively. the most frequent skin papillomas of man and until recently these viruses generated little clinical or scientiﬁc interest, since the typical lesions were a

PHYSICAL AND CHEMICAL PROPERTIES cosmetic nuisance and the viruses were not thought to

be involved in serious disease. However, 20 years ago Structure the ﬁrst papillomavirus isolated from invasive carci-

noma of the cervix was described (Durst et al., 1983). The capsids of the papillomaviruses have icosahedral Since then papillomaviruses have been linked with

symmetry containing 72 capsomeres with a diameter of other squamous cell carcinomas (SCCs) of mucosal

52–55 nm (Figure 22.1).

and cutaneous epithelia and interest in these viruses has been stimulated, resulting in signiﬁcant advances in our understanding of the natural history and patho-

Genome genic process.

The human papillomavirus (HPV) virion contains a double-stranded DNA molecule of 5610 6 Da molecu- CLASSIFICATION

lar weight with an average of 7900 base pairs. The DNA, when in the virion, has a supercoiled circular

At present papillomaviruses are classiﬁed into ﬁve

conﬁguration.

super-groups: A, genital human papillomaviruses, The molecular organisation of the papillomavirus (HPV); B (associated with epidermodysplasia verruci-

genome is well conserved between viruses of various formis); C (ungulate ﬁbropapillomaviruses); D (bovine

species and an example of one of the common genital papillomaviruses, causing true papillomas); and E

isolates is shown in Figure 22.2A. In this ﬁgure the

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

Table 22.1 Classiﬁcation of the Papillomavirus subfamily

Super-groups

Group A

*R, rhesus monkey papillomavirus; P, pygmy chimpanzee papillomavirus; B, bovine papillomavirus; Cr, cottontail rabbit papillomavirus; Co, canine oral papillomavirus; D, deer papillomavirus; E, elk papillomavirus; Ro, rabbit oral papillomavirus. Adapted from the Los Alamos National Laboratory HPV database: http://hpv-web.lanl.gov/

so are read in the same direction, remembering that transcription is in the 5’ to 3’ direction. HPV-16 codes for ﬁve early and three late proteins. There is an upstream regulatory region (URR), which contains the major early promoter at base 97 and enhancer elements for transcription of early genes and also contains the origin of replication. A late promoter for the transcription of the capsid protein mRNA has been identiﬁed and lies in the E7 ORF at 670, although some of the late transcripts may initiate around this nucleotide.

Figure 22.1 Electron micrograph of human papillomavirus

Viral Coded Proteins

from a genital wart (negative stain; 6154 000; bar=100 nm). Courtesy of Dr J. D. Oriel

There are ﬁve proteins coded for by the early region of the HPV genome and three late proteins. Because there are diﬀerences in the pathogenesis of various HPV

genome is represented as a linear molecule with the types, this is reﬂected in some of the functions of the boxed areas indicating the open reading frames

early proteins. Therefore, for brevity a short summary (ORFs) and for convention is divided into two areas,

of the functions of HPV-16 (Figure 22.2) early (E coding for early (E) and late (L) proteins. Because of

preﬁx) and late (L preﬁx) proteins is presented and the overlapping nature of the ORFs, the mRNAs

their molecular weights are shown in Table 22.2. transcribed are complex and it is not always clear

Three of the early proteins, E6, E7 and E5, have which proteins each transcript codes (Figure 22.2B).

properties which are consistent with the virus having to All the ORFs are transcribed from the same strand and

stimulate the infected cells into S-phase, so the viral

PAPILLOMAVIRUSES

Figure 22.2 (A) Schematic of the HPV-16 genome organisation. The early genes are solid grey and the late genes are speckled. The base pairs of the open reading frames are shown and the ATG start codon is shown in italics. E4 is spliced to the N-terminal of E1 (E16E4) since it has no ATG of its own. The poly A+ signals for the early and late mRNA are shown (pA). The origin (Ori) of replication and the upstream regulatory region (URR) or the main early promoter region are indicated. (B) Determination of mRNA species detected in many cases by RT-PCR, so some of the 5’ and 3’ ends are uncertain. E4 is always expressed as a fusion of E16E4

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

Table 22.2 Molecular weights of HPV-16 proteins function may allow cells to divide in an uncontrolled manner. E7 has also been shown to bind to other

Protein Molecular weight (kDa) a cellular proteins, such as various members of the AP-1 E1 70 family of transcription factors, and to upregulate their

E2 45 activity (Antinore et al., 1996) and also some members E4 17 of the basal transcription machinery, such as TATA- E5 8 binding protein (TBP; Phillips and Vousden, 1997).

E6 16 E7 11 E1 and E2 are involved in the replication of the HPV L1

55 genome (see section on Viral Replication, below). E2 L2

50 has additional functions in that it can positively and

negatively regulate transcription from the early pro-

Molecular weight on SDS-PAGE gels may be diﬀerent, e.g. E7 runs at 15–16 kDa because of charged amino acids in the N-terminus of the

moter. The full-length E2 protein contains a

protein, but codes for a protein of 11 kDa and L1 runs at 75 kDa.

transactivation domain at the 5’ end and a DNA binding domain in the 3’ half, and the active complex is

a dimer. Negative regulation of the HPV early DNA has the cell’s replicative machinery available for

promoter is due to the fact that one of the E2 binding propagation of the genome. It has to be remembered

sites lies next to the TATA box of this promoter and so that the virus is attempting to replicate in cells that are

E2 binding sterically inhibits binding of the TATA- programmed to diﬀerentiate and so will have little or

binding protein and therefore inhibits initiation of the no replicative enzymes available to the virus. Both E6

early transcripts.

and E7 are important for the eﬃcient immortalisation E5 is a membrane-associated hydrophobic protein of human keratinocytes and have functions which

with transforming activity for rodent ﬁbroblasts. The

E5 protein inhibits the acidiﬁcation of endosomes sion. E6 has been shown to bind the human p53

disrupt the normal control of G 1 to S phase progres-

(Straight et al., 1995) by binding to the smallest protein and cause its rapid degradation through the

subunit (16 kDa) of the vacuolar ATPase, a multi- ubiquitin proteolysis pathway (Scheﬀner et al., 1990).

component proton pump (Conrad et al., 1993). In It has been shown to bind a number of other cellular

human keratinocytes this results in the delay of the proteins (McMurray et al., 2001), but the biological

epidermal growth factor receptor (EGFR) degradation consequences of the binding are unclear. For instance,

and a hyperstimulated cell. Since the EGFR is the E6 has also been shown to bind to a Ca 2+ binding

major growth factor receptor on keratinocytes, this protein called E6BP, which has homology to a cellular

activity may be important for stimulating cells into S protein, ERC-55, of unknown function (Chen et al.,

phase for viral DNA replication. E5 can transform 1995) and also binds to proteins with a PDZ domain,

rodent ﬁbroblasts and in the presence of the epidermal such as membrane-associated guanylate kinase homo-

growth factor there is an increase in the eﬃciency of logues (MAGUKs), which are found at the membrane

transformation (Leechanachai et al., 1992). and are thought to be involved in intra- and

The late proteins L1 and L2 are the major and minor intercellular interactions (Thomas et al., 2001). The

capsid proteins, respectively, of the virion. The DNA biological consequences of these interactions have not

and amino acid sequences are highly conserved been delineated, although it appears that binding to

between HPV types, especially in the L1 protein. The MAGUK proteins may correlate with the ability of E6

amino acid homology can be as high as 76% between to cooperate with E7 for immortalisation. E7 binds

HPV 16 and 33 (group A9). However, even within another cellular protein, the retinoblastoma gene

groups of PVs type-speciﬁc epitopes predominate, product (pRB; Dyson et al., 1989), and derepresses

although some weak cross-reactivity has been the inhibitory activity of Rb for transcription factors

observed, so it is possible to diﬀerentiate between that are important for expression of genes, whose

types on a serological basis (see next section). products are essential for DNA synthesis. Repression

The mRNA for the E4 protein is spliced, with the 5’ of transcription may be due to the binding by Rb of a

portion coded for by the ﬁrst few base pairs of E1, histone deacetylase protein (Brehm et al., 1998), which

spliced to the E4 open reading frame. The message is functions to condense chromatin and restrict the access

detected late in infection, although it has the preﬁx of of transcription factors to DNA. E7 appears to

an early gene. E4 interacts with cellular microﬁla- compete for the binding site of the deacetylase on

ments, causing them to collapse (Doorbar et al., 1991), Rb. Both these cellular proteins are negative regulators

but the motive for this interaction is not clear, of the cell cycle and so interference with their normal

although it has been suggested that this might allow although it has been suggested that this might allow

There are host-coded histone proteins, H2a, H2b, H3 and H4, associated with the viral DNA within the virion.

SEROLOGY The expression of the major capsid protein, L1, in

yeast and insect cells resulting in the folding and production of virus-like particles (VLPs) has permitted serological studies on the relatedness of HPVs and to determine serological responses in infected patients. VLPs of human papillomaviruses consisting of L1, were synthesised using baculovirus vectors and insect cells, allowing the formation of an icosahedral structure similar to that of the virion (Kirnbauer et al. , 1992). VLPs can also be produced which contain both the major (L1) and minor (L2) capsid proteins, and the latter appears to stabilise the icosahedral structure and also has been shown to possess neutralising epitopes. Antiviral antibodies have been produced to virions of HPV-11 and -16 and these antibodies recognise VLPs of the L1 protein from HPV-11 and -16, respectively (Rose et al., 1994) but do not cross-react. Therefore, VLPs are recognised by antibodies raised to infectious virus particles, and vice versa. The antibodies to VLPs or virions detect conformational epitopes, unlike earlier serological studies, where disrupted particles, or fusion proteins of L1 or peptides of regions of L1 were used either as targets or to raise antibodies to the L1 protein, and produced extensive cross-reactivity. This cross-reactivity was so complete that antibodies to disrupted BPV-1 particles recognised linear epitopes in most HPV types. Therefore, it was only with the production of VLPs that the serological diﬀerences between HPVs, even those closely related, became apparent, e.g. within one group, say A9 containing HPV-16, -31 and -33, polyclonal antibodies only react well with the type to which the antibodies were raised, although some cross- reactivity has been observed. The level of cross- reactivity is very low and is probably not biologically relevant in cross-protection. Monoclonal antibodies to virions or VLPs have shown that there are predominantly type-speciﬁc epitopes, although in closely related viruses such as HPV-6 and -11, where L1 is over 80% homologous at the amino acid level, cross- reactive epitopes have been observed. While there is little cross-reactivity at the antibody level, there appears to be more at the T cell receptor level, as

lymphoproliferative assays show some cross-reactivity between types such as HPV-6 and -16.

VIRAL REPLICATION

Infection of tissue culture cells with papillomavirus particles and subsequent propagation of infectious virus has not been achieved. The major problem is that HPV virion production depends on differentiating epithelial cells and by their nature these cells do not grow in vitro. However, recently (Frattini et al., 1996) it has been possible to transfect human keratinocytes, the natural host cell, with HPV-31 DNA, select stable cell lines containing episomal copies of HPV-31 DNA, and then differentiate the cells using the raft culture system and show that virus particle production occurs in a few cells in the uppermost part of the differentiated epithelium. HPV-11 DNA has also been replicated in keratinocytes by the same laboratory, although because HPV-11 is unable to immortalise human keratinocytes, the cells senesce and so the replicating DNA is lost. The distribution of the virus particles is the same as that observed in infected epithelium (Figure 22.3). The level of virus production is very low and not enough to produce virus particles for serology or infectivity studies. However, propagation of HPV types has been successful in a nude mouse kidney capsule system (Kreider et al., 1985) and in human skin grafts onto the epithelium of SCID mice (Bonnez et al., 1998). In the former system human foreskin tissue fragments are mixed with a viral suspension of HPV and then the tissue is transplanted under the kidney capsule of a nude mouse. Over the next 60 days the tissue fragments grow and produce infectious HPV virus. Both HPV-11 and -16 have been propagated in this animal model; however, the system is obviously complex, requiring skills and animal facilities not available to all. In the alternative method, the tissue fragments are placed under the skin of the immunocompromised SCID mouse and so the growth of the tissue can be monitored and, while the surgical skills are less demanding, the model is not routine.

The origin of replication of HPV-11 and -31 (Frattini and Laimins, 1994; Lu et al., 1993) has been mapped extensively and the region is conserved in other HPV types. The mapping has been achieved by cloning the origin region into a bacterial vector and transfecting mammalian cells, along with plasmids expressing the E1 and E2 proteins of the respective virus. The E1 and E2 proteins are the only HPV- speciﬁc products necessary for the replication of the origin-containing plasmid. The origin region of the

PAPILLOMAVIRUSES

665

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

Figure 22.3 A section of biopsy containing CIN 1, showing nuclear staining of HPV-16 L1 antigen using a monoclonal antibody, followed by alkaline phosphatase-tagged antimouse secondary antibodies. Note that the dark-staining nuclei are found in the outer, diﬀerentiated part of the epithelium

DNA has been mapped to a region at the 3’ end of cell carcinomas develop in nearly one-third of patients the URR (Figure 22.2a) and both E1 and E2 have

with EV, usually in areas of skin exposed to the sun speciﬁc DNA binding sites at the origin. In addition

(face, neck and hands being most commonly aﬀected); to binding at the origin, E1 and E2 can bind

HPV-5 and -8 are commonly found in these lesions together and E1 has been shown to have ATPase and helicase activities, which are typical of viral

Table 22.3 HPV types isolated from patients with the proteins, such as SV 40 large that are involved in the

genetic disease epidermodysplasia verruciformis (EV) and initiation of viral T DNA replication.

associated diseases HPV type

Lesions 5 Warts/SCC

NATURAL HISTORY OF HPV INFECTIONS

8 SCC 9 Warts

HPVs infect and replicate in squamous epithelium on

12 Warts

both keratinised and mucosal surfaces. Most people

14 SCC

15 are infected with certain cutaneous viruses, such as the Warts 17 Warts/SCC ones causing hand and foot warts (HPV types 1, 2, 3,

19 Warts

4), during childhood and adolescence. A small group 20 Warts/SCC of individuals with the rare autosomal recessive disease

21 Warts

epidermodysplasia verruciformis (EV) harbour a

22 Warts

number of virus types not often isolated from normal

23 Warts

24 people (Table 22.1, groups B1 and 2). Two common Warts

25 Warts

presentations in EV patients are multiple warts, which

36 Warts

may be so numerous as to produce coalescent areas

46 Warts

and dry, scaly ﬂat lesions, which may be red or heavily

47 Warts

pigmented. These latter skin lesions, although not having a wart-like appearance, contain many of the SCC, squamous cell carcinoma. HPV types -38 (melanoma), -41 (SCC)

and -48 (SCC) were isolated from immunosuppressed patients. This

unusual types of HPV in groups B1 and B2. Squamous

table was assembled with the help of Dr C. Wheeler.

667 while types -14, -17 and -20 (Table 22.3) account for a

PAPILLOMAVIRUSES

cervix, and in the next section there will be a discussion smaller number. While the viruses found in EV

of the pathogenesis of the genital HPV types. patients are not isolated from normal individuals, some types, such as -5 and -8, have been detected in squamous cell carcinomas in allograft recipients.

PATHOGENESIS Because the disease EV is rare, and with the isolation

of some of the EV-associated HPVs from transplant HPVs cause benign and malignant changes in epithelial patients, it suggests that these viruses must be circulat-

cells. It is the latter property that will be dealt with in ing in the community, infecting normal individuals

this section, since certain HPV types are the most perhaps without any associated clinical lesions.

important component of the aetiology of genital As discussed above, there are mucotropic HPVs

cancers and cervical cancer is one of the most common which mostly infect the genito-urinary tract. Common

causes of cancer-related death in women worldwide. isolates like HPV-6 and -11, which cause benign condyloma, can also infect the oral cavity, particularly the larynx. Infection of the larynx is rare, but usually

Oncogenic Potential of Papillomaviruses requires several episodes of surgery or laser treatment for removal of recurrent lesions. Frequent recurrences

Until recent epidemiological and laboratory-based may be due to the fact that only the visible lesions are

studies, most of the evidence for an oncogenic treated, although healthy-looking areas of mucosal

potential of HPVs came from research with animal tissue may harbour HPV genomes. The oral cavity is

papillomaviruses. Work in the 1930s showed that the also infected with other HPV types associated with

cottontail rabbit papillomavirus (CRPV) produced oral warts and hyperkeratosis.

benign tumours in this animal, its natural host, and Transmission is thought to occur from one epithelial

that these benign tumours in 25% of cases would surface to another in exfoliated cells containing

become malignant after 12 months. Benign tumours infectious virus, rather than free virus particles. There-

produced in domestic rabbits became malignant more fore, direct contact is the most eﬃcient way to transmit

frequently and within a shorter time. Also, application infection. Direct contact through sexual intercourse is

of hydrocarbons or tar produced in both animal the most important mode of transmission of genital

species a higher and more rapid malignant conversion. viruses. However, an infected mother may transmit

The viral DNA was detected in both the benign and virus to her neonate during delivery through the birth

the malignant lesions. These results suggested that the canal. This route of infection is thought to be a major

CRPV produced the benign lesion, but other factors, cause of larynx warts in babies and young children. It

genetic and environmental, may be necessary for is possible that other modes of transmission occur for

production of malignant disease. More recently, the genital viruses, since a number of studies have

oesophageal, intestinal and bladder papillomas pro- detected DNA in the genital and perianal regions of

duced by bovine papillomavirus type 4 (BPV 4) were young children. However, the route of transmission,

shown to become malignant when cattle were fed on a apart from those mentioned above, is unclear.

diet of bracken. In this case the BPV 4 DNA was Infection of the genital mucosa is common and

detected only in the benign lesion and was not involves hundreds of thousands of new cases each year,

detectable after malignant conversion. Recently a usually among sexually active individuals, with 18–30

rhesus monkey papillomavirus type 1 has been isolated year-olds having the highest incidence (cf. common

from a lymph node metastasis of a penile carcinoma. hand warts). The transmission rate was ﬁrst described

This virus is sexually transmitted and is associated with back in the 1970s when Oriel (1971) showed that 64%

both penile and cervical cancers. This animal virus may of partners of individuals with genital warts also

serve as a good, if expensive, model to investigate the developed warts. Subsequent studies have shown that

natural history of papillomavirus infections. up to 90% of male partners have shared common HPV

In humans, one-third of patients with EV develop types with their partner’s cervical isolates. Therefore,

squamous cell carcinoma, usually in sun-exposed transmission of HPV types is common in the relatively

areas. Over 30% of these lesions contain HPV DNA, stable setting of partners, although the frequency of

most commonly types -5 or -8. This suggests that, transmission during casual sex with diﬀerent partners

given the right environmental or genetic conditions, is still unclear.

benign lesions may develop into carcinoma with the Infection with certain of the genital HPV types

help of HPVs. Other evidence of a helper function increases the risk of malignant disease, especially of the

associated with malignant conversion concerns associated with malignant conversion concerns

It is the genital papillomaviruses that are responsible for the most common HPV-associated cancers and these types will be discussed in more detail in the following sections.

Genital Cancers Associated with HPVs The association between the genital cancers, especially

cervical cancer, is now known to be causal rather than casual, but not everyone infected with an oncogenic HPV type will develop cancer. In fact, it appears that people can be infected and have transitory disease, which spontaneously regresses, or perhaps have no obvious lesions resulting from infection. It is not clear why the outcome from infection can be so varied, but the immune response may play an important role, as well as other less well-described factors, such as genetic background and response of the epithelial cells to infection.

HPV Infection and the Normal Cervix It has been observed for some time that HPV DNA

could be detected in cervical cells from women with a normal cervix, as assessed by normal cytology, and no visible lesion upon colposcopic examination. What is not clear, however, is whether the cervix epithelium is really normal, or whether there are micro-lesions present which harbour the virus. So far no histologically normal cervix has been shown to harbour oncogenic HPV DNA. The rate of detection of HPV DNA in cervical cells varies dramatically, depending on the method used to detect the DNA, the age and demographics of the group studied. The most sensitive technique, the polymerase chain reaction (PCR; see section on Diagnosis, below), which is prone to cross- contamination, has recorded levels of 80% positivity in women with a normal cervix in cross-sectional studies. These results are high and were carried out on a very

small number of individuals; they have not been reproduced in studies using much larger numbers. However, HPV infection of the genital tract is common, although there is variation in isolation rate, depending on the age and lifestyle of the individuals studied. In studies of young women aged 18–25 years, up to 46% had detectable HPV DNA by PCR (Bauer et al. , 1991) in epithelial cells from a normal cervix. HPV types -16 and -18, which are commonly found in malignant disease, account for about one-third of the viruses detected. This infection rate has been observed in other populations of young sexually active women. While similar studies on large populations of males have not been carried out, it is clear from smaller studies that males are infected at similar rates, a ﬁnding that would be expected with a sexually transmitted disease. Therefore, a large number of young women may be infected with HPV types, which cause malignant disease. It is clear that while many individuals will develop transitory premalignant disease, only a small number will ever develop malignant disease, but recognising those at risk is not possible at present. HPV detection decreases in older women aged

4 40 years and is usually in the 5–10% range. While the level of infection is lower in older women, it has been shown that they are more likely to have underlying disease (see next section).

HPVs Infection and the Abnormal Cervix The most common genital lesion caused by HPV

infection is the benign genital wart (condylomata acuminata). HPV-6 and -11 are the predominant types associated with these lesions, which are benign and where the rare malignant conversion has only been documented in patients with an underlying immune deﬁciency. These warts are distributed throughout the female genital tract on the cervix, vaginal wall, vulva and perianal region. In males the lesions are found on the penis, scrotum and perianal region.

The premalignant lesions associated with HPV can occur on the same sites as described above for warts, although the cervix is the site where malignant conversion is most often observed. The premalignant lesions of the cervix are called intraepithelial neoplasia and are graded, according to the Bethesda system (Kurman et al., 1994), as low-grade squamous

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

Table 22.4 Genital HPVs and their associated risk of cancer Low risk

6, 11, 40, 42–45, 53–55, 57, 59, 61, 67, 71, 74, 82 High risk

669 intraepithelial lesions (LSILs) or high-grade squamous

PAPILLOMAVIRUSES

and out to local lymph nodes. HPV-16 is the most intraepithelial lesions (HSILs). HPV-6 and -11 are

common virus found in intraepithelial and malignant found in LSILs, while the oncogenic types, HPV-16

disease. It has been found in 70% of cases of HSILs in and -18, are found in all grades and in malignant

Germany and the UK and worldwide has been isolated disease. Table 22.4 gives a breakdown of the high- and

in 50–60% of cases of invasive cancer of the cervix. low-risk HPV types. The premalignant cervical lesions

HPV-6 and -11 have not been found in malignant occur almost entirely on the transformation zone, the

disease of the cervix, but have been isolated from local metaplastic zone between native squamous epithelium

invading lesions of the vulva, such as verrucous of the exocervix and the columnar epithelium of the

carcinoma. However, when the genomes of the viruses endocervical canal, and are white in appearance after

were sequenced, it was found that there were duplica- the addition of 5% acetic acid to the surface of the

tions in the long control region, which may be epithelium. Invasive cancer arises from these areas of

associated with the change in pathogenesis. The HPV HSILs and malignant cells migrate up into the uterus

types and associated lesions are shown in Table 22.5. Table 22.5 Genital human papillomavirus types and the site of associated lesions a

HPV type Associated lesion

Site HPV-6a–f

Site

HPV type

Associated lesion

Condylomata acuminata

Vulva

39 LSIL/HSIL

Cervix and penis

Malignant carcinoma

Penis

40 LSIL/HSIL

Cervix and penis

Shaft

42 LSIL/HSIL/VIN

Cervix and vulva

Prepuce

43 LSIL/HSIL

Cervix

Urethral meatus

44 LSIL/HSIL

Cervix

Perianal

45 LSIL/HSIL

Cervix

Larynx

Malignant carcinoma

LSIL/HSIL b Cervix

51 LSIL/HSIL

Cervix

VIN I–III c Vulva

Malignant carcinoma

Cervix HPV-11a, b Condylomata acuminata

PIN I–III d Penis

52 LSIL/HSIL

Vulva

Malignant carcinoma

Cervix

53 Normal cervix

55 Bowenoid papulosis Penis LSIL/HSIL

Larynx

Cervix PIN I–III

Cervix

56 LSIL/HSIL

Malignant carcinoma 16 Condylomata acuminata

Penis

Vulva, cervix and

57 Intraepithelial neoplasia Oral cavity, cervix

Cervix LSIL/HSIL

penis

58 LSIL/HSIL

Malignant carcinoma VIN I–III

Cervix

Vulva PIN I–III

Vulva

59 VIN

Vulva Bowenoid papulosis

Penis

61 VIN

Cervix Malignant carcinoma

Vulva and penis

66 LSIL/HSIL

Cervix, vulva and

Malignant carcinoma

67 Intraepithelial neoplasia Cervix 18 LSIL/HSIL

penis

Vulva Malignant carcinoma

Cervix and penis

Cervix 31 LSIL/HSIL

68 LSIL/HSIL

Malignant carcinoma Cervix Malignant carcinoma

Cervix

69 Intraepithelial neoplasia Cervix 30 LSIL/HSIL

Vulva 33 LSIL/HSIL

Cervix

70 Condyloma

Malignant carcinoma Cervix Malignant carcinoma

Cervix

71 VAIN e Vagina 34 LSIL/HSIL

Vagina 35 LSIL/HSIL

Cervix

74 VAIN

Cervix Malignant carcinoma

Cervix

82 HSIL

b Adapted from De Villiers (1989) and updated with the help of Dr C. Wheeler. c Low-grade squamous intraepithelial lesion and high-grade squamous intraepithelial lesion of the cervix. d Vulvar intraepithelial neoplasia. e Penile intraepithelial neoplasia. Vaginal intraepithelial neoplasia.

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

There is a diﬀerence in the state of the HPV-16 DNA papillomas. As the recurrence rate of laryngeal warts in premalignant and malignant lesions. In LSILs and

is high, this suggests that the virus is capable of HSILs the HPV DNA is free and unintegrated, while

persisting somewhere in the larynx, respiratory tract or in the majority of malignant cells the DNA is

oral cavity without producing recognisable lesions—an integrated. While integration is random within the

inapparent infection.

chromosomes, the cells, which acquire malignant Also, the viruses associated with the lesions in EV potential, contain the HPV genome integrated in the

patients are not normally found in lesions from E1 or E2 regions of the DNA, resulting in the retention

immunocompetent individuals, yet EV is such a rare of the expression of the E6 and E7 proteins, which

disease that these patients cannot circulate the viruses appear essential for the malignant phenotype. It is not

amongst themselves. It would seem that these viruses clear how important integration is for the development

are circulating in the normal population, causing of invasive cancer, since in a minority of cases the viral

inapparent infections. This is supported by the fact DNA is episomal in malignant cells, but continued

that viruses isolated from warts and SCC in immuno- expression of E6 and E7 appears necessary.

suppressed individuals, are often the same types Malignant disease of the penis while rare in

observed in EV patients.

developed countries is much more common in devel- In addition, during pregnancy genital warts can oping parts of the world. HPV-16 and -18 were

appear on the vulvar epithelium and then disappear detected in over 50% of penile cancers in Brazil

postpartum. It is not known if this is an hormonal (McCance et al., 1986), where in one area of the north-

eﬀect or due to pertubations in the immune response east of the country the incidence of penile cancer is 10

that may accompany pregnancy, or the result of times the frequency seen in Europe. Again, in

acquiring a recent infection from her partner. In malignant disease of the penis the viral DNA is

none of the above situations is there any direct integrated into the host cell chromosomes in the

evidence as to which cells harbour the virus. The majority of cases.

basal epithelial cells are the most likely site, although While HPV types have a role in the aetiology of

there is a considerable turnover of cells. However, not cervical cancer, there are certainly other factors

all cells in the basal epithelium have the same capacity involved that act with the virus to produce invasive

to divide, so the viral DNA may be sequestered in disease, since not everyone infected and exhibiting

quiescent basal cells which, when they subsequently premalignant lesions will develop cancer. It is esti-

divide, may activate replication and produce lesions. mated that up to 25% of women with LSILs will progress to HSILs if not treated, while most will regress over a 3 year period. However, in older women

DIAGNOSIS (440 years of age) infection and persistence are

associated with more serious underlying disease and Apart from the familiar hand and verruca warts found so they are a group to be closely monitored (McCance,

on the hands and feet, respectively, the clinical 1998). The co-factors involved have not been deli-

appearance of papillomavirus infections varies con- neated, although smoking and use of oral

siderably, from the scaly ﬂat lesions on cutaneous contraceptives (45 years) may be such components.

epithelium of individuals with EV to the aceto-white ﬂat lesions on the cervix. The reader is referred to speciﬁc papers for details of the clinical presentation of

Persistent Infections genital lesions (Walker et al., 1983). This section will deal with the laboratory diagnosis of HPV, in

Epidemiological evidence suggests that HPVs can particular the genital isolates. persist in squamous epithelium without producing clinically obvious lesions. Up to 50% of allograft recipients develop cutaneous warts within a year after

Culture Methods transplant, this proportion being high when compared with the incidence in age-matched controls. This

Although several eﬀorts have been made, no easily suggests that transplanted patients experience either

amenable cell type has been capable of supporting new infections or reactivation of persistent virus, the

replication, with production of infectious papilloma- latter being supported by the ﬁnding of HPV DNA

virus particles. The nude mice kidney capsule model, sequences in biopsies of normal areas of larynx from

or the SCID mouse subcutaneous system has been individuals who have had episodes of laryngeal

used to propagate some HPV types, but they cannot be used to propagate some HPV types, but they cannot be

Serological Methods Recently, with the advent of yeast and baculovirus

produced virus-like particles (VLPs), it is now possible to detect antibodies by an ELISA technique in the serum of patients infected with HPV. At present there is a limited number of HPV types that have been used in these assays, but the initial results suggest that antibodies are detected in approximately 50% of infected individuals, which is less sensitive than DNA-based detection methods. These assays have only been carried out for some of the genital isolates. The reasons for the low positivity rates in infected people are unclear, but it has to be remembered that the virus is confined to the stratified epithelium of the genital tract and few cells in a lesion support viral particle production. In addition, the mature viral particles are only produced in the outer layers of the epithelium, where the immune response is at its least effective. Therefore, serological assays may not be very sensitive for diagnosis or screening purposes. However, the serological data and the natural history of the virus suggest that either the virus is not very immunogenic, or possibly induces an inappropriate response.

Polymerase Chain Reaction (PCR) The most sensitive method for the detection of HPV

infection is by PCR. This is a powerful technique, which ampliﬁes a speciﬁc piece of DNA from a small amount of template. The advantages of this method are: (a) the extreme sensitivity; (b) the versatility, in that it can be used to detect more than one type of HPV when degenerate primers are used—in fact, it is possible to detect even unknown HPV types (ones that have not been cloned and sequenced); (c) it is possible to test large populations. The major disadvantage is that, because of the sensitivity of the method, it is possible to amplify contaminating sequences and so have false positives. This was a major problem in earlier studies but, now that this is recognised, investigators have been more careful and included strict controls.

Several sets of partially degenerative or degenerative primers have been used to detect HPV types in the

DNA extracted from lesions, and are directed to the L1 open reading frame. The ﬁrst described, MY09/ MY11 (Bauer et al., 1991), has been improved (PGMY09/PGMY11) and used with a ﬁlter containing

27 HPV types (Gravitt et al., 1998), meaning that the PCR products can be directly hybridised to the filter, allowing for a quick method to detect specific HPV types. Other sets, such as GP5 + /GP6 + (de Roda Husman et al., 1995) and SPF-10 (Kleter et al., 1998), have also been used successfully to detect multiple HPV types in clinical samples. The main difference between the sets of primers is that some detect certain HPV types better than others, but since these types are of low frequency in the populations studied, in reality there is little difference in the sensitivity of detection between the sets (van Doorn et al., 2002). None of the PCR sets have been licensed for use as diagnostic tools, although the Hybrid Capture Method described next has been approved for diagnostic use in the USA.

Hybrid Capture Method

This method is commercially available as a kit from Digene Diagnostics, MD, USA, and uses type-specific RNA probes to detect viral DNA in samples. This method is not as sensitive as PCR, but does not have the problems of false positives associated with PCR, since it does not rely on the PCR-type amplification of the signal. In practice, although not as sensitive as PCR, the method is sufficiently sensitive to detect HPV DNA and has been shown to be superior to one cytologic test in detecting HPV from lesions diagnosed as atypical squamous cells of unknown significance (ASCUS) or above. Cytological smears diagnosed as ASCUS are difficult to interpret, since after biopsy more serious disease may be detected. HPV typing has been shown to be helpful in these situations. This test detects 16 mucosal types, whereas PCR can theoretically detect all HPV types.

TREATMENT

Although in most cases warts are a cosmetic nuisance and will eventually disappear spontaneously, they are notoriously diﬃcult to treat. However, since premalignant lesions, especially on the cervix, may lead to malignant disease, treatment to eliminate disease is important. This section will concentrate on the treatment of genital areas, as others (Bunney, 1982) have dealt extensively with common hand and plantar warts.

PAPILLOMAVIRUSES

671

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

Podophyllin removed intact and the tissue can be used for pathological staging without having to biopsy the

Podophyllin is a resin mixture obtained from the roots lesion ﬁrst. In addition, the margins of the lesion are of Podophyllum (American mandrake) and is an

intact and so the extent of lesion removal can be irritant on cutaneous and mucous surfaces. It is an

assessed. Also it allows the lesion to be used for other antimitotic agent and should be used with care. It is

studies, such as HPV typing. painted carefully onto the surface of warts and should remain for no longer than 6 h and then be washed oﬀ. It is poorly adsorbed by cutaneous surfaces and so has

Surgery

a limited effect. Several treatments are required and the continual inflammation produced can lead to fibrosis

Curettage of common warts is not a common mode of of the areas treated without getting rid of the lesions.

treatment. In any case, not all warts are suitably sited Podophyllin is even less eﬀective in treatment of

for surgical removal. Furthermore, if all the abnormal plantar warts and should never be used for treatment

tissue is not moved, small islands of warts can recur of hand warts.

around the site of the initial lesion.

Cryotherapy Interferon Liquid nitrogen (71908C) and dry ice (solid carbon

Interferon has been used to treat recurrent laryngeal dioxide, 7508C) can be applied to warts to produce

warts and cervical neoplasias. In the former cases, local destruction of the lesion. Care should be taken to

tumour load was ﬁrst reduced by surgery or by CO 2 limit application to the lesion and not surrounding

laser, interferon then being given parenterally. areas, as this will lead to pain and blistering of the

Although recurrences were rare within 2 years after healthy area. Cryoprobes are used to apply these

starting the interferon course, it was necessary to cryogens to the cervix.

maintain patients on interferon to prevent new lesions. The expense of this regime and possible side-eﬀects associated with interferon administration provide

Electrodiathermy drawbacks to this method of treatment. Cervical premalignant lesions have also been treated with interferon, but variable results have been reported.

This is used in treating mucosal lesions, such as those on the cervix, to destroy the diseased tissue by heat.

Prevention by Vaccination Laser Evaporation

There is now a major thrust to develop a vaccine to try and protect against infection. The major capsid protein

The carbon dioxide laser has been used to treat lesions of papillomaviruses, L1 can be produced in yeast or on mucosal surfaces (cervix and vaginal wall) (Singer

insect cells and folds into an icosahedral structure, and Walker, 1985) as well as cutaneous lesions. The

called a virus-like particle (VLP), since it is similar in success rate of laser treatment of the cervix is very

structure to the complete infectious virion. In animal good, with cure rates of 85–90%. However, its eﬃcacy

studies, the antibodies raised to the VLPs are con- with cutaneous lesions has not as yet been assessed

formational and recognise the virion and can adequately. The diﬃculty with these lesions is knowing

neutralise infection. Vaccine trials have taken place how deep to vaporise to eliminate diseased tissue.

to immunise dogs against oral papillomavirus infection (Suzich et al., 1995). The dogs were immunised intramuscularly with VLPs of canine oral papilloma-

Loop Electrosurgical Excision Procedure (LEEP) virus and then challenged with live virus placed on scariﬁed areas of the oral cavity. Protection was 100%,

This is a relatively new procedure for the removal of indicating that it is possible to immunise against cervical lesions and makes use of a heated loop, which

infection with intramuscular inoculations. Phase I very precisely removes the complete lesion. One

trials have been carried out in humans using VLPs advantage over laser evaporation is that the lesion is

from HPV-6 and -11 and good antibody responses

673 were detected in all patients at all doses used. These

PAPILLOMAVIRUSES

Durst M, Gissmann L, Ikenberg H and zur Hausen H responses were to conformational epitopes on L1, as

(1983) A papillomavirus DNA from a cervical carcinoma was the case in the study using dogs.

and its prevalence in cancer biopsy samples from diﬀerent geographic regions. Proc Nat Acad Sci USA, 80, 3812–

At the time of writing there are Phase II trials

investigating the response to VLPs of the virus types - Dyson N, Howley PM, Munger K and Harlow E (1989) The

16 and -18. There are also plans for Phase III trials to human papillomavirus 16 E7 oncoprotein is able to bind to test the eﬃcacy of these vaccines. At the time of writing

the retinoblastoma gene product. Science, 243, 934–937. it is not clear what will be considered a successful

Frattini M, Lim H and Laimins LA (1996) In vitro synthesis outcome. Will the end-point be protection against of oncogenic human papillomaviruses requires episomal templates for diﬀerentiation-dependent late expression. infection or disease? Normally it is protection against

Proc Nat Acad Sci USA , 91, 3062–3067. disease that is the end-point, but since it may take

Frattini MG and Laimins LA (1994) Binding of the human years for HPV-associated lesions to be obvious,

papillomavirus E1 origin-recognition protein is regulated protection against infection would be a faster way to

through complex formation with the E2 enhancer-binding determine eﬃcacy. If absence of disease is the end- protein. Proc Natl Acad Sci USA, 91, 12398–12402. Gravitt PE, Peyton CL, Apple RJ and Wheeler CM (1998) point, then it will need to be a long trial (5 years or

Genotyping of 27 human papillomavirus types by using L1 more) and will need a large number of participants

consensus PCR products by a single-hybridization, reverse (perhaps 20 000). No matter, these are exciting times

line blot detection method. J Clin Microbiol, 36, 3020–3027. for HPV vaccine biology, and other possible vaccines

Kirnbauer R, Booy F, Cheng N et al. (1992) Papillomavirus are being developed and may be in trial before the next L1 major capsid protein self-assembles into virus-like particles that are highly immunogenic. Proc Natl Acad Sci edition of this book.

USA , 89, 12180–12184. Kleter B, van Doorn LJ, ter Schegget J et al. (1998) Novel short-fragment PCR assay for highly sensitive broad- spectrum detection of anogenital human papillomaviruses.

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23 Human Polyomaviruses

Kristina Do¨rries

Julius-Maximilians University, Wu¨rzburg, Germany

CLASSIFICATION AND DETECTION VP1, VP2 and VP3. The shell surrounds a DNA molecule that is stabilised by cellular histones in

The human polyomaviruses were previously grouped chromatin structure. From studies on SV40 and in the large group Papovaviridae with papilloma-

Murine polyomavirus it has been established that 360 viruses, polyomaviruses and SV40, the so-called

molecules of the major capsid protein VP1 are vacuolising agent. However, molecular studies showed

associated with approximately 30–60 molecules of that Simian virus 40 (SV40) is closely related to the

each of the minor capsid proteins, VP2 and VP3. The other polyomaviruses and, with growing knowledge on

icosahedron is composed of 72 pentamers. Each the genomic structure and function of proteins, the

consists of ﬁve VP1 molecules and one molecule of previous subfamily is now classiﬁed as independent

VP2 or VP3. VP1 is the most related capsid protein family, Polyomaviridae. The classical human viruses

among the three primate viruses. This complicates the are now grouped as Polyomavirus hominis (huPyV),

development of highly speciﬁc serologic assays, which type 1 (BKV) and type 2 (JCV). For both viruses,

are important for addressing a possible involvement of diﬀerent genotypes have been described. The small

polyomaviruses in cancer, as well as spread of SV40 in non-enveloped viruses of about 40 nm in diameter

the human population.

contain single genomes of covalently closed super- Virus preparations contain at least two kinds of helical double-stranded DNA. Although Murine

particles. In vitro passage of polyomaviruses at high polyomavirus was detected almost 50 years ago, the

multiplicity is accompanied by the generation of existence of primate polyomaviruses was not realised

considerable amounts of empty capsids. In addition, until the 1960s, with the detection of SV40 in monkeys.

defective viral genomes containing deletions, duplica-

10 years later, JCV and BKV were described. JCV was tions and rearrangements of viral genetic information isolated from brain tissue of a patient with progressive

can be encapsidated, if they are within the appropriate multifocal leukoencephalopathy (PML), employing

size limits. In vivo, the amount of empty shells is human fetal brain spongioblast cultures. BKV grew

considerably lower, indicating highly eﬀective virus in cell cultures after inoculation with urine from a

growth under natural conditions. Complete virus renal transplant recipient (Padgett and Walker, 1976;

particles form a band at a density of 1.34 g/ml in Zu Rhein, 1969).

CsCl 2 equilibrium density gradients, whereas empty capsids have a density of about 1.29 g/ml (Figure 23.1). Although the structure of the huPyV capsid has not been completely established, it has been reported that VIRION STRUCTURE AND COMPOSITION

recombinant JCV VP1 self-assembles into pentameric capsomeres and, under appropriate conditions, these

Polyomaviruses are non-enveloped viruses with icosa- molecules will further assemble into virus-like empty hedral capsids containing three virus-encoded proteins,

capsids (Chang et al., 1997). Calcium ions are required

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

Figure 23.1 JCV purification out of PML brain tissue. Middle: Optical profile of the centrifugation with localisation of virus particles in two bands; intact virus particles at a density of 1.345 g/ml CsCl 2 . Left: Restriction endonuclease cleavage of DNA extracted from the major peak of the gradient followed by Southern blotting and JCV-specific radioactive hybridisation. 1–4 cleavage with single cut enzymes EcoRI, BamHI, HpaI, PstI generating linearised form III DNA of about 5 kb in length; 5,6 cleavage with single and more cut enzymes in combination (BamHI/PvuII, BamHI/HindIII, respectively) generating DNA fragments 5: A–D and 6: A–E. Right: Electron micrograph of negatively stained JCV icosahedral virus particles of the small virus peak representing empty shells without encapsidated viral DNA

for capsid stability and disulphide bonds may exist early protein large T antigen (TAg) early post- between capsid proteins, as reducing agents are

infection, TAg initiates replication of viral DNA. required to disassemble virus particles. The human

Shortly after the onset of DNA replication, virus polyomaviruses are able to haemagglutinate erythro-

multiplication enters the late phase, which includes cytes at high virus concentrations. Each virus also has

expression of late mRNAs. Translation of the late distinct antigenic epitopes and can be distinguished by

regulatory agnoprotein and the production of viral neutralisation and haemagglutination-inhibition assays

capsid proteins are followed by virion assembly. (Pass and Shah, 1982).

Regulated by complex interactions of viral promoter elements with cellular factors and the viral regulatory proteins, late genes are expressed eﬃciently only after DNA replication. In contrast, early genes continue to

be expressed at late stages of infection, then serving as viral transcription factors. The functions of TAg and Polyomavirus expression is essentially divided into

VIRUS LIFE CYCLE

agnoprotein are controlled by consecutive phosphor- three major phases. After expression of the regulatory

ylation events. However, the tropism of huPyV is ylation events. However, the tropism of huPyV is

In comparison to other polyomaviruses, JCV has a long lytic life cycle with an early transcription and DNA replication phase lasting about 5 days followed by continuing initiation of late RNAs for 15–20 days. The virus has a stringent cell specificity and replicates efficiently in vitro only in primary human fetal glial cell cultures, rich in spongioblasts, a precursor cell of oligodendrocytes (Walker and Padgett, 1983). In such other cell lines as embryonic kidney, amnion or urine- derived epithelium virus growth is rather limited. In contrast, BKV has broader cell specificity and can be grown in a wide range of human cell types.

Molecular Structure of the Genome The genomic structure is closely related among the

primate polyomaviruses. The supercoiled, circular, double-stranded DNA of the human viruses is about 5100 bp in length. The genome is divided into two regions, encoding multiple overlapping genes. Each DNA strand carries about half of the genetic information. Early and late mRNAs are synthesised bidirectionally from opposite strands of the genome. Protein coding sequences consist of open reading frames for the early and late proteins. The non-coding region directs the activity and speciﬁcity of virus multiplication. It is divided into two regulatory segments with a single origin of DNA replication (ORI) and the transcriptional control elements (TCR) within the promoter region (Do¨rries, 1997; Kim et al., 2001; Raj and Khalili, 1995) (Figure 23.2).

The coding sequences exhibit high DNA sequence homology. Homology between the human viruses is greater in all proteins than between JCV and SV40 (69%). JCV DNA shares 83 and 59% amino acid homology with BKV in the case of large TAg and agnogene, respectively. The rates are even higher in functionally active regions of the virus genes. The early region codes for the tumour or TAgs. Both human

polyomaviruses encode two major proteins, small t (tAg) and large TAg, based on size. The multi- functional TAg directly controls the virus life cycle and interacts with key cellular regulatory circuits. The huPyV proteins have not been studied to the same extent as those of SV40, but extensive sequence homology points to similar functional activities. This includes eﬀects on nuclear localisation, viral DNA replication by direct DNA binding, helicase activities and binding to DNA polymerase-a, which initiates DNA replication. Interaction with cellular tumour suppressor proteins is associated with cellular transformation and TAg as a transcription factor is known to be crucial for the regulation of early and late gene transcription. Many of the activities depend on the TAg viral chaperone domain, which interacts with cellular chaperones to orchestrate functions that require the rearrangement of multiprotein complexes (Sullivan and Pipas, 2002).

The tumour antigens are generated from a common pre-mRNA molecule by one alternative splicing event leading to identical N-termini and different C- terminal DNA sequences. Recently, three additional early virus proteins, T’135, T’136, and T’165 were characterised in JCV-infected cells. All T proteins use the first alternative splice donor site leading to the same 132 N-terminal amino acids (AA). For T’ proteins, a second splice donor site is combined with alternate acceptor sites. Whereas T’165 shares the C-terminus with TAg, T’135 and T’136 have unique ends encoded by an alternative reading frame. Expression of the proteins is modulated during the virus life cycle and appears to be related to TAg- mediated DNA replication and transcriptional control. T’ proteins may utilise the shared tumour suppressor and chaperone binding domains to enhance DNA replication by differential interaction with pRB and related proteins or the transcription factor Tst-1. Significant differences in the splicing patterns of JCV-infected and JCV-transformed cells and increased expression of the ‘17 kDa’ species suggest correlation with the transforming activity of JCV. Additionally, it has been proposed that the apoptotic function of TAg might be negatively influenced by T’ proteins (Kim et al., 2001).

The late region of the viral genome encodes two minor (VP2 and VP3) and a major (VP1) capsid protein. Late mRNAs are generated from a common precursor by alternative splicing. The sequences of the minor proteins VP2 and VP3 overlap. VP2 contains the entire VP3 sequence at its C-terminus and an additional sequence of approximately 400 AAs at its N-terminus. In contrast, the major protein VP1 is

HUMAN POLYOMAVIRUSES

677

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

Figure 23.2 Circular map of the human polyomavirus JC subtype GS/B genome. Coding DNA segments are represented by shaded arrows. Numbers designate the ﬁrst nucleotide of the start and stop codons for early (large T Antigen, small t Antigen, T prime proteins T’ 165, T’ 135, T’ 136) and late proteins (agnogene, virus capsid proteins VP1, VP2, VP3). Position of splice donor and acceptor sites are indicated by nucleotide numbers. The position of the unique BamHI and EcoRI restriction sites are indicated on the inner circle

generated by an alternative reading frame. The three interaction with cytoskeletal proteins, and appears to primate polyomaviruses all encode the non-structural

deregulate cell proliferation by association with the regulatory agnoprotein in an open reading frame in the

tumour suppressor p53. Although studies on agnopro- late leader region. The sequences are homologous in

tein function are only at the beginning, the protein is the ﬁrst two-thirds of the amino-terminus but diver-

obviously a central regulator of the late virus life cycle gent in the carboxy-terminal one-third. The protein is

(Table 23.1).

probably not necessary for virus multiplication but The non-coding part of the genome is framed by the rather takes part in the orchestration of eﬃcient

start codons for early and late genes. The ORI is progeny virus production. It is produced late in the

located between the TATA box and the initiation virus lytic cycle and physically and functionally

codon for the early genes. This segment includes the interacts with the early key regulator TAg and the

conserved binding sites for large TAg. Corresponding cellular transcription factor YB-1. Recently, it became

to similar DNA replication strategies, the polyoma- obvious that agnoprotein plays an important role in

virus ORI DNA segment is highly conserved in the viral life cycle by modulating viral gene transcrip-

sequence and structure. In contrast, the promoter tion and DNA replication. Likewise, it is suspected to

region to the late side of the control region reveals

be involved in the morphogenesis of virus particles, the extensive diﬀerences. Heterogeneity of structure and localisation of the major capsid protein to the nucleus,

sequence of individual transcription factor binding may regulate intracellular vesicular transport by

sites is reflected in the divergent cell type specificity and sites is reflected in the divergent cell type specificity and

Control of Viral Gene Expression DNA Replication

Regulatory mechanisms leading to DNA replication are closely related among the primate polyomaviruses. This is reflected in an ORI that is constructed by protein binding elements with comparable sequence and spacing requirements. Bidirectional replication takes place in the presence of the core ORI and TAg, proceeding from the ORI elements with the TAg binding sites terminating at a site about 1808 from the initiation site. The features shared by all ORI regions are an inverted repeat on the early side, a GC-rich palindrome in the centre and an AT sequence on the late side of ORI. All core ORIs contain these elements and replication studies on the human polyomaviruses have confirmed that, besides TAg binding, site II and the inverted repeat are essential for DNA replication. Whereas the presence of flanking sequences stimulates DNA replication, the TCR does not affect DNA replication activity directly. Interestingly, the efficiency of DNA replication directed from the JCV ORI is substantially lower than from the SV40 and BKV ORIs. TAg produced in each of the viruses considerably varies in its ability to support replication from the homologous or heterogeneous origins. This became clear as BKV TAg was found to activate replication from the JCV ORI, but JCV TAg failed to drive replication from the heterogeneous BKV ORI. It is likely that

diﬀerences of ORI sequences or spacing among the primate polyomaviruses are responsible for these observations (Kim et al., 2001) (Figure 23.3).

Transcriptional Expression The transcriptional control region (TCR) of the

human polyomaviruses is composed of a great number of different regulatory protein-binding motifs. Promo- ter activity is mediated bidirectionally, and often independently from the binding motifs, by multiple interactions of transcription factors and associated cellular and viral proteins stimulating basal, cell type- specific and, in response of external stimuli, induced functions of the viral promoter (Raj and Khalili, 1995; Sweet et al., 2002). The JCV early (JCVE) and late (JCVL) promoters have been intensively examined in recent years, and it can be assumed that the mechanisms leading to huPyV expression are highly related. For better understanding of the complicated regulatory pathways controlling huPyV growth and pathogenicity, the function of the JCV promoter is more closely examined. The JCV control region has been artificially dissected in the ORI domain and following four TCR subdomains (A–D), in each of which a clustering of early and late interactive promoter sites is observed (Figure 23.4).

Outside of the TCR domain, on the early site of ORI, a binding sequence is located for the potent transcriptional enhancer nuclear factor kB (NF-kB). The site bidirectionally increases transcription from the late and to a lesser extent from the early promoter in glial cells. NF-kB is constitutively expressed in B lymphocytes, one of the sites of JCV persistence. NF- k

B can be retained in the cytoplasm by an inhibitor kB protein. This binding can be released through stimulation by a number of agents as tumour promoters, such as phorbol–myristate–acetate or inﬂammatory cytokines. It results in the transport of NF-kB to the nucleus, where the binding to the consensus DNA sites exerts activity. Upon PMA treatment, both JCV promoters are responsive to NF-kB induction, leading to increased JCV activity. In addition, tumour necrosis factor a (TNFa) is able to enhance binding of NF-kB to the JCV kB site. This stimulated the idea that activation of the transcriptional control in vivo is dependent on the expression of immunological active factors involved in immunologically regulated signalling pathways to modulate JCV expression.

It had been shown that interference with kB function in late gene expression decreased activity but did not

HUMAN POLYOMAVIRUSES

679 Table 23.1 Human polyomavirus early and late proteins

Protein BKV

JCV

Size (aa) Mol wt (kDa) Size (aa) Mol wt (kDa) Early

Late Agno

a Molecular weight varies considerably between laboratories. aa, amino acids; NR, not reported.

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

Figure 23.3 Alignment of the DNA sequence encompassing the origin of DNA replication in the non-coding genomic region of the primate polyomaviruses SV40, BKV and JCV. All sequences are given in the sense of the early coding strand. TAg consensus pentanucleotide recognition sites common to all virus strains are in boxes

abolish it. This suggested that additional TCR ele- Although these cytokines may use diﬀerent signal ments might confer inducibility to the JCVL promoter.

transduction pathways for protein activation, the At present, it is thought that cross-communication

nuclear milieu will in consequence contain the factors between motifs is mediated by a 40 kDa protein

leading to expression by interaction with a promoter complex distinct from the classical NF-kB subunits.

containing the respective contact sites. In the presence of classical NF-kB binding proteins,

Most JCV TCR subtypes contain SP1 binding site this complex may not eﬀectively interact with the kB

one (SP1-I), just upstream from the TATA box. It sequence. However, if the NF-kB activators are not

appears to activate early promoter activity. SP1-II and expressed, the 40 kDa protein may bind and down-

III are located downstream from the TATA box. SP1- regulate JCVL gene transcription. This may come into

II is important for TAg-mediated transactivation of the eﬀect during the persistent state of infection.

early promoter, but is not involved in the regulation of Additionally, the GRS motif within ORI might

basal expression. Interestingly, SP1 associates with interact with the kB motif. GRS is similarly inducible

Pur a and regulates myelin basic protein expression by PMA and inﬂammatory cytokines. The responsive

during brain development, whereas TAg downregu- region interacts with the protein GBP-i, which is

lates its transcription by interacting with Pur a in an induced in a wide range of cell types; thus, it could play

animal model; thus, a close association of these

a role in mediating JCV activation at all suspected sites proteins with SP-1 can be assumed (Kim et al., 2001). of persistence. Comparable to the NF-kB-class of

Domain C contains the JCV early minimal core proteins, the GBP-i complexes probably represent a

promoter (MCP), which is constituted by the TATA combinatorial assembly of various protein species,

box region and the immediately adjacent binding site which is changed upon induction. Duality of function

for the transcription factor Tst-1/SCIP/Oct-6, a could involve the basal transcriptional machinery and

member of the POU-domain protein family. In glial other transcription proteins associated with late

cells, Tst-1 was found to be one of the permanently promoter activity. One potential factor is transforming

produced cell type selective transcriptional regulators. growth factor b (TGFb), acting through the GRS and

Both JCV promoter orientations are stimulated by the NF-1 sites. In such a model, the status of the viral

DNA binding of Tst-1 in glial cells to sequence motifs promoter could be modulated through the GRS and

involving the TATA box and overlapping in part with variable interaction of cytokine-induced proteins.

adjacent OP-1 and YB-1 binding sites.

HUMAN POLYOMAVIRUSES

Figure 23.4 JCV transcriptional control region with most prominent protein binding sites and respective interaction of cellular and viral transcription factors. Transcriptional control region (TCR) structure of prototype JCV Mad-1 is shown between the start codons (arrows) for early and late genes (agnogene). ORI origin of DNA replication; TA TATA box; TR tandemly repeated promoter elements; similar shading represents identical DNA sequences; A–D, dissected promoter domains; MCP, minimal core promoter; LCE, lytic control element. Binding sites and the respective proteins are indicated below the promoter domains. Filled circles indicate interaction of transcription factors. TAT, TAR HIV-1-related transactivation domains. Arrows indicate interaction of inducing agents with the respective proteins. V, position of SP1 binding site in JCV TCR type II genomes

Like most POU proteins, Tst-1/SCIP/Oct-6 is an downstream of the Tst-1 binding site at the poly(dA) intrinsically weak transcriptional regulator. To com-

stretch, contacts with an overlapping pentanucleotide pensate for this weakness, Tst-1 has to rely on viral or

motif (penta). Recently, another cellular nucleic acid glial speciﬁc co-activators. TAg has been identiﬁed as

binding protein Cnbps has been identiﬁed that nega- the viral co-activator that stimulates the function of

tively regulates the JCV early promoter, and TAg Tst-1 synergistically by direct interaction. The modular

binding protein p53 is discussed as a transcriptional synergism of the two proteins does not require DNA

silencer. The OP-1 motif also interacts with the binding of JCV TAg in presence of a functional

adjacent NF-1 site. OP-1 and NF-1 form a composite binding site of Tst-1. In addition, TAg can activate the

element that increases JCV early activity and reduces high mobility group proteins HMG-I/Y as cellular co-

JCV late activity (Kim et al., 2001; Raj and Khalili, activators. Although not a transcriptional regulator by

itself, HMG is believed to serve as a promoter-speciﬁc The penta motif also diﬀerentially binds members accessory factor, modifying the transcriptional func-

of the lytic control element binding protein family, tion. The JCV AT-rich region with the Tst-1 site

LCP-1, recognising alternative DNA structures. This provides a binding site for HMG proteins, and HMG

factor bears remarkable similarity to Pur a and binding substantially stimulates Tst-1 binding to the

inﬂuences early and late promoter functions. There- Tst-1-responsive element.

fore, the region is designated the JCV lytic control Although Tst-1 and HMG are able to synergistically

element (LCE). Subsequent studies have demon- activate JCV gene expression, the TST-1 site alone is

strated that this element interacts with Pur a and not enough to mediate eﬃcient cooperativity. It is

YB-1. YB-1 is a member of a gene family, originally assumed that additional cross-talk occurs with the glial

isolated from a human B cell expression library, cell-speciﬁc late transcriptional silencer OP-1, which,

which is responsive to a wide variety of stimuli,

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

including stress signals drug and interleukin-2 treat- The B domain in the early orientation positively ment in T cells. The essential contact site is located in

contributes to expression in glial cells and is also the C/T-rich sequence on the early strand of the LCE

important for transcriptional activation of JCVL without aﬀecting the adjacent NF-1 binding site. YB-

genes. The central motif responds to glial factor 1

1 binds to double-stranded and single-stranded DNA (GF-1) and appears to be part of the human SmbP-2 and activates transcription from both the early and

factor (Kim et al., 2001). GF-1 protein expression is late viral promoters. The function is attributed to

most abundant in brain tissue. Interestingly, the both its interactions with DNA and its ability to

amount of GF-1 is higher in kidney cells than in interact with other proteins.

other cell types. Therefore it is suggested that the level The single-stranded DNA- and RNA-binding pro-

of GF-1 in kidney cells could be responsible for the tein Pur a binds to penta on the late LCE strand.

ability of JCV to replicate in urogenital tissue. Binding Cross-communication with the proteins on the LCE

to the GF-1 site stimulates transcription from the mediates reduced or increased binding to YB-1,

JCVL promoter and to a lesser degree from the JCVE thereby inﬂuencing early activation. Although TAg

promoter.

has no binding capacities to LCE sequences, close As mentioned above, pronounced high-aﬃnity NF-1 association of YB-1, Pur a and TAg and the complex-

binding sites, NF-1 A/B, are located just upstream ity of binding events suggests that the interplay is

from the A/T rich region. Three more NF-1 sites, C, D important for a broad spectrum of diﬀerent functions

and E, are found in domain A and further into the late during the JCV life cycle. The model of Pur a and YB-

region. Closely associated are three overlapping bind-

1 interaction in TAg-mediated transition of JCV ing sites for the nuclear factor Jun. A consensus early-to-late promoter activity is based on the follow-

sequence similar to that of activation transcription ing ﬁndings: Pur a increases early promoter activity

factor ATF or cyclic AMP-responsive element (CRE) with minor eﬀects on the late sequences; TAg

is localised on the latter half of NF-1 site A/B. A increases late activity considerably; and YB-1 elevates

sequence with partial homology to ATF/CRE sites basal activity on the early and late promoter, with a

overlaps NF-1 site E. NF-1 site D coincides with a site higher rate in the late phase. Increasing concentrations

resembling the sequence for the activator protein AP-1. of TAg results in a gradual decrease in Pur a early

Interaction at the binding sites involves NF-1 or NF-1- enhancement.

like factors, ATF/CRE-related factors and Jun-related In contrast to early expression, cooperative action of

(AP-1-like) factors. Isolated binding sites do not Pur a and TAg leads to interference of Pur a with the

mediate activity, suggesting an essential role of the TAg late stimulatory eﬀect, thus suggesting that Pur a

surrounding binding sites and interactions of related and TAg each exert antagonistic eﬀects on the other’s

proteins for the activation process. transcriptional activity. The mechanism by which the

It was reported that NF-1 sites A/B are involved in two regulators inﬂuence each other’s activities is not

basal as well as in glial cell-speciﬁc modulation. This yet clear; however, TAg may indirectly destabilise the

discrepancy was recently explained by the detection of association of Pur a with the LCE. In this respect, TAg

an activating glial cell-speciﬁc NF-1 form. It became might cause dissociation of Pur a from the LCE by

clear that the level of transcriptional activity and increasing the rate of YB-1 binding.

speciﬁcity of the NF-1 sites is due to the homodimeric– From these ﬁndings it was proposed that at the

heterodimeric nature of diﬀerent NF-1 molecules, and initial phase of infection strong binding of Pur a to the

to the combinatorial interaction of NF-1 with adjacent LCE preferentially stimulates transcription of the early

proteins (Monaco et al., 2001). Apart from the genome. As the lytic cycle proceeds, the composition of

prominent activity conveyed by NF-1 onto the DNA–protein complexes in the LCE is altered. The

JCVE, the sites are additionally involved in JCVL increasing level of TAg facilitates YB-1-mediated

cell-speciﬁc activation by TAg transactivation. BAG-1, dissociation of Pur a from the pentanucleotide repeat

a novel factor, interacts with anti-apoptotic Bcl-2 by stabilising the association of YB-1 with DNA.

protein. Binding to the NF-1 site activates the JCVE Removal of Pur a from the LCE results in a

and JCVL promoters. As BAG-1 is ubiquitously substantial decrease in early promoter activity. At the

expressed, this might be an important regulator in same time, YB-1-associated Pur a release of the late

persistence (Devireddy et al., 2000). promoter allows TAg to enhance the expression during

Cell-speciﬁc activity in glial cells include the cyclic the late phase of infection. Thus, it appears likely that

AMP (cAMP)-responsive element (CRE) overlapping Pur a, YB-1 and TAg are involved in the transition

with the AP1 site. The second messenger cAMP and from early to late gene expression.

forskolin substantially increase JCVE expression.

683 Activity is mediated by a CRE-binding protein

HUMAN POLYOMAVIRUSES

with immunomodulators has not yet been clariﬁed (CREB) and is not inﬂuenced by the NF-1 site.

(Sweet et al., 2002).

However, this does not exclude the possibility that the binding of other proteins to CRE may modulate expression without directly interacting with JCVE sites. Additionally, a glial cell-speciﬁc modiﬁcation of

Heterologous Transactivation of Virus CREB might be responsible for activation. Thus, it is

Transcription

likely that a signiﬁcant contribution to the enhanced glial cell-speciﬁc expression of JCV comes from

Additional to activating eﬀects of cellular transcription inducible transcription factors interacting with

factors, viruses are considered to inﬂuence JCV unique motifs on the JCV promoter. Importantly,

expression through the direct or indirect interaction the mechanisms of induction for the factors are

of a heterogeneous gene product. In general, transac- through diﬀerent signal transduction pathways. This

tivation can occur at any step of protein synthesis, combination allows a highly ﬂexible JCV transcrip-

beginning with the initiation of transcription and tional response to a large number of environmental

ending with posttranslational modiﬁcations. In the signals.

case of JCV, promoter activity and DNA replication At present, in domain A two potential promoter

were aﬀected.

elements have been identiﬁed, a transactivator element The question of whether HIV-1 may transactivate associated to HIV-1 TAR homologous sequences and

JCV is discussed, since it has become clear that PML is Tst-1 binding sites. The D domain is located close to

one of the most prevalent opportunistic infections in the start codon of the late proteins in the leader of the

AIDS patients. The HIV-1-encoded transregulatory late RNAs. It spans binding sites for cellular proteins

protein Tat has been found to be a potent activator of NF-1, c-Jun, YB-1 and a NF-kB-responsive region

the JCVL promoter. Tat is a transcriptional activator (Raj et al., 1995). YB-1 exhibits the ability to modulate

and an essential component for the establishment of a basal and activated levels of transcription of JCVL

productive HIV-1 infection. The transacting respon- through its inﬂuence on p65 on the NF-kB site. This

sive region (TAR) mediates activation of the HIV-1 points to a potential role for YB-1 as a transcriptional

promoter. In the JCV promoter, at least two respon- co-activator for regulation from the NF-kB site. The

sive elements, the TAT element in the ORI domain YB-1 site on the B domain does not account for such

and the TAR element, have been identiﬁed. Transacti- pronounced eﬀects, thus the D domain probably

vation occurs in vitro at the level of transcription by represents the optimal site for interaction of YB-1

HIV-Tat induction of the JCVL promoter. Corre- and NF-kB/rel subunits.

spondingly, BKV TAg is able to transactivate the NF-kB/rel subunits modulate JCV late promoter

HIV-1 long terminal repeat (LTR). Although HIV-1 activity from the D domain, revealing an extensive

expression in oligodendrocytes is regularly low, Tat duality of NF-kB/rel-mediated eﬀects. The subunits

protein appears to be secreted by the HIV-1-infected appear to function mutually antagonistically in terms

cell. Localised to the JCV-infected oligodendrocyte, it of their transcriptional activity from either the D

could induce transcription on Tat-responsive genes. domain or the NF-kB site. Basal uninduced tran-

Recently, co-infection with another retrovirus, HTLV- scription appears to be positively regulated by p50/

1, in PML tissue raised the question of whether

52 from the D domain, whereas induced transcrip- HTLV-1 could activate JCV expression. It was found tion is positively regulated by p65 from the NF-kB

that the JCV promoter is modulated by interaction site. At present, it appears likely that the interplay

with the regulatory protein Tax. Thus, a transactivat- between NF-kB subunits and the D domain is

ing eﬀect comparable to that of HIV-1 Tat protein is dependent on direct and speciﬁc interaction of YB-

conceivable.

1 and the NF-kB/rel subunits, inﬂuencing each Transactivation is also postulated for herpes viruses. other’s binding capacity to their respective target

Human cytomegalovirus (CMV) is highly prevalent in DNAs. Since each of the NF-kB dimers can

men. CMV infection is often activated in immuno- potentially interact with the NF-kB site, multiple

impaired patients, and common target tissues are levels of regulation can be mediated by the complex

kidney, lung, the CNS and lymphoid organs. Mole- interactions of NF-kB/rel subunits. This provides an

cular interaction of CMV with JCV is mediated in vitro exquisite control mechanism over constitutive and

by the HCMV immediate-early transactivator 2 (IE2). induced NF-kB activity. However, the importance of

Stimulation of the JCVE promoter by IE2 leads to the motif in the viral life cycle and in association

increased JCV DNA replication. Similarly, it was increased JCV DNA replication. Similarly, it was

Although co-localisation of HCMV and the human polyomaviruses appears to be possible, neither in AIDS patients nor after kidney transplantation with

a high incidence of CMV infection has a correlation of polyomavirus and CMV viruria been observed. Lack of interaction among CMV and the human polyomaviruses was confirmed by treatment of haemorrhagic cystitis with aciclovir. Although CMV replication decreased, no influence on BKV-associated HC was reported. In contrast, aciclovir therapy was beneficial in single PML cases. Therefore, at present it cannot be decided whether transactivation of CMV contributes to the pathogenesis of polyomavirus-associated disease.

Human herpesvirus 6 (HHV6) infection was co- localised with JCV in oligodendrocytes of PML lesions by in situ PCR. HHV6 has a high prevalence in the adult population, establishing persistent infection in the brain, urogenital tract, lung, liver and peripheral blood cells. HHV6 activation often occurs after transplantation. Obviously, huPyV and HHV6 have

a wide range of target organs in common. Due to putative transactivation mechanisms of other herpesviruses, a comparable interaction of HHV6 with the human polyomaviruses is thinkable.

Although the mechanisms of huPyV transactivation by heterogeneous viruses in vivo are at present not understood, the increasing number of HIV-1 patients with active polyomavirus infections argues for a potential role of concomitantly infecting viruses for the activation of polyomavirus infections in men.

Genomic Heterogeneity of Viral Subtypes In early studies, JCV DNA populations from PML

brain tissue exhibited a remarkable genomic homogeneity within one patient. In contrast, analysis of JCV isolates from different individuals revealed that a large number of JCV subtypes exist. Comparable variations in the BKV and the SV40 genome pointed to a general role of genomic heterogeneity among the polyomaviruses (Do¨rries, 1997; Knowles, 2001). Throughout JCV genomes, numerous single-base changes have been observed and the TCRs exhibit extensive structural differences. Single base mutations in coding genes do not affect the reading frames and most are silent, having no effect on the amino acid composition of the proteins. In contrast to BKV, JCV DNA does not exhibit sufficient sequence variation to generate different serotypes. Therefore genotypes are exclusively

defined by their DNA sequence. The biological significance of the changes remains uncertain, as the role of protein alterations for the viability or cell specificity of viral subtypes is not yet clarified (Pfister et al ., 2001; Sala et al., 2001). Among all viral genes, VP1 shows the greatest degree of variation, whereas the agnogene is the most conserved part. The clustering of mutations on the terminal end of VP1 and TAg proteins provides the tool for V-T subtyping of JCV genotypes in different regions of the world. It is used for virus transmission studies and as a marker for early population migration.

At present, major V-T genotypes are assigned to diﬀerent geographic regions by creation of a phylogenetic tree from isolates all over the world. Three separate JCV genotypes 1 were found in individuals of European origin. Type 2, the so-called European/ Asian type, was traced in a wide region extending from Europe to western and eastern Asia. Additional types (3, 6) were dominantly found in African states and in Afro-Americans. Type 7 is located in the south-east Asia and southern China populations. Although prevalent subtypes vary considerably in the Asian region, most territories are dominated by type 2A, which is closely related to the Japanese types Cy and My. Type 8 is a related JCV clade in the South Paciﬁc region (Agostini et al., 2001; Hatwell and Sharp, 2000). The relationship among genotypes is still under debate and it can be assumed that the number of true genotypes will increase with more populations tested for their JCV strains.

Changes within the non-coding region of JCV are used for classiﬁcation of three major TCR subtypes: class I, class II and the archetype. All major JCV variants isolated so far can be grouped into those basic types. The extensive heterogeneity of the JCV TCR gave rise to the hypothesis that the rearrangements might be involved as a virulence factor in the pathogenic process. It was assumed that JCV TCRs may change from a basic persisting subtype in peripheral organs to a virulent type growing eﬃciently in glial cells (Do¨rries, 1997) (Figure 23.5).

The variable TCR elements are constructed of conserved segments with a high degree of sequence conservation that can be deleted or duplicated and rejoined to new units in individual subtypes. The junctions between these domains are variable in length and are preferentially used as breaking regions, thus serving as a source of rearranged sequence pattern. Type I DNA contains two TATA sequences by inclusion of the TATA box in the repeated element; type II DNA has only one TATA sequence and a

23 bp insertion, which includes a potential enhancer

684

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

HUMAN POLYOMAVIRUSES

Figure 23.5 Structure of the major JCV TCR subtypes. JCV TCR subtype I, American JCV prototype Mad-1; TCR type II, European JCV prototype GS/B; archetype, European isolate JCV GS/K. The sequence is divided into seven boxed segments of identical nucleotide sequence (A, B, C, D, E, F1, F2), diﬀering in nucleotide length. Identical shading represents the same DNA sequence. A represents the TATA box. The initiation codon for the putative agnogene (Agno) is indicated by an arrow. Segment length is given in base pairs; AT, TATA box; Ori, origin of DNA replication

core sequence followed by extensively rearranged was suggested that TCR rearrangements (a) might be a repeats. The archetype, with a single promoter element

rather isolated event or (b) could be preferentially and a 66 bp insertion, does not exhibit any major

associated with early stages of disease; alternatively (c) repetitions.

rearrangements might be generated at activated states Genomic comparison of JCV populations in aﬀected

during persistent infection. In this case, more virulent CNS tissue and persistently infected renal tissue of the

subtypes in heterogeneous TCR populations might same PML patient revealed the presence of homo-

outgrow others, leading to the homogeneous popula- geneous populations of highly related subtypes at both

tions found in PML tissue. Recently, in the active sites of infection. However, in contrast to the brain-

disease process of a long-term PML survivor, only one derived virus type with duplicated promoter elements,

prominent variant was detected. In contrast, at the kidney-derived virus exhibited an archetypal

autopsy additional subtypes were found, which were control region. This provided evidence that a periph-

not related to the ﬁrst sequence. This indicates that eral virus type may invade the CNS during persistence,

some virus types may outgrow others in the active and that duplicated promoter elements may subse-

disease process, but it also suggests that JCV TCR quently be generated anew in each host. The

subtypes can be disseminated to the brain during assumption was evaluated by analysis of JCV DNA

persistent infection from the periphery. populations in PML patients. Heterogeneous genome

Activation of polyomavirus infection is frequently populations could only be found at a low rate in the

observed under impairment of the immune system. active stage of disease. As a possible explanation, it

JCV genomes in such individuals revealed mostly JCV genomes in such individuals revealed mostly

On the basis of these findings, the question for the transcriptional activity of subtype control regions is essential to answer (Ault, 1997). Initial experiments point to a more complicated situation than assumed by the DNA sequence of JCV promoter elements alone. In contrast to all assumptions, it was recently found that the rate of early transcriptional activity mediated by duplicated TCRs can be similar to that of single promoter structures. Additionally, it was confirmed that the overall cell type-specificity of individual subtypes is not affected by structural changes (author’s observations). In contrast to these findings, JCV TCR type 2 was recently detected at increased frequency in PML patients. Therefore, it remains open whether TCR heterogeneity is a determining factor in virus growth rate or is related to pathogenicity (Ferrante et al ., 2001; Pfister et al., 2001; Sala et al., 2001).

Interestingly, JCV European/American genotype 2 genomes linked to PML type TCRs were described in a number of PML patients. In consequence, it was asked whether a specific genotype might constitute a pathogenic strain. As a possible molecular basis for altered neurovirulence, mutations were discussed, which lead to alternative splicing of early mRNA transcripts with expression of T’ proteins. Functional changes in the zinc finger motif of TAg, changes in the late regions of the genome that could be involved in protein folding, virion assembly or virus-receptor binding could also influence pathogenicity (Agostini et al., 2001). How- ever, genotypes 1 and 2 can be linked to both archetypal and PML-type TCR elements, and both

were isolated from PML and persistently infected individuals. Although at present no archetypal genome was described as the only variant in a PML case, from the relationship of archetypal and PML-type regulatory regions, it is conceivable that PML-type JCV TCRs have been generated from single promoter elements. Since geographic clustering of JCV TCR type II genomes cannot be excluded, whether a speciﬁc type of JCV genotype/TCR subtype combination is dominantly associated with PML remains to be further evaluated.

Although genomic analysis of BKV remains rather limited, diverging sequences have been observed, including the TCR and the coding sequences. A geographic association of BKV subtypes has not been performed and attempts to associate BKV subtypes with clinical conditions have not yet been successful. In contrast to JCV, serological groups corresponding to BKV genotypes have been detected. Sequence variation was located to the amino-terminal quarter of the VP1 major capsid protein, with changes in the amino acid sequence and potential implications for the secondary and tertiary structure of the protein. Interestingly, genotypes are often not stable in an individual, but change in multiple or serial samples. The genetic instability appears to be extraordinary; however, the signiﬁcance of this ﬁnding is not certain (Knowles, 2001).

State of Human Polyomavirus Infection in Target

Organs

Primary Infection Primary contact with the human polyomaviruses is

generally asymptomatic. It usually occurs during childhood or early in adulthood, leading to lifelong infection in the immunocompetent host. Viruses normally undergo an initial replication cycle in cells proximal to the site of entry and prior to viraemia. The route of infection has not yet been deﬁned, although BKV seroconversion has been associated with tonsillitis, upper respiratory tract disease, mild pyrexia and transient cystitis. Investigation of nasopharyngeal aspirates and saliva by PCR occasionally demonstrated the presence of BKV DNA, but subsequent virus isolation often was not successful. BKV-speciﬁc IgM antibody in umbilical cord blood or in the sera of children aged 52 weeks suggested prenatal infection. This was supported recently by PCR detection of BKV DNA in maternal and fetal material. In addition, BKV DNA in male and female genital tissue against a

686

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

687 background of a low prevalence of JCV DNA pointed

HUMAN POLYOMAVIRUSES

Despite the knowledge that JCV infection is wide- to possible sexual transmission. However, as the

spread in the population, almost nothing is known detection of BKV DNA was exclusively dependent

about how JCV is transmitted in humans. Studies on on PCR ﬁndings, it remains open whether the

sewage in Europe revealed that human polyoma- oropharyngeal tract or alternative routes of transmis-

viruses are distributed to fresh waters by faecal sion are involved in primary infection. Therefore, the

contamination (Boﬁll-Mas and Girones, 2001). Gen- alimentary tract was proposed as the more likely port

ome analyses in and around Tokyo suggest that of entry (Moens and Rekvig, 2001; Stoner and

population density and environmental conditions may Hu¨bner, 2001) (Figure 23.6).

aﬀect the transmission of JCV. One route of Similarly, the site of JCV entry has not yet been

transmission was elucidated by tracing JCV subtypes localised, although direct infection of B lymphocytes

in Japanese families, in which both parents and and tonsillar stroma favours the respiratory tract

children excreted JCV in their urine. Typing of JCV (Monaco et al., 1996). In contrast, recent detection of

genomes in individual family members revealed the JCV in the colon (Ricciardiello et al., 2000) suggests

presence of diﬀerent viral subtypes (Agostini et al., the lower gastrointestinal tract as a possible entry site.

2001). In about 50% of persons, the JCV strains Irrespective of the site of entry, viraemia is thought to

originated from infection outside families. Children occur, based on the ﬁnding that virus reaches sites of

have many opportunities to come into contact with persistence very early in infection.

urinary JCV infection; therefore, urinary excretion can

Figure 23.6 Course of human polyomavirus infection Figure 23.6 Course of human polyomavirus infection

Persistent Infection The discovery of BKV came from the observation of

cytological abnormalities in the urinary sediment of a kidney transplant patient. Later it became clear that BKV is a urotheliotropic virus aﬀecting the epithelia of the renal calyces, renal pelvis, ureter and urinary bladder. Analyses of prostate biopsies revealed virus infection in 60% of asymptomatic tissue, comparable to the rate of infection in the bladder. Sperm gave an incidence of 95% and cervix and vulvar tissue an incidence of 70%. In contrast, glandular tissue was free of JCV virus and in sperm 21% of the samples were positive for JCV DNA. The high rate of BKV DNA in asymptomatic tissue and semen suggests that these sites might be involved in polyomavirus persistence (Knowles, 2001; Moens and Rekvig, 2001).

Similarly, JCV was detected in the urinary tract of immunosuppressed individuals and pregnant women. PCR analysis suggests that the number of aﬀected individuals closely parallels the percentage of persons with serological evidence for contact with JCV. Thus, the kidney is one site of huPyV infection (Arthur and Shah, 1989). JCV DNA was detected in the kidneys of two children with combined immunodeﬁciency disease who developed PML during primary JCV infection. Additional presence of JCV DNA in spleen, lymph node and lung cells support the thesis that JCV persistence in individual organs is most likely established during primary infection. Repeated detection of JCV DNA in the ureters of non-immunosuppressed patients characterised the renal tract as a site of persistence. The higher detection frequency in the renal medulla indicates that epithelial cells lining collective tubules are the major targets. The presence of JCV in urothelial sediments suggests that they are more often subject to activation processes than other cells in the renal tract. In a study of the presence of JCV DNA in colorectal cancers, JCV DNA was additionally detected by PCR in the colon mucosa of 76% of normal patients, suggesting that the virus is highly prevalent in the human upper and lower gastrointestinal tract of immunocompetent individuals (Ricciardiello et al., 2000).

The dissemination of the human polyomaviruses to

a wide range of organs led to the hypothesis of a possible haematogeneous spread in the host. This was supported by an early report on polyomavirus particles

in lymphocytes of immunocompetent children and induced studies on a general role of lymphoid cells in the establishment of polyomavirus persistence. In tonsils, BKV DNA was associated with the lymphoid tissue of Waldeyer’s ring, thus indicating the involvement of lymphocytes in polyomavirus infection. Lymphoid interaction of BKV was further supported by a stimulatory effect of virus infection on human lymphocytes in cell culture, and the demonstration of specific BKV receptors on the surface of peripheral blood cells. Interestingly, only a small number of cells carried receptors for BKV, which were described as B lymphocytes. Nevertheless, virus protein expression was restricted to less than 1% of the cells, thus indicating attenuated replication in lymphocytes. Although the virus is able to attach to and penetrate into monocytes, expression remained negative. How- ever, the treatment of monocyte cultures with BKV antisera was followed by antibody-dependent enhancement of virus replication. Therefore, it can be assumed that circulating monocytes or tissue-resident macrophages in the normal individual might be permissive for polyomavirus infection. The presence of full-length BKV DNA in the leukocytes of persistently infected individuals confirmed involvement of BKV in peripheral blood infection, similar to that in kidney and brain tissue (Do¨rries, 1997). A large body of analyses has been performed, concentrating on the search of virus DNA in peripheral blood cells (PBMC) by PCR. The results were highly variable, rendering incidences of no BKV DNA present to 60% of BMT patients, 10– 100% of HIV-1 patients or no virus in a group of SLE patients. Similarly, in healthy individuals 53%, 71% and 94% contrasted a finding of no virus DNA, respectively. Even two recent reports with advanced PCR techniques including excessive precautions against contamination demonstrated BKV DNA at rates of 26% vs. 55% (Knowles, 2001). This might be due to geographical differences; however, comparison of selected groups, sample preparation and primer pairs revealed major differences. Most of the studies on the presence of BKV DNA in PBL were performed by nested PCR techniques, often at the limits of detection. The amount of BKV DNA was estimated to be very low, as compared with that of JCV in peripheral blood cells. Taken together the high variability in the detection rate of BKV DNA in haematopoietic cells makes a decision on the role of BKV in blood cell infection difficult. However, there is a body of evidence for a regular lymphotropism of BK virus in the host. This includes detection of BKV-specific DNA and mRNA in PBMC, as well as indirect evidence for BKV susceptibility of monocytes.

688

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

Haematogeneous spread of JCV to the CNS had been suspected in early reports because of the exceptional multifocal distribution of JCV foci in PML. Occasional involvement of the spleen and lymph nodes led to the concept that lymphoid cells might be regularly involved in virus spread. This was conﬁrmed by demonstrating JCV DNA and capsid protein in a small number of mononuclear cells of bone marrow and spleen in PML/AIDS patients. These cells were characterised as B lymphocytes by cellular markers, whereas CD4/CD8 + T lymphocytes were not aﬀected. The presence of JCV infected mononuclear cells in perivascular parenchyma and in Virchow–Robin spaces further supported the haematogeneous route of entry into the brain for JCV. Although JCV DNA could not be detected in the spleens of HIV-1 patients and controls, the detection rate of 40% in PML patients suggests that virus DNA may accumulate in lymphoid organs.

The thesis of lymphocytes as a reservoir for JCV closed a gap in the understanding of viral pathogenesis. It was conceivable that JCV-infected lymphoid cells may act as a vector for JCV dissemination. The role of JCV infection in lymphoid cells was further clariﬁed by the detection of tonsillar B lymphocytes and tonsillar stromal cells as host cells for JCV infection (Monaco et al., 1996). Cultivation of tonsillar cells followed by demonstration of JCV DNA established the susceptibility of tonsillar cells. These ﬁndings not only strongly suggest the involvement of the tonsils in primary infection but also argue for a persistent polyomavirus infection in tonsillar cell types.

The role of lymphoid cells in JCV persistence and pathogenesis was studied more intensively by asking whether JCV DNA is present in circulating peripheral blood cells during periods of immunocompromise. The infection rates in peripheral lymphocytes of PML patients were the highest (30–100%), irrespective of the underlying disease or the aﬀected cell type, the virus load or the number of infected cells (Andreoletti et al., 1999; Do¨rries, 2001; Dubois et al., 1997; Koralnik et al ., 1999). This pointed to a regular involvement of lymphoid cells in the disease process. The association of JCV with PBMC from the bone marrow and blood of leukaemia patients added further support to the idea that polyomaviruses might be lymphotropic.

The question of whether peripheral blood cells play

a role in the dissemination of JCV during persistence was addressed by studies on risk group patients without evidence of PML. Detection of JCV DNA in leukaemia patients at a rate of 10–60% prior to and past BMT revealed that these patients are at a high risk for JCV association with peripheral blood cells.

Interestingly, the amount of virus DNA detected was highly variable and did not correspond to the timespan after BMT. This pointed to an intermittent rather than

a continuous course of virus replication. In HIV-1 infected patients, as the most prominent risk group, comparable detection rates were reported. This allows the assumption that virus replication leading to the detection of JCV DNA in PBMC is independent of the type of immune impairment (Andreoletti et al., 1999; Lafon et al., 1998).

Regular association of JCV with PBMC at a rate of more than 80% was further confirmed by amplification of JCV DNA in PBMC of healthy persons. In situ hybridisation added support to the idea that the human polyomaviruses are associated with peripheral blood cells (Do¨rries et al., 1994). Experimental evidence was added by interaction of JCV with human B lymphocyte cell lines. Susceptibility of haematopoietic precursor cells for virus infection was mediated by enhanced expression of the transcription protein nuclear binding factor NF-1. This additionally argued for the involvement of those cell types in natural JCV infection (Do¨rries, 1997; Monaco et al., 2001). However, PCR studies in different laboratories produced markedly divergent results. Incidence rates ranged from none detected to 59%. In most PBMC samples the concentration of virus-specific DNA was estimated to be in the range of less than one genome equivalent in 20 cells, this probably being the reason for the low rates of detection or even failure to amplify JCV DNA by PCR, as loss of targets by DNA extraction methods appears to be a critical step in the detection of persisting virus genomes.

Further characterisation of the virus–cell interaction addressed the question of the cellular target of JCV in lymphoid tissue or peripheral blood. The first reports pointed to the B lymphocyte in bone marrow and spleen as the only target cell. However, in B lymphocyte-depleted PBMC JCV-specific amplification was similarly reported (Dubois et al., 1997). Subsequent studies suggested that JCV could be associated with several haematopoietic cell types (Do¨rries et al., 2003; Koralnik et al., 1999). Generally, the detection rate in PML patients was higher than in those with HIV-1 infection alone, and healthy individuals had the lowest incidence for JCV in blood cells. Although unsorted cells from normal persons were negative for JCV, virus DNA could be detected in sorted populations from the same patient. This corresponds to reports of a low virus load in PBMC. Virus–cell association could either be detected in all of

B and T lymphocytes, in granulocytes and in monocytes of a given blood sample, or only in a single

HUMAN POLYOMAVIRUSES

689

population or any combination of cell types. Interest- ingly, the most prominent cell affected was the granulocyte, a cell type that may not only be specifically associated with JCV infection, but can also be involved in phagocytosis (Do¨rries et al., 2003). These results suggest that JCV DNA molecules are regularly associated with cell types of different lineages; however, the type of association remains to

be elucidated. Similarly, investigations on JCV expression in haematopoietic cells rendered controversial results. JCV mRNA was detected in PBMC and in B lymphocytes of PML patients. In contrast, no expression could be found in HIV-1 patients and blood donors (Andreoletti et al., 1999; Dubois et al., 1997; Lafon et al., 1998). This discrepancy could be explained by a preferential latent state of virus infection in PBMC that might be activated in PML. Clearly, more experiments are necessary to understand the role of JCV DNA in PBMC for viral persistence and possible consequences for the disease process.

Although the suggestion that haematopoietic cells are a reservoir for the virion in the diseased brain might be questionable, it is conceivable that JCV- infected lymphoid cells may act as a vector for JCV CNS invasion and dissemination during persistent infection (Gallia et al., 1997). Apart from how the virus might be disseminated to the CNS, the pathogenic question, of whether PML results from cytolytic invasion of the CNS under severe immunosuppression or as a consequence of a preceding persistent infection, is controversially discussed (Do¨rries, 1997). Although JCV can easily be demonstrated in disseminated areas of PML autopsy material virus DNA or protein, it has not been detected in the healthy CNS by classical methods. However, application of PCR revealed frequent JCV infection in the brain of patients without evidence for PML. In contrast to fetal brain tissue, JCV DNA sequences were present in about 30–70% of adults, depending on the experimental groups. JCV obviously has no topographical preference, because dissemination patterns are comparable to those in PML. Cloning of genomic JCV DNA molecules in brain tissue resulted exclusively in unique, full-length, episomal JCV genomes. Com- pared to thousands of genome equivalents present in aﬀected PML samples, the amount of virus-speciﬁc DNA in asymptomatic individuals is much lower, with an estimated range of 1–100 genome equivalents/

20 cells, and even less. Thus, persistent polyomavirus infection in the CNS is probably restricted to isolated cells and most likely represents chronic infection and not the early stage of disease (Elsner and Do¨rries, 1998).

The ﬁndings prove that human peripheral polyomavirus infection is associated with subclinical virus entry into the CNS, probably long before the development of clinically overt PML. Which cell type is targeted by the virus is not yet known; however, virus activation may lead to an increased number of infected cells and a higher detection frequency in cases of severe immunosuppression, in patients with malignant diseases and in the elderly, this being in line with the assumption that impairment of immune competence favours involvement of the CNS in polyomavirus infection.

Concomitant JCV and BKV Infection Double infection of JCV and BKV was particularly

established after organ transplantation, in HIV-1 infected patients, in pregnancy and in a small number of immunocompetent individuals by urinary excretion or antibody rise against both viruses. Molecular detection of JCV- and BKV-speciﬁc DNA conﬁrmed that concomitant persistence frequently occurs in kidney tissue. Extensive homologies of the genomic structure, similarities of virus spread and state of infection very early started the discussion on possible other sites of dual JCV and BKV infection. After PBMC were found to be a target of polyomavirus infection, the presence of JCV and BKV DNA in blood cells of the same individuals was not astonish- ing. A high rate of concomitant infection was evidenced molecularly in both healthy and immunosuppressed individuals. As JCV protein expression was detected in lymphoid cells of immunosuppressed patients, and BKV virus is able to replicate in lymphocytes in vitro, it is likely that both huPyV infections are periodically activated in persistently infected PBMC.

Although BKV was not expected to invade the CNS at a high rate, the cell specificity of BKV is less stringent than that of JCV, therefore several laboratories searched for double infection in the CNS. Cloning from CNS gene libraries and PCR revealed frequent BKV dissemination to the brain. Indirectly, BKV infection of the human CNS was confirmed by the report of a subacute BKV-associated meningoencephalitis in an AIDS patient. The physical state and genetic complexity of BKV genomes was comparable to that of JCV DNA; however, a low concentration of brain-derived BKV-specific PCR products suggests

a considerably lower activity of BKV in the CNS than that of JCV. The detection of ampliﬁcation products belonging to both polyomavirus species

690

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

691 gives strong evidence for concomitant polyomavirus

HUMAN POLYOMAVIRUSES

was due to BKV, with an incidence of up to 50% in the infection in the CNS. Additionally, it demonstrates

post-transplant period. Although these ﬁndings point that not only JCV but also BKV is neurotropic in the

to a signiﬁcant role of the immune state for the level of human host, and frequently establishes persistent

polyomavirus expression in the kidney, an enhance- CNS infection.

ment of urinary excretion by HIV-1-induced The rate of adult healthy individuals found positive

immunosuppression was not detectable. Similarly, for concomitant infection was astounding, but the data

PML patients do not necessarily have concomitant correspond to recently discussed rates of infection with

JCV viruria. Aggressive chemotherapy does not BKV of almost 100% by the age of 10 and JCV

increase virus frequency, and in other immunosup- reaching more than 90% in adulthood. Therefore, the

pressive diseases viruria can be intermittent, with number of positive cases probably reﬂects the true

sparsely distributed infected cells in cytologically incidence of polyomavirus infection in the European

positive urine, similarly pointing to a rather low rate population.

of virus production (Arthur and Shah, 1989). Activation at other sites of persistent infection has been less intensively examined. This is due to reduced virus expression in asymptomatic transient activation

Asymptomatic Activation of Infection states and to the low virus load in persistently infected healthy individuals. Although the presence of JCV

Transient polyomavirus viruria probably occurs at the DNA in PBMC appears to be a common event, time of primary infection; however, in most instances

activation can so far only be deduced from the the presence of virus in the urine is due to activation

presence of higher amounts of virus DNA in immu- processes. It was assumed that activation of infection

nologically impaired individuals. Additionally, in HIV/ in healthy individuals was predominantly related to

PML patients mononuclear cells expressing JCV- alterations of immunocompetence in the older age

speciﬁc proteins were characterised in PML tissue. groups. Recently, PCR was used to show that JCV

Thus, it appears likely that a possible JCV infection in DNA was present in urine of about 30% of American

lymphocytes is activated under as-yet unknown and 60% of Japanese and European healthy indivi-

circumstances (Gallia et al., 1997). duals. The incidence of excretion depended on age,

The presence of JCV DNA in the brain tissue of with lower rates in the young, then a gradual increase

immunocompetent patients in a distribution compar- with age. Because of the high frequency of JCV

able to that in PML, and increasing incidence of excretion throughout adulthood, the authors suggest

detection with age and in patients with malignancies, that JCV infection may regularly not be in a latent

point to viral activation in the CNS. The detection of persistent state but in a productive one. Since all other

virus-speciﬁc protein in a limited number of glial cells data were accumulated by less sensitive methods, it is

in non-PML brain, and PCR ampliﬁcation of JCV- conceivable that virus expression is permanently

speciﬁc products in the CSF of the same patients’ maintained at a basic level.

groups, argues for asymptomatic JCV activation in Pregnancy is among the most common conditions

persistently infected brain tissue (Do¨rries, 1997). that have been linked to viral activation. The incidence

If the current knowledge is summarised, it must be of viruria, as detected by periodic cytological examina-

assumed that activation of BKV and JCV infection tion, was about 3–7% in the case of JCV and 15% in

occurs species-speciﬁcally and as a result of immune the case of BKV. The onset of viruria was late in the

system alterations induced by pregnancy, older age, second trimester and during the third trimester of

malignant tumour growth or AIDS. Some of those pregnancy. In excreters, virus shedding, once it was

individuals may undergo sporadic activation as a established, continued intermittently to term and then

consequence of their genotypes or of incidental ceased in the postpartum period. The detection rates

transactivation events by other viruses. In general, parallel high or rising antibody titres in a comparable

however, virus growth appears to be dependent on the study population, and therefore represent probably the

impairment of immunological control, resulting in a true rate of activated persistent infections.

diﬀerentially regulated activation pattern in the target Renal transplant recipients experienced viruria at a

organs. Although related mechanisms are so far higher rate (9–19%), compared to a rate of 2.4% in

unknown, the higher frequency of deﬁciencies corre- pregnant women with similar detection sensitivities.

lated with T cell function in PML patients points to Duration periods may be months or even years. In

deﬁcient cellular immunity as a major virulence bone marrow transplant recipients, almost all viruria

factor.

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

Polyomavirus-associated Diseases Strong evidence for BKV-induced systemic and CNS disease came from AIDS patients. Histopathological

Activation of huPyV infection may either represent evaluation revealed association of BKV with aﬀected transient asymptomatic events or pathological pro-

lung, kidney, and CNS tissue. The major pathological cesses. The induction of fatal disorders, however, is

ﬁndings were tubulointerstitial nephropathy, intersti- almost exclusively observed under long-lasting severe

pneumonitis and subacute impairment of the immune system. BKV is a urothe-

tial

desquamative

meningoencephalitis. In the kidney alterations princi- liotropic virus, almost exclusively linked to urogenital

pally consisted of focally accentuated tubular necroses. tract diseases (Arthur and Shah, 1989). Mild courses

Virus products were detected in epithelial cells along are reported in primary BKV infection, whereas BKV

the entire nephron. Alterations in the lung were disease is rather associated with persistent BKV

characterised by intra-alveolar aggregates of desqua- infection, often diagnosed in lymphoma and AIDS

mated pneumocytes. Virus products were detected in patients (Moens and Rekvig, 2001). Virus-associated

exfoliated pneumocytes, epithelial cells and smooth pathogenic eﬀects are observed in BKV-associated

muscle cells of the bronchioli. Occasionally, isolated nephropathy (BKN) and ureteral stenosis as complica-

endothelial cells in the lung carried virus protein. A tions of renal transplantation (RT), as well as in

common feature in both organs was focal interstitial haemorrhagic cystitis in bone marrow transplant

ﬁbrosis with a mild inﬂammatory response. Whereas (BMT) patients. After BMT, BK viruria is detected

ﬁbrocytes were infected, inﬂammatory cells were free often during episodes of cystitis, leading to severe

of virus products.

haemorrhage in about 25% of BMT patients. Hae- In the CNS, no pathological changes apart from morrhage and viruria are most likely due to viral

mild inner atrophy were described. Mononuclear cells activation in the uroepithelium. Recent studies suggest

indicating chronic inﬂammation loosely inﬁltrated that interstitial tubular nephritis is the most frequent

thickened ﬁbrotic leptomeninges. In the cortex and disease after RT. Clinical features may mimic graft

adjoining white matter, endematous tissue alterations rejection or drug toxicity, but histopathological exam-

were found. Reactive astrocytes were localised in the ination almost always demonstrates BKV infection.

outer layers of the cerebral cortex. The ventricular Immunosuppressive therapies increase the incidence of

system exhibited focal degeneration of its ependyma BKV-associated disease, thereby conﬁrming a close

and spongiform destruction of subjacent brain tissue. relationship of virus growth and immunomodulation

The choroid plexus showed ﬁbrosis of the stroma (Table 23.2).

and atypical epithelial cells. In some areas, necrosis Table 23.2 Human polyomavirus-associated diseases

Patients

BKV infection Disease

JCV infection

Cell type involved Immunocompetent

Cell type involved

Disease

None observed

Mild respiratory disease –

Mild pyrexia Transient cystitis

Immunocompromised Urogenital system

None observed

Ureteral stenosis

Epithelial cells

Haemorrhagic cystitis

Fibrocytes

Tubulonephritis

Lung None observed

Interstitial pneumonitis

Epithelial cells Pneumocytes Endothelial cells Fibrocytes

CNS Progressive multifocal

Subacute meningoencephalitis Epithelial cells leukoencephalopathy

Oligodendrocytes

Fibrocytes (PML)

Astrocytes

Peripheral blood

Endothelial cells

mononuclear cells

Astrocytes

B lymphocytes

Ependymal cells Peripheral blood

mononuclear cells

693 and exfoliation of the plexus epithelium occurred.

HUMAN POLYOMAVIRUSES

Table 23.3 Causes for JCV activation and induction of Small inﬁltrates were seen in association with the

PML prior to the AIDS epidemic lesions.

Conditions associated with activation The dominant target cells of BKV infection were PML (% of cases*

except AIDS) ﬁbroblasts of the loose reticular connective tissue,

of persistent infection

endothelial and smooth muscle cells of blood vessels,

None astrocytes and inﬁltrating macrophages in pons and

Age

None medulla oblongata. Epithelial cells in the choroid

Pregnancy

None plexus and astrocytes of the subependymal brain tissue

Inﬂammation

Transplantation

Rare

Rare were additionally infected. The only glial cell type

Diabetes

62.2 involved in BKV-associated CNS disease was the

Lymphoproliferative disease

6.5 astrocyte, whereas oligodendrocytes and nerve cells

Myeloproliferative disease

2.2 were not aﬀected. The high number of cell types

Carcinomatous disease

16.1 involved in BKV infection in vivo demonstrates the

Immune deﬁciency states

Granulomatous/inﬂammatory disease 7.4 broad BKV cell speciﬁcity and points to a higher

*Brooks and Walker (1984).

relationship of BKV to SV40 than to JCV. The detection of a BKV-associated disease aﬀecting the

gradual, but each new functional impairment becomes kidney, lung and CNS is in line with the involvement

progressively more severe. Once clinical signs appear, of those organs in BKV persistence.

the disease usually progresses steadily. Early neurolo- In contrast to BKV, JCV was never observed as an

regularly indicate multiple aetiologic agent in urogenital tract or lung disease. The

gical

symptoms

disseminated lesions in the brain. The extent and most prominent disease associated with JCV is the

topography of the lesions correlate well with the CNS disorder PML (Berger and Major, 1999; Walker

duration and symptomatology of the illness and Padgett, 1983; Zu Rhein, 1969). Although SV40

(Table 23.4).

was described as the cause of PML in three American The pathognomonic feature of the disease is the and Japanese patients, it later became clear that most

striking alteration of oligodendrocytes in all lesions. probably JCV was the only agent responsible for the

Oligodendrocytes are located in the peripheral rim disease.

surrounding the zone of myelin loss. The central area is PML is a demyelinating disorder occurring as a late

composed of reactive astrocytes, including giant cells complication of pre-existing systemic diseases that

in mitosis and astrocytes resembling the malignant impair immunological competence. Several years of

cells of pleomorphic glioblastomas. Infection of treatment precede PML in diseases such as rheumatoid arthritis, chronic asthma, sarcoidosis, lupus erythema-

Table 23.4 Development of PML lesions tosus and chronic polymyositis. Prior to the AIDS era,

Early Lesion

malignant proliferative diseases were the dominant Altered oligodendrocytes in areas of demyelination basic disorders in PML in about half of the cases. In

Nuclei enlarged (two- to three-fold) HIV-1-infected patients, a steadily increasing number

Regular chromatin pattern is lost of cases was associated with AIDS prior to the highly

Nuclei tint deeply with basophilic dyes active antiretroviral therapy (HAART). Although

Irregular basophilic nucleic inclusions longer PML survival times can be observed under Seldomly activated pleomorphic microglia Pinhead size (mm) lined up the cortical ribbon

HAART, the rate of PML patients remains stable at about 5% of AIDS patients (Antinori et al., 2001).

Aging Lesion

Additionally, a higher frequency of PML can be Reduced number of altered oligodendrocytes in the centre

of demyelination

observed in correlation with immunomodulatory Shrinkage of nuclei at the rim of lesions therapies and is probably due to higher JCV activation

Widely distributed larger foci with coherent tissue rates (Table 23.3).

Late Lesion

The onset of PML is often insidious. In the earliest Altered oligodendrocytes at the peripheral advancing zone manifestation, multiple pinhead-sized demyelinating

surrounding the area of myelin loss in the centre lesions are described beneath the cortical ribbon. New

Very sparse or absent oligodendrocytes small foci are continuously being added in neighbour-

Singly scattered reactive hypertrophic astrocytes ing tissue as the growth centres of new lesions. This

Giant astrocytes in mitosis

concept of the histological evolution of the disease is Astrocytes resembling cells in pleomorphic glioblastomas forming irregular necrotic patches (cm)

supported by the clinical evolution: the onset might be supported by the clinical evolution: the onset might be

The basic cause of tissue destruction is a cytolytic JCV infection of the oligodendrocyte. Destruction of these cells results in loss of myelin, tissue breakdown and impairment of brain function. Infected oligodendrocytes mediate highly eﬀective virus growth in the

range of 410 10 virus particles/g tissue. Viral expression products and virions are found in the nuclei and cytoplasm of the oligodendrocytes. Bizarre astrocytes may contain JCV; however, the question of whether astrocytes may actively support infection has not yet been answered. On the grounds of morphological changes and occasional reports of intracellular virus particles, it is suggested that astrocytes may represent a semipermissive cell type, mediating reduced JCV expression or, alternatively, being sensitive to virus transformation.

In contrast to classic PML, in the AIDS patient frontoparietal and grey matter involvement increases and inflammatory reaction with perivascular mononuclear cell infiltrates is often observed. Mononuclear cells in the Virchow–Robin spaces occasionally contain JCV-specific DNA and capsid antigen, and in the subcortical white matter adjacent to blood vessels the density of infected cells appears to be increased. In addition, beneath the ependymal layers, JCV-infected cells are detected. This indicates a close topographical association of infected cells and the ventricular system. These findings might be explained by an essentially faster progress of the disease, or by differences in the immunological control of JCV infection in HIV/PML (Mazlo et al., 2001).

The duration of disease after the onset of neurological symptoms is reported to be 4–6 months on average (Berger and Major, 1999). However, there are cases of 412 months duration, with an intermittently progressive or a subclinical course over years. These cases are most often found in combination with HIV-1 infection in patients receiving HAART. Since the introduction of HAART, the definition of HIV/PML cases has had to be adapted to changes in the disease pattern, probably resulting from HAART-induced immune reconstitution, in about half of the patients. For these two groups a terminology is newly proposed for disease outcome. It is based on diagnostic criteria for definitive, probable and possible PML, with a definition of PML outcome based on disease activity. Disease progression can be classified as ‘active’ or ‘inactive’, based on clinical, radiological, virological and pathological criteria. Patients in ‘active PML’ have high diagnostic evidence of disease activity through the course of illness. The great majority of

these patients die within a few months. Patients with ‘inactive PML’ lack evidence for disease activity. Stabilisation of PML may occur at any time during disease progression; however, usually it is achieved in a few months after ﬁrst diagnosis. There is virtually no progression over years of follow-up, even after withdrawal of HAART (Cinque et al., 2003). Use of these criteria as standard terminology in the description of clinical and biological studies would greatly assist understanding of the mechanisms of pathogenesis and therapeutic intervention.

Oncogenicity of the Human Polyomaviruses

Cancer is thought to progress through multiple stages, and with each step cells with an increasingly malignant phenotype may arise. Progression can be driven by mutations in tumour suppressor genes or oncogenes; it can also be dependent on newly acquired transforming proteins. These genes encode proteins regulating genome stability, cell cycle proliferation or apoptosis. The polyomaviruses with the large TAg provide a prototype of viral oncogenes targeting cellular regulatory proteins, thereby interfering with cellular functions. The ﬁrst 121 AA comprise the shortest fragment to induce transformation. Here it binds to the retinoblastoma family of tumour suppressors and a number of other cellular proteins believed to ultimately activate the p53 tumour suppressor pathway. Activa- tion leads to elevated steady-state levels of p53, ending in a block of the cell cycle. In addition apoptotic genes are induced. Association of TAg with or binding to p53 results in inactivation of its transcriptional activation potency, preventing the growth-inhibitory functions of genes downstream from p53. In cases where TAg expression is followed by malignant transformation, a general correlation is observed between production of higher levels of TAg and the appearance of tumours. Animal models are useful tools to ascertain molecular markers as indicators for tumour induction and progression. In contrast, in humans these markers can be used to prove hypotheses on polyomavirus oncogenicity, even in the absence of viral protein expression (Sullivan and Pipas, 2002).

The discussion of a possible oncogenicity of a huPyV in humans had its origin not only in the oncogenic potential of large TAg but also in the bizarre pleomorphism of astrocytes within lesions of PML. This was described as a hallmark of PML in the initial description of the disease and later oligoden- droglioma and multifocal glioblastoma were reported,

694

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

corresponding topographically to the demyelinated lesions in PML. Thus, oncogenicity of JCV and BKV was examined almost from the beginning in experimental animal systems. Developing tumour types reflected the cell type specificity of the viruses. Corresponding to the specificity of JCV for glial cells, predominantly brain tumours were observed. The highest yield was found after intracerebral inoculation of newborn Syrian hamsters, whereas peripheral nervous system tumours are prominent after intraocular virus administration. Central nervous system tumours are located in the cerebrum, cerebellum, brainstem and spinal cord. Mesenchymal tumours within the cerebral meninges are classified as malignant meningiomas. Ependymomas are the dominant type of intraventricular tumours, and occasionally choroid plexus tumours develop. The most common neoplasms were medulloblastomas at a rate of 95% in newborn hamsters. These are followed by malignant astrocytomas, glioblastoma multiforme, neuroectodermal tumours and pineocytomas. The neuro-oncogenic potential in primates was confirmed by the occurrence of malignant glial brain tumours in about 50% of adult monkeys after intracranial infection. The predominant type was characterised as astrocytoma grade 4. JCV TAg expression in tumour cells suggests that the early region of JCV DNA was present in most tumours, and rescue of JCV from tumour tissue was occasionally successful. The virus genome was regularly integrated in tandem copies at single or multiple sites, comparable to the pattern seen in hamster tumours.

The majority of JCV-induced CNS tumour types in hamsters have their human counterpart in tumours of infants, older children or young adults. In view of the giant glial cells being key features in PML, the tendency to form malignant astrocytomas and giant cells appears to be more a characteristic of JCV than that of the animal species inoculated. Although the course of PML does not allow extensive tumour growth, it can therefore be hypothesised that a semipermissive persistent JCV infection might provide the cellular background for transformation and tumour induction in the human host.

The oncogenic potential of BKV appears to be less pronounced, as it only induces tumours in rodents but not in non-human primates. The most prominent cell types affected by BKV infection in men—epithelial cells, fibrocytes, ependymal cells, astrocytes and endothelial cells—are reflected by different tumour types induced in experimental animal models. The type essentially depends on the route of infection, on the amount of virus inoculated and on the BKV isolate

used. In addition, as co-factors the age and immunocompetence of the host inﬂuences the rate of malignancy.

After intraperitoneal inoculation tumours are rarely observed; subcutaneous infection yielded sarcomas with an incidence of 2–12%. Intravenous application results in ependymomas. They are sometimes associated

with peripheral tumours, insulinomas, osteosarcomas and tumours of the intestine. In contrast, intracerebral inoculation resulted in choroid plexus papillomas and papillary ependymomas without a sign of peripheral tumours in 88% of the animals. The disseminated localisation of tumours demonstrates a marked tissue tropism and multi- oncogenicity of BKV in hamsters. Genetic diﬀerences of virus stocks with high transforming capacities and predilection for speciﬁc tumour types could be mapped to the BKV transcriptional promoter elements, suggesting that the transforming capacity might depend on TCR function.

In the tumours BKV DNA is either tandemly integrated in the cellular genome or in a free episomal state. In most tumours complex integration patterns of multiclonal origin were observed. Most cells express nuclear TAg, virus often can be rescued by cell fusion with permissive cells and tumour-derived cell lines grow permanently, maintaining their tumour-induct- ing potential. Selection of deﬁned integration pattern and decrease in the amount of BKV DNA during prolonged tumour cell culture may point to rearrangements of integrated BKV DNA in the course of tumour growth. This could give rise to cells with simpler integration patterns and loss of viral DNA. Even an intact TAg gene is not necessarily needed for maintenance of cell growth, once the transformed state is established.

The spectrum of BKV-induced tumour types is comparable in all sensitive animal species. Thus, speciﬁcity of viral transcription might be one of the major selection criteria for huPyV-associated transforming activity in animal systems. In view of this assumption, it is discussed that human polyomaviruses might also be responsible for the respective tumour types in their natural host. Consequently, a large number of laboratories have evaluated the role of huPyVs in the aetiology of human tumours with most virological and molecular methods available. Despite the oncogenicity of these viruses in animal systems, association with human tumours remains controversial (Del Valle et al, 2001; Do¨rries, 1997; Major et al., 1992; Stoner and Hu¨bner, 2001). However, with the introduction of PCR and the better understanding of molecular steps leading to malignant transformation,

HUMAN POLYOMAVIRUSES

695

human tumour induction by polyomaviruses ﬁnds more and more arguments.

TAg expression was analysed immunohistologically in medulloblastomas, oligodendrogliomas, astrocytomas, glioblastomas, ependymomas, choroid plexus and urinary tract tumours. The results were inconsistent. Antibody titres to TAg in sera from tumour-bearing patients exhibited no significant difference to sera of normal people. From these findings the conclusion can

be drawn that TAg is not regularly expressed in human tumours. Since TAg must not be expressed necessarily at all stages of tumour growth, the presence of huPyV- related DNA sequences was analysed repeatedly in a large number of tumours. So far, in one laboratory JCV DNA sequences were detected in 57% of oligodendrogliomas, about 75% of various astrocytomas, 60% of glioblastomas, 83% of ependymomas and 87% of medulloblastomas. Examination of the p53 level in tumours expressing TAg revealed that the number of tumour samples often closely paralleled those with p53 immunoreactivity. In addition, double labelling suggested close association of both proteins within the tumour cell (Del Valle et al., 2001). JCV DNA sequences have also been detected in colorectal tumours. Although this does not represent a known site of persistence, the demonstration of JCV DNA in normal mucosa adjacent to these tumours points to the colonic epithelial cell as a new target for JCV infection (Laghi et al, 1999; Ricciardiello et al., 2000).

BKV DNA was only occasionally detected by classical methods in series of human tumour types that were frequently described in animal models. This included brain tumours, osteosarcomas, insulinomas and Kaposi’s sarcoma. Analyses of the state of BKV DNA revealed mostly episomal virus DNA, even if virus was rescued, this being rather more indicative of

a persistent state of infection than of an association with transforming activities. After the introduction of PCR, BKV DNA was demonstrated in a variety of human tumours, including brain, pancreas and various urinary tract neoplasms (Corallini et al., 2001; Moens and Rekvig, 2001). There are studies accumulating that are not solely based on PCR results, viz. demonstration of DNA sequences using Southern blot analysis and DNA sequencing indicate that BKV DNA can be present in the integrated state. Interestingly, rearrangements of the respective viral DNA sequence probably interfere with BKV DNA replication and productive infection. This may lead to accumulation of TAg and transformation events. Presence of TAg in neuroblastomas and its interaction with cellular proteins was conﬁrmed by immunohistochemistry and by immunoprecipitation of TAg/p53 complexes.

In contrast to these findings, the presence of JCV or BKV DNA or protein in tumour tissue could not be confirmed by others (Moens and Rekvig, 2001). This may be explained either by technical differences among laboratories or by different handling of samples. However, even detection of virus DNA in tumour tissue makes a differentiation of persistent infection and transformation difficult. First, it cannot be excluded that blood-derived virus may be found by highly sensitive detection methods in tumour tissue. Since the state of DNA in tumour cells is not yet unequivocally characterised, the presence of episomal DNA does not exclude persistent infection.

Furthermore, it cannot be ruled out, that polyomaviruses interact synergistically with other factors to induce malignant growth in human cells. In that case, the presence of virus DNA in tumour tissue could be a consequence of an event occurring early in the natural history of the tumour, when the cell cycle might be deregulated by TAg and its interaction with cellular factors. After genetic alterations are established, the virus genome and transcription products might be dispensable. In such a setting, the role of huPyV remains uncertain and a more detailed analysis of tumour tissues and the steps leading to transforming events in the animal models will be required to answer the question of whether or not JCV and BKV are involved in human tumorigenesis.

DIAGNOSTIC EVALUATION OF POLYOMAVIRUS-ASSOCIATED DISEASE

Diagnosis of PML by Biopsy

The most important disease linked to the human polyomaviruses is PML. The classic method of PML diagnosis involves neurological evaluation and neuroimaging of the brain followed by definitive laboratory diagnosis on biopsy material (Do¨rries et al., 1998b). Open surgery for PML diagnosis has been almost completely replaced by stereotactic biopsy. Topogra- phical selection of samples at the outer rim of the lesions provides the best material for virus detection. Diagnosis is based on the identification of virus products and typical cellular changes in glial cells. The extraordinary multiplication rate of the virus in diseased tissue allows detection of viral nucleic acids and proteins by classical immunohistological methods. Molecular detection of JCV by PCR in biopsy samples is usually confirmed by histopathology. As pathological criteria for the term ‘active PML’ in AIDS, the presence of JCV-infected glial cells, bizarre astrocytes

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PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

and lipid-laden macrophages in the context of demyelination are essential. In contrast, ‘inactive PML’ is characterised by the presence of demyelinated areas without JCV proteins, detected by immunohistochemistry. Regression of astrocytic and oligodendroglial changes and the disappearance of macrophages and JCV proteins was already described in one case of biopsy-proven PML in the pre-AIDS era (Cinque et al ., 2003).

It is accepted that polyomavirus DNA is present in the CNS of adults with former virus contact (Do¨rries, 1997; Major et al., 1992; Weber and Major, 1997). In the asymptomatic state of infection virus DNA is regularly not detectable by diagnostic PCR analysis of single brain samples. If the infection is activated in immunoimpaired individuals, the virus load might increase and could then be detectable. However, compared to the thousands of genome equivalents present in PML tissue, the amount of virus is considerably lower. Consequently, in cases with doubt-

ful results quantitative PCR analysis of any type diﬀerentiates a persistently activated and a PML- associated JCV infection. At present, a combination of stereotactic biopsy and PCR techniques ensures a rapid diagnosis of PML with the highest sensitivity and speciﬁcity available.

Demonstration of Polyomavirus DNA in CSF In view of the high frequency of PML in AIDS patients

and the future development of immunomodulatory therapies, introduction of less invasive methods with a comparable detection rate in early diagnosis is urgently required (Do¨rries et al., 1998b; Major et al., 1992; Weber and Major, 1997). Although the concentration of JCV in affected tissue is extraordinarily high, virus load in CSF is considerably lower and PCR is the only technique available for virus detection, although undoubtedly the most sensitive method, PCR on CSF, often causes divergent diagnostic results. Reports on the test specificity vary (10–80%). Since polyomavirus PCR has not yet been standardised, variable specificity rates are in part caused by technical differences. With the increasing number of reports, factors such as primer quality, sensitivity of the detection system, extraction methods and sample volume are eliminated. Even the sensitivity of the test systems was enhanced by a second nested PCR amplification of the primary products with internal primer pairs, providing detection limits in the range of about 10 genomes. However, nested PCR on the CSF

of a high-risk HIV-1 patient without PML similarly revealed positive JCV amplification. Thus, increase of the detection limits by nested PCR may lead to an increasing number of false-positive PML diagnoses and reduces the prognostic significance of the technique. This finding is explained by asymptomatic activation of JCV CNS infection in states of immunocompromise. Whether this is indicative for an early state of disease or might represent a timely restricted activation is not yet known.

In addition, even in nested PCR reactions, CSF samples remained negative in autopsy-veriﬁed PML cases. Serial sampling revealed that the virus load is low, often almost not detectable early after ﬁrst diagnosis and may increase at late stages of disease. In these cases repeated CSF sampling at time intervals depending on clinical and radiological progression of disease appear to be an essential factor for virus detection. Nevertheless, there are cases remaining negative even at late stages of the disease. Concentra- tion of virus, destruction of tissue, therapy and disease history is comparable to that of patients shedding JCV either continuously or intermittently into the CSF (Eggers et al., 1999). At present, an explanation for the absence of JCV in those cases cannot be given.

PCR diagnosis is the most sensitive method for the detection of virus in body fluids; however, in view of asymptomatic activation of JCV, persistent infection under immunosuppression, differential diagnosis of PML and subclinical infection by PCR analysis alone cannot, at present, be recommended for routine diagnosis. Further data on the amount of JCV shed into the CSF in the course of PML in comparison to that in CSF of non-PML patients have to be accumulated and the techniques used among laboratories have to be standardised. None of the presently available clinical, radiological and virological parameters can be used as a stand-alone criterion for the diagnosis of PML. Thus, to date, PCR can only be understood as a supporting diagnostic tool. The new definition scheme for PML is based on criteria for ‘definite PML’ involving: (a) progressive uni- or multifocal neurological disease; (b) typical magnetic resonance imaging (MRI) lesions; associated with (c) typical brain biopsy features with confirmation of JCV specific products. ‘Probable PML’ represents cases with typical clinical and imaging findings, with amplification of JCV DNA in CSF but without bioptic diagnosis. Absence of histological confirmation and JCV demonstration in CSF in a setting of typical PML clinical and radiological findings leads to a diagnosis of ‘possible PML’. If either pathological diagnosis can be performed or JCV can be detected in

HUMAN POLYOMAVIRUSES

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PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

CSF in serial CSF samples, this may later classify for a titre changes. Use of the enzyme immunoassay (EIA) ‘deﬁnite PML’ diagnosis. Incidentally discovered white

for the detection of huPyV-speciﬁc antibodies reveals a matter lesions or JCV ampliﬁcation in CSF without

better sensitivity, with geometric mean titres 10 times active clinical or radiological ﬁndings should not be

higher than that of HAI; however, the quality of the considered as representative for PML (Cinque et al.,

results is identical to that of preceding studies (Weber 2003).

et al ., 2001). Although recombinant proteins as antigens are a prerequisite for the diagnostic EIA, it is not yet clear whether species-speciﬁc huPyV Ig class G antibodies can be diﬀerentiated from group-

Polyomavirus-specific Humoral Immune Response speciﬁc antibodies with recombinant virus capsids. Extensive studies on possible cross-reactions among

Attempts to establish additional diagnostic procedures the recombinant primate polyomavirus antigens are concentrate on the virus-speciﬁc humoral immune

under way.

response. Reports focus on the determination of The first polyomavirus-specific IgM assays were virus-specific antibodies and their dynamic titre

performed with BKV antigen. Cross-reactivity of BKV changes in acute disease. The predominant polyoma-

with JCV IgM was not evaluated, and whether virus- virus subgroup-speciﬁc antigenic sites on the major

speciﬁc IgM can be distinguished in double infections capsid protein VP1 are accessible only after disruption

remains unresolved. However, the prevalence of BKV- of virions, in virus-infected cells or on puriﬁed VP1

speciﬁc IgM in children was consistent with age protein. Consequently, virus-speciﬁc antisera risen

distribution of primary BKV infection. The occurrence against intact virus particles are species-speciﬁc.

of BKV IgM at approximately 5% in sera from Species-speciﬁc antibodies can be distinguished from

healthy blood donors is consistent with the ﬁnding that one another by neutralisation and haemagglutination-

BKV activation is uncommon in healthy adults. Thus, inhibition (HAI) tests, with a good correlation of

possible cross-reactive IgM antibodies might be in the virus-speciﬁc HAI and neutralising antibody titres

minority. JCV-speciﬁc IgM sero-antibodies were (Pass and Shah, 1982).

detected in 15% of healthy blood donors. In the The prevalence of BKV-speciﬁc antibodies in sera is

same group almost all sera contained HAI antibodies, about 50% by the age of 3 years and nearly all

suggesting that blood donors harbour the virus in a individuals are seroconverted by the age of 10 years

persistent state. The high prevalence of JCV infections (Knowles, 2001). The incidence of JCV antibodies is

in adults led to the assumption that the presence of about 50% during adolescence and more than 80% by

IgM is frequently associated with JCV activation in the adulthood. The rates diﬀer slightly according to

healthy. In about half of PML patients, a rise of IgM demographic data and geographical distribution.

antibodies with increasing levels was observed under Further discrepancies can probably be explained by

progression of the neurological illness. At present, the diﬀerential sensitivity of the detection techniques.

high prevalence of IgM-positive sera in healthy The range of polyomavirus-speciﬁc HAI antibody

persons does not allow a correlation of IgM presence titres is not dependent on age or sex, but in pregnant

with acute disease (Knowles et al., 1995). women rising titres indicate an incidence of active

In early studies the CSF was usually found to be infection of more than 25%. Since virus-speciﬁc

unremarkable in PML, and JCV HAI titres were only antibodies in normal persons and age-matched

rarely detected. Thus, in the past the humoral immune patients with various tumours and lymphomas exhib-

response was regarded as essentially unhelpful for ited similar geometric mean titres, this is believed to be

diagnosis and studies on JCV antibodies and their the result of activation processes mediated by preg-

dynamic changes in the CSF of PML patients were nancy. Titres in patients with malignancies remain

limited to single cases. However, recently a slight stable even under multidrug- or immunotherapy, thus

increase in CSF protein was reported in about one- demonstrating that JCV antibody titres in sera are not

quarter of PML patients, an elevated IgG albumin markedly inﬂuenced by these diseases or associated

index in about one-ﬁfth, and a slight pleocytosis in therapies. Equally, under PML, the range of JCV-

other patients (Berger and Major, 1999). Further speciﬁc serum and HAI titres cannot be distinguished

analyses revealed moderate blood–brain barrier from those in the general population. This is explained

impairment in PML patients. JCV-speciﬁc intrathe- by the severe basic illnesses abrogating a normal

cally synthesised HAI antibodies and oligoclonal antibody increase, but it cannot be excluded that the

bands were detected in the CSF of 67% conﬁrmed low sensitivity of the HAI test may not pick up modest

PML cases. Additionally, changes of HAI titre in the

699 CSF were observed under PML treatment, suggesting

HUMAN POLYOMAVIRUSES

lesions, thus indicating an intact antigen presentation. that JCV-speciﬁc antibody titres might be responsive

In HIV/PML patients, immune reconstitution by to therapeutic intervention. Thus, JCV intrathecally

HAART is related to a rise in helper CD4 + T produced HAI antibodies appear to be suggestive of

lymphocyte counts as well as a drop of HIV-1 load active JCV multiplication within the CNS. This was

and clinical improvement of JCV-related neurological further conﬁrmed by EIA analyses with recombinant

symptoms. However, about 50% of HIV/PML JCV VP1 capsid protein as antigen. An intrathecal

patients do not respond to HAART, despite reduction immune response was detected in 78% of PML

of HIV-1 load and rise in CD4 + counts. Therefore it patients vs. 3% of controls. Detection of JCV-speciﬁc

appears likely that the CD4 + T cell subset does not oligoclonal bands was slightly less sensitive. Interest-

play an important role in PML development. ingly, a JCV-speciﬁc intrathecal immune response

Studies on cytotoxic T lymphocytes revealed the against JCV VP1 evolved in HIV-1 patients during

presence of JCV-speciﬁc circulating cells directed therapy. At present, it appears likely that a response

against the early TAg and the capsid protein VP1 in evolves over time in the majority of PML patients

survivors of PML. Comparison of the CTL response (Weber et al., 2001).

demonstrated that a strong JCV-speciﬁc CTL response The range of titres in individual PML patients

is related to the outcome of disease. Recently, a appears to be highly dependent on the state of the

cytotoxic epitope of JCV VP1 was used to characterise disease and may additionally reﬂect the type of

the CTL in PML patients and control subjects. A CTL underlying disease, with characteristics diﬀerently

response was detected in patients with a prolonged aﬀecting parameters of the immune system. Analyses

disease course but in none of the classic PML patients, of virus-speciﬁc, intrathecally produced antibodies

or in non-PML HIV-1 patients, or healthy controls. and oligoclonal bands are well matched in PML vs.

These data conﬁrm that the JCV-speciﬁc cellular non-immunosuppressed cases. It can be assumed that

immune response plays an important role in PML. In

a mild intrathecal immune response with presence addition, it is conceivable that the JCV-speciﬁc CTL of oligoclonal JCV-speciﬁc antibodies indicates

response may provide a useful diagnostic and prog- intraCNS growth of JCV. It remains to be determined

nostic tool in the therapeutic management of these whether the ﬁnding of intrathecally produced JCV-

patients (Du Pasquier et al., 2001; Koralnik et al., speciﬁc antibodies and oligoclonal bands is indicative

for acute disease and PML only, or might also be characteristic for a persistent activated state under immune impairment of other diseases. A thorough examination of the humoral immune response to JCV

TREATMENT OF POLYOMAVIRUS-ASSOCIATED in the high-risk groups has to be performed in order

DISEASES to define specificity and frequency of intrathecal JCV- specific antibodies, prior to a decision on the

Treatment of huPyV infections concentrated for years usefulness of these assays as a diagnostic tool in

on PML. Diﬀerent treatment regimens have been PML.

proposed on the basis of molecular ﬁndings and small series of patients. However, randomised therapeutic trials were introduced only recently and the observation of stabilised PML or even remission highlights the

Polyomavirus-specific Cellular Immune Response inadequacies of early anecdotal reports suggesting the value of a speciﬁc therapy (Berger and Major, 1999;

JCV activation occurs in the context of immune Major et al., 1992; Weber and Major, 1997). impairment, and humoral immunity obviously is not

Nucleoside analogues have been found to interfere able to control JCV spread. Therefore, from the early

with viral DNA synthesis in virus infections, and beginnings breakdown of cellular immunity was

several compounds have been tried in the treatment of suspected to play a major role in the pathogenesis of

PML. Cytosine arabinoside (ARA-C, cytarabine) has polyomavirus diseases. However, studies on the

been used from the early beginnings, with various immune responses are limited and did not contribute

degrees of improvement being reported. Recently to a general understanding of polyomavirus-associated

acquired molecular data showed that cytosine-b- D - mechanisms of immune control. Recently, it was found

arabinofuranoside suppressed JCV replication in tissue that the major histocompatibility complex (MHC)

culture, thus supporting the potential interference of classes II and I were expressed at high levels in PML

ARA-C with PML. However, the study on eﬃcacy of

ARA-C by the AIDS Clinical Trials Group, trial 243, showed no benefit (Hall et al., 1998). In contrast, in a small group of non-AIDS PML patients the treatment stabilised PML in 36% of the patients for 1 year (Aksamit, 2001). However, in the presence of a demonstrable in vitro effect of ARA-C, it was hypothesised that the failure of the drug might be due to insufficient delivery of the drug through conventional intravenous and intrathecal routes. The so-called

‘convection-enhanced

intraparenchymal

delivery’ was developed which may enhance the eﬃcacy of ARA-C (Levy et al., 2001). Other analogues, such as adenosine arabinoside (ARA-A, vidarabine), iodo-desoxyuridine or zidovudine, similarly do not appear to have an eﬀect in the treatment of PML.

The use of highly active antiretroviral therapy (HAART) in patients with HIV-related PML is associated with disease stabilisation. Prolonged survival in about 50% of the patients is associated with decrease of JC viral load in the CSF. The survival rate significantly increased in patients receiving a protease inhibitor-containing regimen. The effect of HAART on PML is believed to be the result of improved immune defence. However, in half of the patients PML progressed despite virological and immunological response to HAART. Failure to respond to HAART, as demonstrated by high plasma viral loads, has been associated with poor PML prognosis and a course of disease similar to that in absence of antiretroviral therapy. Nevertheless, the use of HAART has significantly improved survival time, although the prognosis of HIV/PML patients is still severe (Antinori et al ., 2001; Cinque et al., 2003).

Cidofovir diphosphate, a structural analogue of deoxycytidine triphosphate, is known as a potent inhibitor of human herpes and papillomaviruses. It demonstrated a significant inhibitory effect on replication activity in vitro and was therefore introduced as a potential drug in PML treatment. Anecdotal reports pointed to clinical improvement and following studies reported beneficial effects, either alone or in combination with HAART, in AIDS-associated PML. However, new pilot studies did not report a beneficial effect in patients receiving potent antiretroviral agents, therefore the efficacy of the drug remains controversial.

The use of immune modulatory agents in PML therapy is based on the ﬁndings of extended survival in patients with improvement of the immunological competence. a-Interferon has established eﬃcacy in the treatment of other polyomavirus-associated diseases, and prolonged survival was reported. In

contradiction to these results, other studies reported no significant enhancement in survival time. Similar discrepancies were found with the combination of adenine arabinoside and b-interferon or transfer factor revealing no efficacy, whereas interferon alone was associated with modest improvement in the clinical picture and on MRI. Recently, an effect on survival after interleukin-2 treatment of a lymphoma patient after bone marrow transplantation was contra- dicted in another patient. In addition, treatment with low-dose heparin sulphate was suggested to prevent seeding of JCV to the CNS by activated lymphocytes. Other agents have been tried, either alone or in combination. No effect has been found with cortico- steroid therapy or tilorone, an immune enhancer. The use of antisense oligonucleotides to prevent virus- specific protein expression has been proposed, but awaits development of the respective drugs. Thus, unequivocal effective therapy of PML as yet remains elusive, and further investigations and collaborative efforts are desperately needed to decide which of the different approaches might be effective for the treatment of PML.

In recent years the incidence of BKV-associated diseases has increased. Diseases of the urothelia are predominantly found in a setting of therapeutic immunosuppression schemes, states which also favour transient asymptomatic BKV activation. Among renal transplant patients, up to 60% have BKV reactivation, as shown by the presence of infected cells in the urine, whereas BKV has been implicated as a cause for BKV- associated nephropathy (BKN) in about 5%. Careful reduction of the doses of immunosuppressive therapy helped to stabilise or improve the disease. It has been suggested that the new immunosuppressive drugs, such as tacrolimus or mycophenolate mofetil, played an active role in promoting BKV infection. Remission could be achieved by decrease of high-dose therapy or replacement by cyclosporin A. Similarly, BKV was more frequent in patients with quadruple therapy than in those with double therapy. A reduction of immunosuppressive therapy in renal transplant patients was also successful in ureteric stenosis. Cidofovir has been successfully used in bone marrow (BMT) recipients with BK viruria and concomitant cystitis. Treatment with vidarabine appears to have some beneﬁt in BKV- associated haemorrhagic cystitis but, as with prosta- glandin E2 treatment, gave variable results (Moens and Rekvig, 2001).

Although for the treatment of polyomavirus diseases

a number of drugs and therapeutic regimens were associated with some beneﬁcial eﬀect, an established antiviral therapy is not available. The high prevalence

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PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

701 of these viruses in humans and the pathogenic

HUMAN POLYOMAVIRUSES

(eds Khalili K and Stoner GL), pp 409–430. Wiley-Liss, potential makes intensive analyses of viral activation

New York.

mechanisms and following development of speciﬁc Devireddy LR, Kumar KU, Pater MM and Pater A (2000) drugs an essential aim for the near future. BAG-1, a novel Bcl-2-interacting protein, activates expression of human JC virus. J Gen Virol, 2, 351–357. Do¨rries K (1997) New aspects in the pathogenesis of polyomavirus-induced disease. Adv Virus Res, 48, 205–261. Do¨rries K (2001) Latent and persistent human polyomavirus

ACKNOWLEDGEMENTS

infection. In Human Polyomaviruses. Molecular and Clinical Perspectives (eds Khalili K and Stoner GL), pp 197–237.

Work in the author’s laboratory was supported by

Wiley-Liss, New York.

Graduate College 520, Immunomodulation, of the Do¨rries K, Arendt G, Eggers C et al. (1998b) Nucleic acid detection as a diagnostic tool in polyomavirus JC-induced Deutsche Forschungsgesellschaft and by Grant

progressive multifocal leukoencephalopathy. J Med Virol, 1999.061 of the Wilhelm Sander-Stiftung.

54 , 196–203. Do¨rries K, Sbiera S, Drews K et al. (2003) Association of human polyomavirus JC with peripheral blood of immunoimpaired and healthy individuals. J Neurovirol (in

press). Do¨rries K, Vogel E, Gu¨nther S and Czub S (1994) Infection Agostini HT, Jobes DV and Stoner GL (2001) Molecular

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24 Human Parvoviruses

Kevin E. Brown

National Heart, Lung and Blood Institute, Bethesda, MD, USA

INTRODUCTION hepatitis B virus surface antigen (Cossart et al., 1975). In 1983 the chemical nature of the agent was

Parvoviruses are, as their name suggests, small viruses described (Summers et al., 1983) and the virus was (from the Latin, parvum meaning small), with a single-

classiﬁed initially with the autonomous parvoviruses. stranded DNA genome. To date there is only one

However, more recently B19 parvovirus was reclassi- known human pathogen (parvovirus B19) in this entire

ﬁed as the ﬁrst member and type species of the genus family of viruses. The family Parvoviridae consists of

Erythrovirus . Subsequently, three simian parvoviruses two subfamilies, the Densovirinae and the Parvovirinae:

have been identiﬁed in cynomolgus monkeys, pig- the Densovirinae are all viruses of insects, and the

tailed macaques and rhesus macaques (Green et al., Parvovirinae are viruses of vertebrates (Berns et al.,

2000; O’Sullivan et al., 1994). These erythroviruses 1995; Table 24.1). The Parvovirinae is further subdi-

share with B19 up to 60% homology, similar genome vided into three genera, based on the transcription map

organisation and similar biological behaviour in and their ability to replicate eﬃciently, either autono-

natural hosts. Interestingly, among the Parvovirinae mously (Parvovirus), with helper virus (Dependovirus) or

in general, the sequence homology suggests that B19 in erythroid progenitor cells (Erythrovirus).

parvovirus (genus Erythrovirus), minute virus of mice The genus Parvovirus contains a wide range of

(genus Parvovirus) and adeno-associated virus (genus viruses of mammals and birds, some of which cause

Dependovirus ) are equally diﬀerent from each other, major diseases in their animal hosts, but some, ﬁrst

suggesting that they diverged at about the same isolated as contaminants of cell cultures, have

evolutionary point in time.

unknown primary hosts. No member of this family is Although parvovirus-like particles have been known to infect humans. Dependovirus spp. have been

described in human stool specimens (Paver et al., described in a number of mammalian and avian

1973), these particles have not yet been fully char- species. To date at least eight diﬀerent primate

acterised and so their classiﬁcation must await dependoviruses have been described (Gao et al.,

deﬁnitive molecular studies. Moreover, similar parti- 2002) and adeno-associated viruses -2, -3 and -5 are

cles may on occasion be found in the faeces of common human infections. Although AAV DNA has

asymptomatic individuals, so the precise pathogenic been detected in some fetal abortion tissues (Friedman-

role of these agents remains unclear. More recently, Einat et al., 1997; Tobiasch et al., 1994), none of the

workers in Japan found evidence of single-stranded dependoviruses have been linked deﬁnitively with

DNA virus in the serum of a patient with hepatitis of disease in either humans or animals.

unknown aetiology (Nishizawa et al., 1997). Although Parvovirus B19 was discovered initially in the serum

thought originally to be a parvovirus, subsequent of an asymptomatic blood donor (coded 19 in panel B)

studies have shown that this virus, named TTV, is a as a cause of false positive results in counter-

member of a large group of related circoviruses (small immunoelectrophoresis tests for the detection of

DNA viruses with a single-stranded circular genome).

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

Table 24.1 The Parvovirinae Subfamily

Host Parvovirinae

Aleutian mink disease virus

Minks

Canine parvovivus (CPV)

Dogs, foxes

Mice minute virus (MVM)

Mice

Porcine parvovirus (PPV)

Pigs

Cats Dependovirus

Feline parvovirus (FPV)

Adeno-associated virus 1–6 (AAV)

Humans, primates

Avian adeno-associated virus (AAAV)

Birds

Canine adeno-associated virus (CAAV)

Dogs

Cattle Erythrovirus

Bovine adeno-associated virus (BAAV)

Parvovirus B19 (B19)

Humans

Simian parvovirus (SPV)

Primates

Rhesus parvovirus (RhPV)

Primates

Pigtailed macaque parvovirus (PtPV)

Primates

As with the human adeno-associated viruses, infection density of *1.41 g/ml in caesium chloride gradients, with these TTV-like viruses is common, and has not

whereas the empty particles have a density of 1.31 g/ml. been associated with any disease.

The particles do not contain lipids or carbohydrates. As with all parvovirus particles, the infectious particle is stable over a wide range of pH and resistant to lipid

PARVOVIRUS B19 solvents. It is not quite as resistant to heat as other parvoviruses (Blumel et al., 2002), but still only shows

a 2.5 log reduction in infectivity when heat-treated at Structure

508C for 8 h (Miyagawa et al., 1999). At high In electron micrographs of negatively stained prepara-

concentrations, infectious virus is still present after tions, parvovirus B19 appear as non-enveloped,

808C treatment for 72 h (Bartolomei Corsi et al., 1988). icosahedral particles with a diameter of 18–25 nm,

B19 can be inactivated by formalin, b-propiolactone, and often both ‘full’ and ‘empty’ capsids are visible

oxidising agents and g-irradiation. (Figure 24.1). The infectious particles have a buoyant

Genome Organisation Parvovirus virions consist of a linear single strand of

DNA with a molecular weight of approximately 1.8610 6 . In contrast to the autonomous parvoviruses, which preferentially encapsidate single-stranded DNA of negative polarity, parvovirus B19 encapsidates both positive and negative strands with equal frequency. The genome of B19 virus is one of the largest among the parvoviruses, with *5500 nucleotides. All parvovirus genomes have palindromic sequences at both 5’ and 3’ termini. These segments of the DNA fold back on themselves to form hairpin loops, which are stabilised by hydrogen bonding. The hairpin termini of B19 virus are identical at the 5’ and 3’ end, and substantially longer than those of most parvoviruses, with terminal repeat sequences of 365 nucleotides in length. There is some mismatching in the middle of the

Figure 24.1 The appearance of B19 virus particles by hairpin, leading to the occurrence of two diﬀerent immune electron microscopy showing the icosahedral structure

sequence configurations referred to as ‘flip’ and ‘flop’,

705 which is typical of parvovirus genomes (Deiss et al.,

HUMAN PARVOVIRUSES

Viral Proteins 1990).

The transcription map of B19 distinguishes it from The only unspliced transcript encodes the non- other Parvovirinae. There is a single strong promoter at

structural protein, a 78 kDa phosphoprotein (Ozawa the far left side of the genome (map point 6, or p6) and

and Young, 1987). Consistent with its role in viral unusual polyadenylation signals in the middle of the

propagation, the protein has DNA-binding properties genome. As is usual with parvoviruses, the 5’ end of the

(Leruez-Ville et al., 1997), adenosine and guanosine genome codes for the non-structural protein and the 3’

triphosphatase activity (Momoeda et al., 1994) and end of the genome for the capsid proteins. There are

nuclear localisation signals (Momoeda, Young and nine RNA transcripts in infected erythroid cells, and

Kajigaya, unpublished observations). Expression of the three major viral proteins, one non-structural

the non-structural protein causes host cell death protein and two capsid proteins, are produced by

through induction of apoptosis (Moﬀatt et al., 1998; alternative splicing from the promoter and its accom-

Ozawa et al., 1988).

panying leader sequence (Figure 24.2; Ozawa et al., There are two structural proteins in B19 virus, VP1 1987). The relative quantities of the major and minor

(84 kDa) and VP2 (58 kDa), which diﬀer only in that capsid proteins are in part regulated by the presence of

VP1 has an additional 227 amino acids at the amino- multiple upstream AUG codons situated before the

terminus (Shade et al., 1986). The capsid of B19 is authentic transcription initiation codon (Ozawa et al.,

composed of 60 capsomeres: VP2 is the major capsid 1988). In addition, there are transcripts for several

protein, with only *5% VP1 in the infectious particle. smaller peptides of 7.5 and 11 kDa (Luo and Astell,

Expression of VP2 capsomeres alone will self-assemble 1993; St Amand and Astell, 1993) of unknown

to form recombinant empty capsids that resemble B19 function.

particles morphologically and antigenically (Kajigaya

Figure 24.2 Transcription map of parvovirus B19 Figure 24.2 Transcription map of parvovirus B19

Structural studies of B19 have been undertaken for B19 using both crystal diﬀraction and cryo-EM studies. They indicate that, as seen for the autonomous parvovirus, B9 has the central structural core of eight antiparallel b-sheets, but the prominent spikes on the three-fold axis of canine and feline parvoviruses are absent from B19 capsids (Agbandje et al., 1994; Chipman et al., 1996).

Virus Variation No antigenic variation in B19 has been demonstrated,

and there appears to be a single, stable antigenic type of B19 virus. Infection is followed by lifelong immunity, which also indicates a single neutralisable type. Minor variation between diﬀerent isolates of the virus in their reactivity with mouse monoclonal antibodies has been detected but this has so far not proved to be of epidemiological or diagnostic signiﬁcance.

Similarly, the genome of B19 virus shows a constancy that is typical of viruses with DNA genomes, with *2% sequence variability between isolates. Small changes of nucleotide sequence have been detected by several investigators using restriction enzyme analysis or direct sequence analysis, but there is no correlation to specific disease presentation. In a study of 12 viruses isolated in Japan at two different times, 1981 and 1986–1987, the genome type differed during each time period, but B19 viruses with similar genome types disseminated widely in Japan during each time period (Umene and Nunoue, 1990).

In 1998, a B19 isolate, tentatively termed V9 was identified in a French child with transient aplastic crisis, which on sequence analysis was seen to be markedly different (>10% nucleotide difference) from other B19 sequences (Nguyen et al., 1999). Subsequent studies have identified both similar sequences, and a third group of B19 isolates with 10% sequence difference from both classical B19 sequences and V9 sequences (Nguyen et al., 2002; Servant et al., 2002). It is unknown whether these different genotypes will have different disease characteristics. The prevalence of these viruses in the general population is also unknown currently, with no variant B19 sequences detected in plasma pools from more than 120 000 Danish blood donors (Nguyen et al., 2002). In addition, despite the differences in the DNA sequences, the capsid proteins sequence is conserved, and V9 capsids show serological cross-reactivity with B19 capsids (Heegaard et al., 2002).

PATHOGENESIS

For a number of years following its discovery, B19 virus appeared to be associated with, at most, a mild, non-specific, febrile illness accompanied by self-limiting leukopenia. In the early 1980s the central role of B19 virus in the aetiology of aplastic crisis in chronic haemolytic anaemias (Pattison et al., 1981) was identified and then, in 1983, erythema infectiosum (fifth disease) began to emerge as the common manifestation of B19 virus infection (Anderson et al., 1983). Subsequently the virus has been shown to be responsible for a significant percentage of cases of hydrops fetalis (Anand et al., 1987) and to cause chronic infection in the immunocompromised (Kurtzman et al., 1988, 1989a). It is now clear that the pathogenesis of B19 virus-associated disease involves two components. The first is due to the lytic infection of susceptible dividing cells and the second is dependent upon interaction with the immune response.

Volunteer Studies

Two studies of volunteers infected experimentally have allowed the kinetics of infection to be elucidated (Anderson et al., 1985a; Potter et al., 1987). In both cases volunteers (nine and three, respectively) were infected by intranasal inoculation of a saline solution contain-

ing approximately 10 8 virus particles. The volunteers were followed daily and clinical, biochemical, haematological and viral studies undertaken (Figure 24.3).

Viraemia was ﬁrst detected from day 6 and reached

a peak at days 8–9 at about 10 11 particles/ml, a level comparable to that seen in natural infections in blood donors and patients with aplastic crisis (Anderson et al., 1985b). Virus was also detected in throat swabs and gargles at the time of viraemia only. Virus was not found in urine or faeces. On days 6–8 the volunteers showed the typical symptoms of a viraemia, with headache, myalgia and chills, associated with pyrexia. These features are generally thought to be due to the production of inﬂammatory cytokines. During the viraemia reticulocyte numbers fell to undetectable levels, recovering 7–10 days later, and there was a consequent temporary fall in haemoglobin of about l g/dl in a normal individual. Lymphopenia, neutropenia and thrombocytopenia also occurred, although slightly later, with the lowest numbers being recorded 6–10 days after inoculation and not so consistently as the changes in reticulocyte numbers and haemoglobin concentration.

706

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

HUMAN PARVOVIRUSES

Figure 24.3 Virological, haematological and clinical events during B19 virus infection of volunteers

In the second study, bone marrow morphology was for 2–4 days and the joint symptoms for 4–6 days. In examined at intervals (Potter et al., 1987). On day 6

the second study none of the patients developed the post-inoculation the marrow appeared normal. How-

second phase symptoms. It may be signiﬁcant that in ever, at day 10 there was an almost total loss of

the second study all (3/3) the volunteers were male and erythroid precursors at all stages of development. The

that joint symptoms in natural B19 infection appear to erythroid progenitor cells (BFU-E) from bone marrow

be much less frequent in male patients. and peripheral blood were reduced at this time. The

All the volunteers who received <10 6 virus particles myeloid compartment of the bone marrow appeared

in the inoculum and were B19 antibody-negative normal, but myeloid precursors from peripheral blood

(IgG<0.3 au) developed viraemia and an antibody were reduced as soon as 2 days from the onset of the

response to the virus. Volunteers with a positive B19 viraemia.

IgG (IgG<2 au) did not become infected. One of the The viraemia resolved as the patients developed a

pilot volunteer patients had ‘equivocal’ levels of IgG and detectable antibody response (Figure 24.3) and a

appeared to develop a modiﬁed infection, with minimal second phase of the illness began in most of the

viraemia and a boosting of his immune response. volunteers at day 15–17 (Anderson et al., 1985a), as the

In both studies, viraemia was monitored by the IgM response peaked and IgG became detectable,

relatively insensitive dot–blot hybridisation technique. characterised by pruritis followed by the development

Studies with PCR, especially nested PCR, indicate that of a ﬁne maculopapular cutaneous eruption. The rash

B19 DNA can be detected for many weeks or months extended over the limbs and was accompanied by

following acute infection (Clewley, 1989; Musiani et al., arthralgia and a mild arthritis. The rash was present

1995; Patou et al., 1993).

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

Cell Tropism philic nuclear inclusion bodies. These cells have been recognised for more than 50 years and were originally observed in patients with haemolytic anaemia and

Parvovirus B19, like the autonomous parvoviruses, aplastic crisis (Owren, 1948). By electron microscopy depends on certain host cell factors or functions only

virus particles are seen in the nucleus and lining

cytoplasmic membranes, and infected cells show mitosis for replication. However, B19 also has a very

present in cells transiting the late S or early G 2 phase of

marginated chromatin, pseudopod formation and narrow target cell range and can only be propagated in

cytoplasmic vacuolation, all typical of cells undergoing human erythroid precursor cells. When bone marrow

apoptosis (Morey et al., 1993). However, not all the or peripheral blood cells are cultured to develop

giant pronormoblasts contain virus particles, and part erythroid colonies in the presence of B19 virus, the

of the cytopathy probably reﬂects the expression of the formation of colonies is inhibited. For erythroid cells

non-structural protein. Megakaryocytopoiesis is also from bone marrow, susceptibility to parvovirus B19

inhibited by B19 parvovirus in vitro but this is in the increases with diﬀerentiation; the pluripotent stem cell

absence of virus replication and almost certainly due to appears to be spared and the main target cells are

the cytotoxic eﬀect of non-structural protein (Srivastava the BFU-E and CFU-E cells (cells capable of giving

et al. , 1990).

rise to erythroid colonies in vitro) and erythroblasts (Takahashi et al., 1990).

Erythroid progenitors from a variety of sources have Immune Response been used to study in vitro viral replication, including human bone marrow (Ozawa et al., 1987), fetal liver

The pattern of parvovirus disease is strongly inﬂuenced (Brown et al., 1991b; Yaegashi et al., 1989) and

by the immune response. Bone marrow depression in peripheral blood. B19 will also replicate in a limited

parvovirus infection occurs during the early viraemic number of erythropoietin-dependent cell lines, includ-

phase and under normal conditions is terminated by a ing UT7/Epo (Shimomura et al., 1993) and KU812Ep6

neutralising antibody response. The pathogenesis of (Miyagawa et al., 1999). However, the yield of virus

the rash in erythema infectiosum and polyarthropathy from all these cultures is poor, and they cannot be used

is almost certainly immune complex-mediated. In as a source of antigen for diagnostic tests.

volunteer studies, the rash and joint symptoms Erythroid speciﬁcity of parvovirus B19 is due to the

appeared when the high titre of viraemia had dropped tissue distribution of the virus’ cellular receptor,

signiﬁcantly and coincident with a detectable immune globoside, also known as blood group P antigen

response (Anderson et al., 1985a). (Brown et al., 1993). P antigen is found on erythroid

Persistent B19 parvovirus infection is the result of progenitors, erythroblasts and megakaryocytes. Puriﬁed

failure to produce eﬀective neutralising antibodies by P antigen (globoside) blocks the binding of virus to

the immunosuppressed host. Antibodies to parvovirus, erythroid cells and these cells can be protected from

as determined in immunoassays or ELISA, are not infection by preincubation with monoclonal antibody

present in most patients, but a pattern of antibody to globoside. P antigen is also present on endothelial

response suggestive of early infection (weak IgM cells, which may be targets of viral infection involved

antibody and IgG antibody directed to the major in the pathogenesis of transplacental transmission,

capsid protein) may be found in patients with possibly vasculitis and the rash of ﬁfth disease, and on

congenital immunodeﬁciency (Kurtzman et al. , fetal myocardial cells (Rouger et al., 1987). Rare

1989b). A poor reaction on immunoblot testing is a individuals who genetically lack P antigen on erythro-

consistent ﬁnding and correlates with poor neutralising cytes are resistant to B19 infection, and their bone

activity for the virus in erythroid colony assays marrow cannot be infected with B19 in vitro (Brown

(Kurtzman et al., 1989b). Conﬁrming that the role of et al. , 1994b). However, erythroid speciﬁcity may also

the humoral response appears to be dominant in

be modulated by speciﬁc erythroid cell transcription controlling human parvovirus infection, administra- factors (Liu et al., 1992).

tion of commercial immunoglobulins can cure or The virus is cytotoxic, producing a cytopathic eﬀect

ameliorate persistent parvovirus infection in immuno- with characteristic light and electron microscopic

deﬁcient patients: ﬁfth disease symptoms can be (Young et al., 1984) changes. Infected cultures are

precipitated by treatment of persistent infection with characterised by the presence of giant pronormoblasts

immunoglobulin (Frickhofen et al., 1990). (Figure 24.4), 25–32 mm in diameter, with cytoplasmic

The infected fetus may suﬀer severe eﬀects because vacuolisation, immature chromatin and large eosino-

red blood cell turnover is high and the immune

709 response deﬁcient. During the second trimester there is

HUMAN PARVOVIRUSES

studies predict case-to-case intervals of 6–11 days,

a great increase in red cell mass. Parvovirus particles which accords well with case-to-case intervals observed can be detected by electron microscopy within the

both in outbreaks of erythema infectiosum and in haematopoietic tissues of liver and thymus (Field et al.,

aplastic crisis. Transmission between siblings is com- 1991). B19 DNA and capsid antigen have been

mon, and in families where more than one child is detected in the myocardium of infected fetuses (Porter

aﬀected by chronic haemolytic anaemia all susceptibles et al. , 1990), and there is evidence that the fetus may

should be monitored for 2 weeks if one develops an develop myocarditis (Naides and Weiner, 1989),

aplastic crisis.

compounding the severe anaemia and secondary cardiac failure. By the third trimester, a more eﬀective fetal immune response to the virus may account for the decrease in fetal loss at this stage of pregnancy.

Transmission EPIDEMIOLOGY

Although spread from respiratory tract to respiratory Parvovirus B19 is a common infection in humans, and

tract is the common route of transmission of B19 virus, serological studies indicate that infection is worldwide,

the high-titre viraemia which occurs during infection with infections occurring in all populations, apart from

can lead to transmission by blood and blood products. some isolated groups in Africa (Schwarz et al., 1989)

Virus is found in donated units of blood, although the and Brazil (de Freitas et al., 1990). In temperate

incidence of high titre is low (1/20 000–40 000 in climates infection occurs throughout the year but

epidemic periods; Cohen et al., 1990), as would be outbreaks are more common in late winter, spring and

expected of an acute infection circulating mainly the early summer months. These outbreaks of infection

among children. However, testing donated blood are often centred on primary schools, where up to 40%

samples by more sensitive PCR technique suggests of the school may be clinically aﬀected by the rash

that B19 can be detected in *1/3000 units (McOmish illness of erythema infectiosum. If one includes

et al. , 1993) and perhaps in >1/150 samples in one subclinical infection then the attack rates may be as

study (Yoto et al., 1995). Blood-borne transmission high as 60% of the susceptible population in a school

has been shown to occur in recipients of whole blood (Plummer et al., 1985). During these outbreaks,

and of factor VIII concentrates (Mortimer et al., 1983). susceptible adults (parents and teachers of cases)

The frequency of B19 seropositivity among haemo- frequently become infected.

philiacs is signiﬁcantly higher than in normals, except In addition to seasonality, the virus exhibits longer-

for those haemophiliacs who have received factor VIII term cycles: in Jamaica, peaks of incidence (monitored

concentrates that have been heated to 808C for 72 h as cases of aplastic crisis) occur every 3–4 years. In the

(Williams et al., 1990). Even so, B19 virus can be UK, the cycle seems somewhat longer, with peaks

transmitted by treated blood products (Lyon et al., occurring every 4–5 years.

Clinical observations of the frequency of cases of erythema infectiosum among different age groups reflect the serological profile of the population;

Table 24.2 Disease manifestations and persistence of antibodies are most commonly acquired between ages parvovirus B19 infection in diﬀerent host populations

4–10 years, after which the frequency continues to rise,

Host but more slowly. By age 15 years approximately 50%

of children have detectable IgG. Infection also occurs in adult life, so that more than 90% of the elderly have

Fifth disease

Acute

Normal children

Normal adults detectable antibody (Cohen and Buckley, 1988).

Patients with increased version rate of 1.5% (Koch and Adler, 1989). Studies

Women of child-bearing age show an annual serocon-

Transient aplastic

Acute

erythropoiesis in diﬀerent countries (USA, France, Germany, Japan)

crisis

Immunodeﬁcient show similar patterns.

Persistent anaemia

Chronic

/immunocompromised Case-to-case intervals, determined by the time

patients

elapsing between acquisition and excretion of the Fetus (<20 weeks)

Hydrops fetalis/

Acute/

congenital anaemia chronic

virus, are independent of the type of disease. Volunteer

CLINICAL FEATURES The consequences of B19 virus infection range from

the wholly asymptomatic to serious and potentially fatal conditions in a minority of the population that is particularly predisposed. The spectrum of clinical consequences of infection depends in part on the natural variation in symptomatology that occurs, and in part on recognisable host factors (Table 24.2).

Minor Illness Combined clinical and laboratory studies of infection

in children, in whom B19 infection is most common, have indicated that about half of all infections are asymptomatic. Non-speciﬁc respiratory tract illness is the next most common consequence of infection, at least in boys (Grilli et al., 1989). This can be mild or severe enough to mimic inﬂuenza and these respiratory tract illnesses coincide with the viraemic phase of the illness.

Rash Illness/Erythema Infectiosum Erythema infectiosum is most common in children

aged 4–11 years and was probably first described by Robert Willan in 1799 and illustrated subsequently in his 1808 textbook (van Elsacker-Niele and Anderson, 1987). In 1905, Cheinisse classified it as the ‘fifth rash disease’ of the classical exanthema of childhood (Cheinisse, 1905), and this name, ‘fifth disease’, remains in use to this day. The link between B19 virus infection and erythema infectiosum was made in 1983 (Anderson et al., 1984). During the last 10 years the use of specific laboratory tests for the diagnosis of B19 virus infection has revealed a spectrum of rash illness, due to B19 infection, in which the classic features of erythema infectiosum occupy a central position and parvovirus B19 is now known to be the only aetiological agent for erythema infectiosum (Anderson et al., 1984).

Fifth disease was well described by clinical investigators prior to the discovery of B19 (Ager et al., 1966; Balfour, 1969; Brass et al., 1982). Infection is characterised by a non-speciﬁc prodromal illness which often goes unrecognised. Fifth disease may be associated with symptoms of fever, coryza, headache and mild gastrointestinal symptoms (nausea, diarrhoea). The exanthem occurs in three stages. Approximately 18 days after acquisition of the virus, and 2–5 days following the prodromal stage of the infection, the classic ‘slapped-cheek’ eruption appears,

a ﬁery red rash on the cheek, accompanied by relative

circumoral pallor (Figure 24.5). The edges of the involved areas may be slightly raised. At this stage the appearance may be suggestive of scarlet fever, drug sensitivity or other allergic reactions, or collagen vascular diseases. The second stage appears 1–4 days later, an erythematous maculopapular rash on the trunk and limbs. This rash is initially discrete but spreads to involve large areas. As this eruption fades it produces a typical lacy or reticular pattern. This third stage of the exanthem is highly variable in duration and may be transient or recurrent over 1–3 or more weeks, with periodic evanescence and recrudescence. The rash can be exacerbated by exercise, emotion, hot baths or sunlight. In addition, there may be great variation in the eruption’s appearance, from a very faint erythema that is easily missed to a ﬂorid exanthema, and it is often pruritic in adults, especially on the soles of the feet (Woolf et al., 1989). While classic cases of erythema infectiosum are easy to recognise clinically, especially during outbreaks, the wide variation in the form of the rash may make individual cases hard to distinguish from other viral exanthema, including rubella.

Erythema infectiosum is essentially a benign disease in which the major complication is joint involvement (see below). Other reported complications include cases of transient haemolytic anaemia, encephalitis with recovery without residua, and encephalopathy in

a 9 month-old boy, resulting in permanent sequelae. Each of these cases occurred prior to the appreciation of B19 virus as the causative agent for erythema infectiosum; in view of the diﬃculty in diagnosing sporadic cases of erythema infectiosum on clinical grounds, it cannot be certain that these cases were due to B19 virus infection.

There have been occasional cases of B19 infection associated with a purpuric rash. In most B19 infections the platelet count is normal (Lefre`re et al., 1985) but thrombocytopenia has been recorded (Mortimer et al., 1985b; Saunders et al., 1986). Although the purpura in most cases of B19 infection is transient, some patients diagnosed with idiopathic thrombocytopenic purpura do have evidence of recent B19 infection (Murray et al. , 1994). Similarly some B19 infections may resemble clinical Henoch–Schonlein purpura, but it is unclear what percentage of cases of this condition are caused by B19 (Heegaard and Taaning, 2002). Rarely, other dermatological presentations are seen; vesicopustular rash (Naides et al., 1988), glove and stocking syndrome (Halasz et al., 1992), other purpuric rashes with or without Koplik spots (Evans et al., 1992) and erythema multiforme (Frank et al., 1996; Lobkowicz et al., 1989).

710

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

Joint Involvement/Arthropathy

In children, B19 infection is usually mild and of short duration. However, in adults, and especially in women, there may be arthropathy in *50% of patients (Woolf, 1990). It is now also clear, with the widespread application of B19 diagnostic tests, that the joint involvement may occur in the absence of any evidence of a rash, although there are no estimates of how frequent this is.

In adults, the most common presentation is with a sudden onset of symmetrical arthralgia and even frank arthritis affecting the small joints of the hand. Proximal interphalangeal and metacarpophalangeal joints are most often affected, followed by wrists, ankles, knees and elbows. Shoulders, cervical spine and lumbar spine as well as the hips may also be involved. There may be pain and stiffness in the joints, which may

be accompanied by minor swelling or synovitis. Resolu- tion usually occurs within a few weeks, but persistent or recurring symptoms can continue for years (Reid et al., 1985). In children the joint involvement may be asymmetrical and symptoms seem more severe than in adults and may be of longer duration.

In the absence of a history of rash, the symptoms may be mistaken for acute rheumatoid arthritis, especially as prolonged symptoms do not correlate with serological studies, such as the duration of B19 IgM response, or persistent viraemia. In addition, B19 infection can be associated with transient autoanti- body production, including transient rheumatoid factor (Luzzi et al., 1985). In one large study of patients attending an ‘early synovitis’ clinic in England, 12% had evidence of recent infection with B19 (White et al., 1985). Three patients would have fulfilled the American Rheumatism Association’s diagnostic criteria for definite rheumatoid arthritis. B19 infection should be considered as part of the differential diagnosis in any patient presenting with acute arthritis. In contrast to rheumatoid arthritis, B19 infection is not generally associated with joint destruction. However, differentiation between early rheumatoid arthritis and B19 arthropathy is important, as immunosuppressive therapy prescribed for rheumatoid arthritis is not indicated in parvovirus B19 infection.

The role of parvovirus B19 in the aetiology of chronic arthritis is unclear. Parvovirus B19 DNA has been found in the synovial ﬂuid of a women with serologically proven B19 infection (Dijkmans et al., 1988) and in synovial ﬂuid cells of a patient with ‘reactive arthritis’ (Kandolf et al., 1989). However,

PCR ampliﬁcation studies should be interpreted with care: in one carefully performed controlled study, although B19 DNA was indeed detected in synovial tissue of 28% of children with chronic arthritis, it was also found in 48% of non-arthropathy controls (Soderlund et al., 1997), indicating that PCR-detectable DNA may persist in synovial tissues for months/years.

It has been postulated that B19 is involved in the initiation and perpetuation of rheumatoid arthritis leading to joint lesions (Takahashi et al., 1998), but these results have not been reproducible by other groups (El-Gabalawy, Goldbach-Mansky and Brown, unpublished observations). In addition, in one study of long-term follow-up, none of 54 patients with B19- associated arthralgia reported persistence of joint swelling or restricted motion, and no evidence of inﬂammatory joint disease was found (Speyer et al., 1998). It seems unlikely, though, that B19 plays a role in classic erosive rheumatoid arthritis, but understanding the pathogenesis of B19 arthropathy may provide insight into the mechanisms by which rheumatoid arthritis develops.

Transient Aplastic Crisis

Transient aplastic crisis is the abrupt cessation of erythropoiesis characterised by a fall from steady-state values of haemoglobin concentration, disappearance of reticulocytes from peripheral blood and the absence of red blood cell precursors in the bone marrow. It is classically seen in patients with haemolytic anaemia, where the B19-induced cessation of erythropoiesis lasts 5–7 days and patients present with symptoms of worsening anaemia, namely fatigue, shortness of breath, pallor, lassitude, confusion and sometimes congestive cardiac failure. The event is serious in most patients and occasionally it is fatal. Blood transfusion is required in the acute phase but after about 1 week the bone marrow recovers rapidly. There is a brisk reticulocytosis and the haemoglobin concentration returns to steady-state values.

Transient aplastic crisis was the ﬁrst clinical illness associated with B19 infection. When stored sera from 600 children admitted to a London hospital were examined, six children had evidence of recent B19 infection (either antigenaemia or seroconversion). All were Jamaican immigrants with sickle cell disease presenting with aplastic crisis. There was a reduced haematocrit, and evidence of aplastic crises on their bone marrow (Pattison et al., 1981). Retrospective studies of sera from Jamaican sickle-cell patients showed that 86% of transient aplastic crises were

HUMAN PARVOVIRUSES

711

associated with recent parvovirus infection (Serjeant et al. , 1981). Studies since then have shown that more than 90% of all cases of aplastic crises in patients with chronic haemolytic anaemia are due to B19 virus infection (Anderson et al., 1982; Serjeant et al., 1993). Most cases occur in children under the age of 20 (Serjeant et al., 2001) but adults who remain susceptible to the virus infection may have B19-associated aplastic crises in later life.

B19-associated aplastic crises are not confined to patients with sickle cell anaemia, but have been described in a wide range of patients with underlying haemolytic disorders, such as hereditary spherocytosis (Kelleher et al., 1983), thalassaemia (Brownell et al., 1986), red cell enzymopathies, such as pyruvate kinase deficiency (Duncan et al., 1983), and autoimmune haemolytic anaemia (Smith et al., 1989). Aplastic crisis can also occur under conditions of erythroid ‘stress’, such as haemorrhage (Frickhofen et al., 1986), iron deficiency anaemia (Lefrere and Bourgeois, 1986) and following kidney (Neild et al., 1986) or bone marrow transplantation (Niitsu et al., 1990). Acute anaemia has been described in normal patients (Hamon et al., 1988) and a drop in red cell count (and reticulocytes) was seen in healthy volunteers (Anderson et al., 1985a), but usually there is sufficient haematopoietic reserve and this is not apparent clinically.

Although suﬀering from an ultimately self-limiting disease, patients with aplastic crisis can be severely ill. Symptoms include not only the dyspnoea and lassitude of worsening anaemia, but the patient may develop confusion, congestive heart failure, severe bone marrow necrosis (Conrad et al., 1988), cerebrovascular complications (Wierenga et al., 2001) and the illness can be fatal (Serjeant, 1992). Aplastic crisis can be the ﬁrst presentation of an underlying haemolytic disease in a well-compensated patient (Cutlip et al., 1991; McLellan and Rutter, 1987).

Community-acquired aplastic crisis is almost always due to parvovirus B19 (Anderson et al., 1982) and should be the presumptive diagnosis in any patient with anaemia due to abrupt cessation of erythropoiesis, as documented by reduced reticulocytes and bone marrow appearance. In contrast to patients with erythema infectiosum, transient aplastic crisis patients are often viraemic at the time of presentation with concentra-

tions of virus as high as 10 14 genome copies/ml, and the

diagnosis is readily made by detection of B19 DNA in the serum. As the B19 DNA is cleared from the serum, B19-speciﬁc IgM becomes detectable. Typical transient aplastic crisis is readily treated by blood transfusion. It is a unique event in the patient’s life, and following the acute infection immunity is lifelong.

Transient aplastic crisis and B19 infection in haematologically normal patients is often associated with changes in the other blood lineages. There may be varying degrees of neutropenia (Doran and Teall, 1988; Saunders et al., 1986) and thrombocytopenia (Inoue et al., 1991). Transient pancytopenia is less common (Frickhofen et al., 1986; Hanada et al., 1988; Saunders et al., 1986). Haemophagocytosis, which can occur after many diﬀerent viral infections, has been noted in acute (Boruchoﬀ et al., 1990; Muir et al., 1992) and persistent B19 infection (Koch et al., 1990).

B19 Infection in Pregnancy

Animal parvoviruses are known to cause congenital infections in a variety of animals, leading to fetal loss in rodents and pigs (Mengeling, 1975) and to congenital infections in cats (Kilham and Margolis, 1966) and dogs (Jeﬀeries and Blakemore, 1979). Thus, the potential for parvovirus B19 causing disease in pregnancy is high, and the occurrence and eﬀects of B19 virus infection in pregnancy have always been of interest.

Initial studies of the relationship between parvovirus B19 infection and pregnancy did not show any adverse relationship. No increase in fetal malformations could

be detected following clinically diagnosed outbreaks of erythema infectiosum (Ager et al., 1966) and in a study of sera taken during the first month of life from infants with birth defects, no B19 antigen or specific IgM could be detected (Mortimer et al., 1985a). However, there have been a large number of case reports of B19 infection in pregnancy leading to an adverse outcome, either miscarriage or hydrops fetalis (Anand et al., 1987). The clinical features have been remarkably similar. In cases where pathological studies were undertaken, the fetuses showed evidence of leukoery- throblastic reaction in the liver, and large pale cells with eosinophilic inclusion bodies and peripheral condensation or marginatum of the nucleus were seen. Parvovirus B19 DNA could be detected by dot– blot or in situ hybridisation, and parvovirus particles seen by electron microscopy (Field et al., 1991). There are however many more reports of favourable outcome after confirmed parvovirus B19 in pregnancy (Kinney et al. , 1988).

To address the frequency of complications associated with parvovirus B19 infection in pregnancy, two prospective studies in the UK followed 190 and 255 women, respectively, with serologically conﬁrmed B19 during pregnancy (Miller et al., 1998; Public Health Laboratory Service Working Party on Fifth Disease,

712

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

713 1990). In both studies there was an excess fetal loss rate

HUMAN PARVOVIRUSES

defect) and it was concluded that the risk of congenital (spontaneous abortion and intrauterine deaths) during

malformations due to B19 infection was less than 1%. the ﬁrst 20 weeks of pregnancy of 9% compared to

In addition, no late eﬀects of B19 infection were seen in controls, with the greatest fetal loss due to B19 during

the children followed-up at 7–10 years. In a similar study weeks 9–16. The fetus was lost most frequently 4–6

in America, again no increase in adverse long-term weeks after the maternal infection, although there was

outcomes was observed. Thus, there is no evidence to no diﬀerence between outcomes following either

date that B19 causes birth defects, although it should be asymptomatic or symptomatic maternal infection. It

remembered that the sample sizes in the studies have thus appears that B19 infection is a signiﬁcant cause of

been too small to detect a rare defect, i.e. one with a rate second trimester fetal loss.

of 1% or less. Nevertheless, there is no reason to In seven of the cases the fetus developed hydrops

recommend termination of pregnancies complicated by fetalis and the risk of hydrops fetalis following B19

laboratory-proven B19 virus infection. infection during weeks 9–20 was calculated at 2.9%.

Infants born with chronic anaemia following a history Three of the hydropic infants survived: two of the three

of maternal B19 exposure and intrauterine hydrops were treated with intrauterine blood transfusions.

have been described (Brown et al., 1994a). In three such Other studies have shown that spontaneous resolution

cases, who at birth were found to have hypogamma- of hydrops following B19 infection does occur (Pryde

globulinaemia, viral DNA was present in the bone et al. , 1992; Sheikh et al., 1992) and there are risks

marrow although absent from the serum. One child associated with intrauterine blood transfusions. How-

died but the other two remained persistently anaemic ever, several studies have shown that intrauterine blood

in spite of immunoglobulin therapy. In a related study transfusions may enhance survival: an observational

of bone marrow from children with Diamond–Black- study from the UK reported survival of 9/12 fetuses

fan anaemia, B19 DNA was found in 3/11 marrow treated with transfusions, compared to 13/26 untreated,

smears, all from children that underwent remission of

a significant difference after allowing for ultrasound their anaemia. Thus, intrauterine B19 infection may be findings and gestational age (Fairley et al., 1995).

responsible for some cases of congenital anaemia, Non-immune hydrops fetalis is rare (1:3000 births)

although the incidence is probably rare. and in approximately 50% of cases the aetiology is unknown. In a study of 50 cases, the majority were due to cardiovascular or chromosomal abnormalities, but parvovirus B19 DNA was detected by in situ hybridi-

B19 Infection in the Immunosuppressed sation in the lungs of four fetuses despite there being

no known epidemic of B19 during the study period

A chronic B19 infection resulting in pure red cell (Porter et al., 1988). Parvovirus B19 probably causes

aplasia (PRCA) occurs in immunocompromised indi- 10–15% of all cases of non-immune hydrops (Yaegashi

viduals who have failed to produce neutralising et al. , 1998), and it is worth investigating B19 as a

antibody to the virus. Chronic infection has been cause of non-immunological hydrops fetalis by speciﬁc

reported in a wide variety of immunosuppressed tests for B19.

patients, ranging from patients with congenital immu- nodeﬁciency (Kurtzman et al. , 1987), acquired immunodeﬁciency (AIDS; Frickhofen et al., 1990),

Congenital Malformations lymphoproliferative disorders (Kurtzman et al., 1988) and transplant patients (Frickhofen and Young, 1989).

Although sporadic case reports have noticed an The stereotypical presentation is with persistent association between genitourinary abnormalities

anaemia rather than immune-mediated symptoms of (Public Health Laboratory Service Working Party on

rash or arthropathy. Patients have absent or low levels Fifth Disease, 1990; Rodis et al., 1990), cerebral

of B19 speciﬁc antibody (Kurtzman et al., 1989b) and abnormalities (Katz et al., 1996) and ocular malforma-

persistent or recurrent parvoviraemia, as detected by tions (Hartwig et al., 1989), the abnormalities reported

B19 DNA in the serum. Bone marrow examination are all relatively common and, without a control

generally reveals the presence of scattered giant population, it is diﬃcult to interpret the abnormalities

pronormoblasts. Administration of neutralising anti- as being due to B19. No systematic studies have shown

body in the form of human normal immunoglobulin evidence for congenital abnormalities following B19

often leads to a fall in virus titre, reticulocytosis and in infection. In the two British studies only one case with

some cases rash illness presumed to be due to immune congenital malformation was seen (a ventral septal

complexes (Kurtzman et al., 1988).

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

Table 24.3 Atypical disease presentations of parvovirus B19 rare, large multicentre trials may be required to substantiate or disprove the causal relationship.

Infection in the healthy host Neurological disorders/encephalitis Myocarditis

LABORATORY DIAGNOSIS

Hepatitis Glomerulonephritis Haematological

Aplastic crisis is the only one of the clinical syndromes Thrombocytopenia

that can be assumed, with some accuracy, to be due to Neutropenia

B19 infection. Even so, in patients with chronic Pancytopenia

haemolytic anaemia moderate to severe degrees of Haemophagocytosis

hypoplasia may be associated with systemic bacterial Rheumatic diseases

infection or marrow-suppressive drugs, and anaemia in Chronic fatigue syndrome

Fibromylagia the immunocompromised may have many other

Myositis causes. Illnesses associated with maculopapular rashes Vasculitis

with or without joint involvement also have a multi- Kawasaki disease

plicity of causes and only a minority of cases of hydrops fetalis will prove to be due to B19 infection. Thus, accurate diagnosis of B19 infection depends

The prevalence of B19-induced anaemia in HIV- upon speciﬁc laboratory tests. seropositive patients is probably higher than recog-

nised at present. In one early study of 50 patients with AIDS, no patients with B19 viraemia were identiﬁed.

Specimens In a larger cohort study, B19 DNA was found in only 1/191 (0.5%) of HIV-seropositive homosexuals. How-

Serum is the principal specimen used for the laboratory ever, B19 DNA was found in 5/30 (17%) of

diagnosis of B19 infection. This approach is suitable transfusion dependent HIV-seropositive homosexuals,

for virus detection in cases of aplastic crisis, persistent and when a haematocrit of <20 was used as a

infection in immunosuppressed patients and persistent criterion, 4/13 (31%) were positive (Abkowitz et al.,

fetal infection. The detection of speciﬁc IgM antibody 1997). In contrast to the earlier studies, the marrow

in serum is the cornerstone of the diagnosis of rash morphology need not be suggestive of PRCA and giant

illness and arthropathy, and is often valuable in cases pronormoblasts may not be present.

of aplastic crisis. Standard blood specimens are all that In less severely immunosuppressed patients (i.e. SLE

are required and no special arrangements are needed on steroid therapy), prolonged anaemia following B19

for transport to or storage in the laboratory. It should infection has also been described (Koch et al., 1990).

be remembered that viraemic samples may contain However, in these patients there was a spontaneous,

high titres of infectious virus, and care should be taken albeit delayed, development of antibodies, and virae-

when handling samples, especially those taken early in mia resolved without therapy. Presumably such

the course of an aplastic crisis, to ensure that patients represent one end of the spectrum of disease

seronegative individuals in the laboratory are not manifestations of B19 in patients with a compromised

infected (probable cases of laboratory-acquired immune system.

infection have been described; Cohen et al., 1988). B19 assays can also be performed on tissue samples. DNA can be extracted from fresh, frozen or ﬁxed Atypical Presentations

samples for detection of parvoviral DNA. Detection of B19 virus by either immunohistochemistry or in situ

A wide variety of symptoms and diseases have been hybridisation can also be performed on formalin-ﬁxed, associated with parvovirus disease (Table 24.3; and see

paraﬃn-embedded tissue, so that standard pathology To¨ro¨k, 1997), generally as case reports or limited small

protocols can be used for dealing with bone marrow or series of patients. Determining the role of B19 in these

fetal tissue.

diseases is often diﬃcult: the diseases are rare and B19 may not be the only cause. In addition, with sensitive PCR-based assays B19 DNA can be detected in bone

Virus Detection marrow (Cassinotti et al., 1997) and other tissues (Soderlund-Venermo et al., 2002; Soderlund et al.,

The culture of parvovirus B19 in erythroid progenitor 1997) from healthy individuals. When the disease is

cells remains a research procedure and is not used

715 in the routine diagnosis of B19 infections. Instead,

HUMAN PARVOVIRUSES

diagnosis of recent B19 infection, although immuno- B19 virus detection relies on the detection of either

ﬂuorescent and Western blot assays are commercially viral antigen or viral DNA. Although ELISA- and

available. Due to the inability to grow B19 in standard haemagglutination-based assays are being developed

cell culture systems, until recently there has been a to detect viral antigen, this is generally in the context of

shortage of viral antigen for any diagnostic assays. blood screening (Wakamatsu et al., 1999). Instead, for

Attempts have been made to develop assays based on diagnostic purposes, DNA hybridisation and/or elec-

the use of synthetic peptides or fusion proteins in tron microscopy are commonly used. The common

Escherichia coli , but the epitopes presented by these clinical situation in which these tests are applied is to

products do not accurately reproduce the epitopes of the acute-phase specimen of cases of aplastic crisis or

the native capsids, and the practical results have immunosuppressed patients with chronic anaemia.

generally been disappointing, with lack of speciﬁcity DNA hybridisation using cloned viral genome labelled

when used on discriminating sera. The expression of with 32 P (Anderson et al., 1985b) will detect about 10 4 B19 capsid proteins as virion-like particles, using the

particles/ml, in specimens taken within 24 h of the baculovirus expression system (Brown et al., 1991a; onset of an aplastic crisis this will yield positive results

Kajigaya et al., 1991), appears to have overcome these in 60% of cases. Immune electron microscopy is only

problems, and assays based on these antigens show slightly less sensitive than DNA hybridisation.

excellent correlation with assays based on native virus The most sensitive technique for the detection of

(Kajigaya et al., 1991). The antigens are relatively easy virus requires ampliﬁcation of the DNA using PCR. A

to mass-produce and are non-infectious, and therefore variety of diﬀerent primers and probes have been

without hazard to laboratory workers. described, the most sensitive assays are capable of

The best assay to detect recent infection with B19 detecting 1–10 virus particles (Clewley, 1993; Patou et

remains the IgM capture assay in which IgM-speciﬁc al. , 1993). This can be a very useful tool, but as with all

antibody in the patient’s serum is bound by a solid PCR, there is also great propensity for cross-contam-

phase coated with antibody to human m-chains. The ination and false positive results. In addition, low

viral speciﬁcity of the bound antibody is determined by levels of viral DNA can be detected for months or even

the addition of B19 virus antigen to the solid phase. In years after acute infection (Cassinotti et al., 1997;

turn bound antigen is detected by the addition of Musiani et al., 1995) and thus the detection of B19

monoclonal anti-B19 antibody, which may itself be DNA by PCR alone cannot be used to diagnose acute

tagged with enzyme. Alternatively, an extra step may B19 infection.

be incorporated, whereby the bound monoclonal anti- The diagnosis of B19 infection in a fetus also

B19 is detected by the addition of labelled anti-mouse depends upon the detection of virus. Maternal infec-

immunoglobulin (Cohen et al., 1983). IgM tests using tion will have occurred some weeks previously and

an indirect format, are less useful for diagnosis due to maternal serum may therefore be B19-speciﬁc IgM-

their lack of both speciﬁcity and sensitivity. negative. In most instances the fetus has also been

Sera containing 10 au/ml or more of anti-B19 IgM found to be speciﬁc IgM-negative but there is

are unequivocally associated with recent infection. frequently a persistent viraemia. Therefore, the diag-

This relatively high threshold is set because sera nosis is best made by detection of virus in fetal blood

containing high concentrations of antirubella virus samples by the techniques described above. Equally

IgM may give low false positive results when tested for virus can be detected in fetal tissues taken at autopsy,

anti-B19 IgM, and vice versa (Kurtz and Anderson, from which DNA has been extracted or detected by

1985). This must be borne in mind when testing sera immunohistochemistry or in situ hybridisation on

taken within 2 weeks of a rubelliform illness. However, formalin-ﬁxed, paraﬃn-embedded tissue sections.

high concentrations of anti-Bl9 IgM usually appear Glutaraldehyde-ﬁxed material can also be used for

within 3–4 days of the onset of symptoms and electron microscopy, but scattered B19 particles within

antibody can be detected in over 90% of cases by the cells may be more diﬃcult to recognise, due to the

third day of transient aplastic crisis, or at the time of large number of ribosomes of similar size.

rash in erythema infectiosum. IgM antibody remains detectable for 2–3 months following infection. In patients presenting with symptoms of aplastic crisis,

Specific Antibody Detection and occasionally in rash-like presentations, speciﬁc IgM may not appear until 7–10 days after the onset.

Standard solid-phase enzyme-labelled immunoassays Therefore, if a negative or equivocal result for anti-B19 (ELISAs) are the preferred method for the serological

IgM is obtained with a serum taken within 10 days of IgM is obtained with a serum taken within 10 days of

B19 IgG can be detected by capture assay or preferably by indirect assay. It is usually present by day 7 of illness and probably is lifelong thereafter. As more than 50% of the population have IgG antibody to B19 infection, detection of B19 IgG in a single sample is not useful for the diagnosis of acute infection, but can be used to document seroconversion.

In 1995, the WHO Expert Committee on Biological Standardization recommended that an international standard (IU/ml) be set for anti-parvovirus B19 testing. The international standard chosen correlates well with the original arbitrary unit (au) scale in the earlier literature. In the 12 months following infection, patients have relatively high (<50 IU/ml) concentrations of anti-B19 IgG and the finding of such concentrations supports the diagnosis of infection during that time. However, there is marked individual variation in the maximum amounts of specific IgG that can be demonstrated in a patient, so that the finding of only low values (>20 IU/ml) does not exclude recent infection. Previous infection (and therefore immunity in most individuals) is indicated by the detection of >5 IU/ml of anti-B19 IgG. However, a negative result cannot be taken to be synonymous with susceptibility.

There is a poor humoral immune response in immunocompromised patients with chronic parvovirus infection and only low concentrations of anti-B19 IgG (and IgM) can be detected in these patients. None of the detectable antibody neutralises virus infectivity and this is taken to be an essential component of the pathogenesis of the chronic infection. The diagnosis of B19 infection in these patients is dependent on the detection of viral antigen or DNA.

TREATMENT AND PREVENTION There is no speciﬁc antiviral chemotherapy for B19

infection. Symptomatic relief of troublesome joint symptoms may be required for B19 virus-associated arthralgia, and blood transfusion may be necessary in the acute phase of aplastic crisis.

The only speciﬁc treatment for B19 infection is the intravenous administration of human immunoglobulin in cases of persistent infection in the immunocompro-

mised. Human normal immunoglobulin preparations are a good source of neutralising antibodies to B19 virus (Takahashi et al., 1991), since at least half the adult population have been exposed to the virus. Controlled trials have not been done but on an empirical basis the administration of 0.4 g/kg body weight/day for 5–10 days has proved eﬀective in reducing viraemia and allowing the haemoglobin to return to near-normal values (Frickhofen et al., 1990; Kurtzman et al., 1989a). Patients sometimes relapse months later but they have been shown to respond to repeat courses.

Prevention of disease by isolating susceptible individuals is impractical because infections may be subclinical, and even symptomatic individuals are most frequently infectious before any sign of illness. Theoretically, susceptible individuals with chronic haemolytic anaemia (or immunocompromised chil-

dren) could be temporarily protected by the administration of human immunoglobulin, but to date this has not been put into practice.

Many animal parvovirus infections are prevented in animals by vaccination, and the prospects for a B19 parvovirus vaccine are good. The immunogen will be recombinant capsid rather than attenuated or killed virus, due to the difficulty of cultivating B19 parvovirus in vitro and the potential dangers of inadvertently modifying, for the worse, the host range of B19 parvovirus by selection in vitro. Baculovirus-produced B19 parvovirus capsids induce neutralising antibodies in experimental animals (Kajigaya et al., 1991), even without adjuvant. The presence of VP1 protein in the capsid immunogen appears critical for the production of antibodies that neutralise virus activity in vitro, and capsids with supranormal VP1 content are even more efficient in inducing neutralising activity (Bansal et al., 1993). Sera from human volunteers immunised with one candidate vaccine had neutralising antibody titres equal to or higher than those observed after natural infection. Phase I trial results appear promising, and phase II trials are planned. However, the targets for such a vaccine remain to be determined. Should only patients at high risk of severe or life-threatening disease, such as sickle cell patients be protected? Or, in view of the wide variety of disease manifestations affecting all strata of the population, should a universal vaccine policy be pursued?

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25 Human Immunodeficiency Viruses

1 2 3 Robin A. Weiss 1 , Angus G. Dalgleish , Clive Loveday and Deenan Pillay

1 University College London and 2 St George’s Hospital Medical School, London, and

3 International Clinical Virology Centre, Great Missenden, UK

INTRODUCTION TO HUMAN RETROVIRUSES molecular biology of animal and human retroviruses (Coﬃn et al., 1997).

Retroviruses occur in numerous vertebrate species and Retroviruses are a single taxonomic group of RNA are associated with a diversity of diseases. Studies of

viruses that encode RNA-directed DNA polymerase animals have shown that retroviruses cause a wide

(reverse transcriptase, RT). Upon infection, this variety of neoplasms, many with human counterparts.

enzyme catalyses the synthesis of a double-stranded Leukaemia and sarcomas in chickens were ﬁrst

virion DNA. The provirus subsequently becomes identiﬁed as having a viral aetiology from 1908, and

integrated into host chromosomal DNA and serves retroviruses were later found to be associated with

as a template for viral genomic and messenger RNA malignant disease in mice, cats, primates and other

transcription by the host cell’s RNA synthetic and hosts, including ﬁsh. Apart from malignancy, retro-

processing systems (Figure 25.1). Other special features viruses are associated with autoimmune disease,

of retroviruses include a diploid RNA genome, high immunodeﬁciency syndromes, aplastic and haemolytic

frequency of intermolecular recombination between anaemias, bone and joint disease (osteopetrosis and

related viruses and the ability to acquire host genes arthritis) and neuropathy (Table 25.1). A comprehen-

which encode functions responsible for neoplastic sive text covers most aspects of the biology and

transformation (oncogenes). There also exist endogenous proviruses in the normal cellular DNA of many vertebrates (including humans), which represent ‘fossil’

Table 25.1 Diseases caused by retroviruses in animals infections of the germline suggestive of prior infection of the human species by retroviruses, and which are passed

Disease Species affected from generation to generation in a Mendelian manner. Leukaemia

Avian, mouse, cat, primates All retroviruses carry at least three genes in the order Lymphoma

Avian, mouse, cat, primates, ﬁsh 5’-gag–pol–env-3’. Gag encodes a precursor protein Carcinoma

Mouse (mammary), chicken (renal) which is cleaved to yield three or four structural core Sarcoma

Rat, chicken, ﬁsh and matrix proteins; pol encodes the reverse transcrip- Anaemia, aplasia

Cat, horse tase, protease and integrase, synthesised from a gag–pol Autoimmune disorders

Cat, primates precursor; and env encodes a precursor cleaved to form Nervous system

Cat, primates, sheep, mouse Immune deﬁciency

Sheep, goat, mouse the two envelope proteins, surface protein (SU) and Osteopetrosis

Chicken transmembrane (TM). The core proteins are often Arthritis

Goat named by molecular weight (e.g. p24 and p17 of HIV). The outer surface protein is glycosylated and is known

SLE, systemic lupus erthematosus. A wide variety of diseases are caused by animal retroviruses with familiar human counterparts of

as gp120 (e.g. for HIV, with an approximate molecular

unknown aetiology. The major associations are listed above.

weight of 120 000) or gp70 (e.g. g-retroviruses). The

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

Figure 25.1 Simpliﬁed replication cycle of a retrovirus. The virion containing two RNA genome copies enters the virus via a speciﬁc cell surface receptor complex. The single-stranded RNA genome is converted into a double-stranded DNA provirus by the virion enzyme reverse transcriptase. The provirus inserts into host chromosomal DNA in the same orientation as the original virion RNA. Transcription of RNA from the integrated DNA provirus is mediated by cellular RNA polymerases, and this RNA serves both as messenger RNA for the synthesis of viral antigens and as genomic RNA, which becomes packaged into progeny virion budding from the cell surface

anchored transmembrane protein, to which the surface viruses, which have not been shown to be pathogenic. protein is bound, is glycosylated in some retroviruses

More recently, retroviruses of higher vertebrates have (e.g. gp41 of HIV) but not others (e.g. p15E of gamma

been reclassiﬁed into seven distinct genera by dividing retroviruses). The DNA proviral genome is capped by

oncoviruses into ﬁve, as they are only distantly related long terminal repeats (LTRs) at both ends and the

by genome sequence and morphology (Figure 25.2, RNA form has a polyadenylate tract at the 3’ end like

Table 25.2). More generally, retroviruses are divided mRNA. The LTRs are responsible for integration of

into those with ‘simple’ genomes, having gag, pol and the DNA provirus with cellular DNA, and contain

env genes and perhaps one other, and those with important promoter and enhancer sequences which

‘complex’ genomes. Members of the latter group bind cellular and viral proteins regulating viral gene

possess regulatory genes, such as tat and rev of HIV expression.

and tax of HTLV, and accessory genes, such as nef, vif and vpr of HIV.

Four groups of retroviruses have been reported as CLASSIFICATION OF RETROVIRUSES

human infections but only the ﬁrst will be described in detail in this chapter:

Retroviruses used to be taxonomically divided into three subfamilies: the Oncovirinae, which include those

1. Human immunodeﬁciency virus types 1 and 2 (HIV-1 with oncogenic potential; the Lentivirinae or slow

and HIV-2) are the lentiviruses that cause acquired viruses, including HIV and the prototype Maedi–Visna

immune deﬁciency syndrome (AIDS). Before the virus (MVV) of sheep which causes progressive

term ‘HIV’ was coined in 1986, HIV was called wasting disease, pneumonia and degeneration of the

lymphadenopathy virus (LAV), HTLV-III or ARV. central nervous system; and the Spumavirinae or foamy

HIV and AIDS are the main topics of this chapter.

723 Table 25.2 Classiﬁcation of retroviruses of vertebrates

HUMAN IMMUNODEFICIENCY VIRUSES

Genus Example Virion morphology a Genome 1. Alpha-retroviruses

Simple Avian leukosis virus 2. Beta-retroviruses

Rous sarcoma virus

Central spherical core; C-type

Murine mammary tumour virus

Simple Simian retrovirus type I

Eccentric spherical core; B-type

Central spherical core; D-type

3. Gamma-retroviruses Murine leukaemia virus

Simple 4. Delta-retroviruses

Central spherical core; C-type

Complex 5. Epsilon-retroviruses

Human T cell leukaemia virus

Central spherical core

Simple 6. Lentiviruses

Fish dermal sarcoma virus

Central spherical core; C-type

Complex 7. Spumaviruses

Human immunodeﬁciency virus

Cone-shaped core

Primate foamy virus Central spherical core; pronounced envelope spikes Complex a Beta-retroviruses and spumaviruses have condensed cores visible in the cytoplasm of infected cells, whereas in the other retroviruses the cores

condense as crescent-shaped bodies during maturation and budding at the cell membrane (see Figure 25.5).

Figure 25.2 Phylogenetic tree of retroviruses

2. Human T lymphotropic virus types 1 and 2 (HTLV-1 the PFV genome is indistinguishable from that of and HTLV-2) are deltaviruses. Type 1 causes adult

the simian foamy virus type 6 of chimpanzees, it T cell leukaemia and neurological disease. They are

may represent a zoonosis (Rosenblum and reviewed in Chapter 25A.

McClure, 1999). Serological studies indicate that

3. Primate foamy virus (PFV) is a spumavirus origin- spumaviruses are not endemic in human popula- ally detected in cultured nasopharyngeal carcinoma

tions (Ali et al., 1996), in contrast to many other of a Kenyan patient (Achong et al., 1971). Because

primate species. Zoonotic SFV infection without primate species. Zoonotic SFV infection without

4. Human endogenous retroviruses (HERVs) are Men- delian loci in human chromosomes representing ‘fossil’ infections of the germline. These endogenous genomes derive from mammalian g and b (HERV- K) retroviruses. No lentiviruses or spumaviruses are known to have become endogenous. HERV genomes are defective, i.e. human endogenous retroviral genomes have not been rescued in infectious form, in contrast to BaEV of baboons and PERV of pigs, which threaten the safety of human xenotransplantation from these sources (Weiss, 1998). Some HERV genomes, however, express envelope and other proteins, e.g. ERV-3 and HERV-W in the human placenta (Mi et al., 2000; Venables et al., 1995), HERV-K in testicular tumours and type 1 diabetes (Conrad et al., 1997; Lo¨wer et al., 1996) and HERV-W (MSRV) in multiple sclerosis (Perron et al., 1997). None of these viral genomes, however, has been unequivocally shown to play a causal role in disease. With the complete version of the human genome available in 2003, it has become apparent that approximately 8% of human DNA derives from fossil retroviruses.

HISTORY OF AIDS The disease AIDS ﬁrst came to the notice of physicians

and epidemiologists in 1981 in the USA, when a handful of homosexual men in cities presented with Pneumocystis carinii pneumonia (PCP) and Kaposi’s sarcoma (KS). These diseases were previously extraordinarily rare in young adults and indicated that some kind of immune deﬁciency was occurring in gay men. The ﬁrst full case description already showed a selective depletion of CD4 + T helper lymphocytes in the peripheral blood (Gottlieb et al., 1981).

It was soon noted that a larger proportion of gay men suﬀered from generalised, extended lymphadenopathy. The disease was initially called gay-related immune deﬁciency (GRID). By early 1982, however, investigators at the Centers for Disease Control and Prevention in Atlanta, USA, detected similar cases of what we now call AIDS among injecting drug users, sex workers and recipients of blood transfusions and blood products, especially of pooled clotting factors administered for haemophilia. These epidemiological observations were familiar to those working with hepatitis B virus in the 1970s and led to the conclusion

that AIDS was not simply a consequence of the gay lifestyle, but was caused by an infectious agent spreading both by sexual and by parenteral transmission. Similar signs and symptoms to AIDS in USA were recorded in Haiti, Europe and Africa.

Retrospective serological surveys indicated that HIV-1 began to spread among American gay men from 1977 onwards, showing a considerable incubation period before the manifestation of AIDS. The earliest known positive blood sample was collected in 1959 in Zaire (Zhu et al., 1998). However, molecular clock analyses of diversity suggests a common origin for HIV-1 (Group M) dating from around 1931 (Korber et al. , 2000) and HIV-2 from around 1940 (Lemey et al., 2003).

CLASSIFICATION OF HIV

HIV-1 was first isolated in 1983 (Barre´-Sinoussi et al., 1983), just 2 years after the identification of AIDS. Further HIV-1 isolates were reported in 1984 (Gallo et al. , 1984; Levy et al., 1984; Vilmer et al., 1984) which, together with serology (Cheingsong-Popov et al., 1984), made a convincing case for HIV as the cause of AIDS. HIV-2 was first isolated in 1986 (Clavel et al., 1986).

HIV-1 and HIV-2 represent two separate epidemics with distinct origins (Hahn et al., 2000). HIV-1 probably started as a zoonosis from the chimpanzee, which harbours a related lentivirus, SIVcpz, whereas HIV-2 came from sooty mangabey monkeys in West Africa. HIV-1 is divided into three groups, the main group (M), the new group (N) and the outlier group (O). These groups may represent three separate zoonotic transfers from the chimpanzee. Groups N and O remain largely conﬁned to a part of West Central Africa (Gabon and Cameroon), although sporadic infection through contact with persons from that region occur. Group M has radiated widely to cause the worldwide AIDS pandemic. The HIV-1 subtypes or clades lettered A–K all belong to group M. The genomic and antigenic variation manifested by HIV-1 groups and subtypes is important for diagnostic virology based on genomic and serological assays.

HIV-1 and HIV-2 strains can also be classiﬁed according to phenotype, which does not relate directly to their major genotypic classiﬁcation. Thus, within each HIV-1 subtype there are virus isolates that are syncytium-inducing (SI) (Figure 25.3) or are non- syncytium-inducing (NSI) for CD4 cells in vitro. Most primary, transmitting HIV-1 strains have an NSI phenotype, while SI substrains tend to appear in

724

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

HUMAN IMMUNODEFICIENCY VIRUSES

725 Table 25.3 Global estimates of HIV infection and AIDS in

adults and children (end of 2003) People living with HIV/AIDS

Global 40.0 million Africa

29.4 million Adults

38.6 million Women

19.2 million Children

3.2 million

New HIV infections in 2003

5.0 million Deaths due to HIV/AIDS in 2003

3.2 million Total deaths from AIDS, 1982–2003

23.0 million

Children orphaned by AIDS

14.8 million

Data from UNAIDS (2002).

practices have also helped to reduce sexual transmission, and antiviral drug treatment has markedly reduced perinatal transmission from mother to child.

Nevertheless, HIV continues to spread, and 30% of new infections in developed countries and over 50% in developing countries aﬀect women. In parts of the developing world HIV infection has been catastrophic. By the end of 2003, estimates of the global burden of

Figure 25.3 Phase-contrast micrograph of a giant syncytial cell formed 6 h after mixing, 10% HIV-1-producing H9 cells

HIV infection were close to 63 million cases, with 42 with 90% JM (Jurkat) indicator cells

million living with HIV and over 23 million deaths since 1981 (UNAIDS, 2003), with an estimated 5.5 million new HIV infections and 3.2 million AIDS-

infected individuals later as they progress to AIDS. As related deaths in 2002 (Table 25.3 and Figure 25.4). In described later, this phenotypic classiﬁcation is related

the UK, the epidemic has been slowly but steadily to cellular tropism for macrophages or T cell lines and

increasing over the last 5 years. This trend extrapolates to which kind of chemokine co-receptor the virus uses

to an estimate of over 49 000 people living with HIV/ to gain entry into cells. Most NSI strains utilise the

AIDS in the UK in 2004 and doubling by the end of CCR5 co-receptor and are known as R5 viruses,

the decade (Communicable Disease Surveillance whereas most SI strains utilise the CXCR4 co-receptor

Centre, 2002).

and are known as X4 viruses (Berger et al., 1998). The greatest burden of AIDS falls on Africa (Figure 25.4), although there has also been rapid spread of HIV in India, the Far East, South America, Russia and

EPIDEMIOLOGY Eastern Europe. Indeed, India has become the country with the second largest estimated number of infected

In the early stages of the AIDS epidemic, the main risk individuals after South Africa. While HIV-1 infection groups for HIV infection in Western countries were

ﬁrst arose in Central Africa, the greatest recent gay men practising anal sexual intercourse, men and

increase is in southern Africa, with 30% or more boys with haemophilia who were exposed to contami-

young adults infected in South Africa, Botswana and nated clotting factors, recipients of blood transfusion,

Zimbabwe. There are large diﬀerences in HIV pre- and intravenous injecting drug users. However, in

valence in diﬀerent cities in Africa for reasons that are Africa (where AIDS was originally recognised as ‘slim’

still not clear, although sexually transmitted diseases disease; Serwadda et al., 1985) and when it spread to

associated with genital ulceration, such as herpes Asia, HIV has from the beginning been heterosexually

simplex type 2 infection, and lack of male circumcision transmitted, aﬀecting men and women alike. With the

are risk factors; women become infected at younger rapid introduction of screening following the develop-

ages than men (Auvert et al., 2001). ment of commercial antibody tests in 1985, HIV

Across the world, approximately 80% of new HIV transmission through blood and blood products

infections among adults are heterosexually trans- virtually disappeared, although this remains a problem

mitted, the remainder being via homosexual in countries where screening is not stringent. ‘Safe sex’

transmission (6%), intravenous drug use (7%) and

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

Figure 25.4 Distribution of adults and children (total 40 million) estimated to be living with HIV/AIDS at the end of 2003. Adapted from UNAIDS (2003)

other modes, including contaminated blood transfu- have become more widely distributed and in the UK sions and contaminated hypodermic needles (7%). A

most subtypes currently occur (Parry et al., 2001). recent suggestion that a higher proportion of HIV

Genetically recombinant viruses derived from more transmission results from non-sterile injection (Gissel-

than one subtype (co-existing in the community) are quist et al., 2003) is hotly contested (Schmid et al.,

becoming increasingly prevalent, and these are called 2004; Walker et al., 2003). In the UK, 28% of new

circulating recombinant forms (CRFs). HIV infection is homosexual, 51% heterosexual and

HIV-2 has a lower transmission rate than HIV-1. In 20% intravenous drug use. In recent years an

West Africa, where HIV-2 remains endemic, HIV-1 increasing proportion has been heterosexual, with

infection has now surpassed HIV-2 in incident infec- most infections caught abroad (Communicable Dis-

tions. HIV-2 has spread elsewhere, including Europe ease Surveillance Centre, 2004). However, this pattern

and India. Mother-to-child transmission of HIV-2 is may change again if a new generation of homosexuals

rare, perhaps owing to a generally lower plasma HIV-2 disregard ‘safe sex’ practices.

viral load.

Up to 10% of all HIV infection occurs in children, almost entirely among infants of infected mothers, so non-sterile injection is not a major route of transmission.

VIROLOGY Transmission is mainly perinatal, and maternal antiviral

therapy shortly before birth greatly reduces transmission rates. Breast-feeding is also a signiﬁcant route of HIV

HIV Culture and Isolation transmission, particularly during primary infection with

HIV can be propagated in short-term cultures of high viraemia in the mother. Thus, a combination of

CD4 + peripheral blood mononuclear cells (PBMCs) maternal treatment, Caesarean section to reduce contact

stimulated by phytohaemagglutinin and interleukin 2 of the baby with maternal genital secretions and blood

(IL-2). The SI strains can adapt to growth in immortal during birth, and abstinence from breast-feeding can

CD4 + T cell lines, which become chronic virus virtually eliminate vertical transmission.

producers. NSI strains, however, need to be propa- HIV-1 clades or subtypes diﬀer in prevalence

gated in PBMCs, where they are cytopathic, and in geographically. In Africa, subtypes A, C, D and F

non-cytopathic macrophage culture. Virus isolation is are frequent, whereas subtype B is the commonest

useful for determining the phenotype of HIV strains, subtype in the West. In Thailand, recombinant subtype

whether they are SI or NSI, and whether they have A/E is most prevalent. In recent years various subtypes

developed drug resistance, although genotyping is

HUMAN IMMUNODEFICIENCY VIRUSES

Figure 25.5 HIV-1 particles produced by CEM cells. Note the crescent-shaped core in budding particles and the condensed cone- like core in mature particles of approximately 100 nm diameter. The black spots are indirect immunogold labelling of antibody from an AIDS patient absorbed to the virus-producing cells

Figure 25.6 HIV-1 and HIV-2 genome maps

more rapid. Figure 25.5 shows HIV particles produced HIV Genome and Proteins by the CEM T cell line in culture.

Isolation of HIV in cell culture can be detected by Figure 25.6 shows the proviral genomes of HIV-1 several means: (a) a cytopathic eﬀect (CPE), including

and HIV-2. The gene maps are similar except that syncytia for SI strains (Figure 25.3); (b) detection of

HIV-2 lacks vpu but carries vpx. The core and viral antigens in infected cells by antibodies, e.g.

matrix proteins are encoded by gag. The Gag immunoﬂuorescence, enzyme-linked immunocytology

proteins of the mature virus are p17, p24, p7 and of infected cells, ELISA for p24 antigen in cells or in

p6, and are processed by cleavage of the p55 supernatant medium from infected cultures; (c) RT

precursor protein by the viral protease. The matrix assay, either by enzyme activity or by ELISA; and (d)

antigen p17 is localised to the inner layer of the viral genome detection, using PCR or Southern blots.

envelope and requires myristoylation, which allows it

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

to be tightly bonded to the inner envelope; matrix have a competitive format, i.e. the antibody in the antigen is indispensable for budding. The core shell

serum sample to be tested needs to displace an enzyme- is made of p24 capsid antigen. This is the viral

labelled standard antibody in the test kit to score a protein most usually detected clinically as a measure

positive result. Microparticle agglutination assays of antigenaemia. The pol gene encodes the enzymes

bearing crude inactivated HIV antigens are also useful. protease, reverse transcriptase and integrase. The env

Strategies for clinical testing are discussed later. gene encodes the gp41 and gp120 envelope glyco-

For mass screening of sera, as in blood banks, proteins, cleaved by cellular enzymes (furins) from

combined tests for HIV-1, HIV-2 and sometimes other the gp160 precursor.

viruses are used. Diﬀerential HIV-1 and HIV-2 tests In addition to gag, pol and env, HIV-1 and HIV-2

can then distinguish between the two types of HIV. carry seven regulatory and accessory genes (Emerman

Antibodies to the various subtypes of HIV-1 are and Malim, 1998). The tat gene encodes a protein that

detected in screening assays with diﬀerent sensitivities, binds to TAR RNA sequences in the 5’ LTR to

although current commercial assays detect all subtypes upregulate viral RNA transcription through complexes

of HIV-1 group M and also the outlier group O, of cellular transcription factors and cyclin T; rev serves

prevalent in Cameroon and Gabon. Group N has not to aid export of long HIV transcripts from nucleus to

been rigorously tested in commercial kits because few cytoplasm through recognition of Rev-response ele-

humans, conﬁned so far to West Central Africa, are ments (RREs) in unspliced or singly-spliced viral

infected with group N viruses. mRNA; nef has multiple functions in signal transduc-

Neutralising antibodies can be measured by reduction and downmodulation of CD4 cell surface

tion of infectious titre of HIV in cell culture assays. expression; vpr allows transport of newly infected

Such assays are not useful for screening or diagnosis pre-integration complexes of the HIV proviral DNA

but are important for testing HIV candidate vaccines into the nucleus for integration (by integrase) into host

and in monitoring progression to AIDS. Immuno- chromosomal DNA; vpr also arrests cells in the G 2 ﬂuorescence assays are no longer used for diagnostic

phase of the mitotic cycle, enhancing virus production;

purposes.

vpu has poorly understood functions; vif is incorpo- Diﬀerential assays for antibodies in sera to speciﬁc rated into virus particles and helps infection in new

HIV antigens are generally performed by Western blot target cells by abrogating restriction exerted by host

on antigens prepared from whole virions, infected APOBECG3 (Sheehy et al., 2003). Although SI strains

cultures or recombinant viral proteins on nitrocellulose of HIV-1 can be propagated in T cell lines with

strips. In the early days of HIV diagnosis, Western deletions or non-functional mutations in nef, vpu or vif,

blots were considered important as conﬁrmatory to such mutations adversely aﬀect HIV replication in

ELISA tests. They still have a place for sera yielding PBMCs and macrophages, so that attenuated infection

indeterminate results, especially at the beginning of or no replication occurs. Thus, all the viral genes are

seroconversion during primary infection, although required for eﬃcient infection and pathogenesis in vivo

genome detection using PCR is now preferred for (Stevenson, 2003).

such cases.

HIV Serology HIV Load and Resistance The major diagnostic method for determining whether

Detection of viraemia was originally performed by tests

a person is infected by HIV is a serological assay for for p24 antigen in plasma (antigenaemia) and more speciﬁc anti-HIV antibodies. A number of ELISA

recently by detection of viral genomes. Molecular assays are commercially available which are sensitive

techniques for detecting HIV, quantifying viral load and speciﬁc (having extremely low false negative or

and typing for drug resistance have become a signiﬁcant false positive rates), and which are relatively inexpen-

part of patient diagnosis and clinical management. sive. Most of these assays are based on detection of

Measurement of proviral DNA is of importance in antibodies bound to HIV antigens in ELISA plates and

detecting early HIV infection before seroconversion detected with enzyme-linked antiglobulin. The antigen

(although p24 antigaemia is also convenient). in modern assays is generally a mixture of recombinant

Measurement of plasma viral load and resistance viral proteins, including envelope (gp41) and core

markers are important for antiviral treatment. (p24) antigens, or including synthetic peptides based

The detection of viraemia through the measurement on immunodominant epitopes. Some ELISA assays

of viral genomes is performed for HIV-1 and HIV-2 by

729 extracting RNA from virions, preparing complemen-

HUMAN IMMUNODEFICIENCY VIRUSES

phase of infection lasting 2–15 years (Figure 25.7). tary DNA and PCR ampliﬁcation of the cDNA

Virologically, however, this does not represent latent product (RT-PCR) (Berry and Tedder, 1999; Semple

infection but rather a high turnover of HIV production et al. , 1993). These processes can be combined and

(up to 10 10 infectious particles/day) and of infected performed with commercial kits. Alternatives to PCR,

lymphocytes (about 10 8 –10 9 cells/day) with equally such as branched DNA (bDNA) and nucleic acid-

active replenishment (Ho et al., 1995; Wei et al., 1995). sequence based ampliﬁcation (NASBA) systems are

Thus, it is remarkable that the overall CD4 T also available. Quantiﬁcation of viral load can be

lymphocyte count does not decline much faster measured by serial dilution of the sample against a

(McCune, 2001; Perelson, 2002). Generally, a high known standard, by competitive PCR, or by real-time

HIV load in the plasma after seroconversion is kinetic methods of detecting ampliﬁed product.

predictive of faster progression to AIDS (Mellors et al., While the sensitivity of HIV genome detection for

1996) and is known as the viral ‘set point’. Lymphoid the major HIV subtypes is well established, detection

tissue is actively infected by HIV throughout the of HIV-2 viral load, and detection of minor HIV-1

asymptomatic period (Stevenson, 2003). groups such as groups O and N, requires further

The constant, high production of virus was dis- development (Berry and Tedder, 1999). The principal

covered through measurement of the perturbation of method of determining HIV infection by a molecular

viral dynamics by antiviral drug treatment (Ho, 1995; technique is the detection of proviral DNA in

Wei et al., 1995). It helps to explain the extraordinary peripheral blood cells or other tissues. PCR ampliﬁ-

rate of evolution of HIV, both within one infected cation of proviral DNA, like RT-PCR of virions in

individual and throughout the human population, plasma, is a sensitive technique, provided that

because the virus undergoes so many replication cycles, inhibitors of the amplifying enzymes are not present

and the RT does not recognise or repair mutations. in the sample. The great sensitivity of PCR ampliﬁca-

There are, however, reservoirs of stable, integrated tion for any diagnostic target means that there is a

provirus in non-proliferating memory T lymphocytes danger of obtaining false positive results unless

and in macrophages. These can re-supply active HIV scrupulous care is taken to prevent contamination

replication when antiviral therapy ceases. Many of the sample. Modern diagnostic kits incorporate

infected cells contain more than one provirus (Jung high containment for the sample but the specimen

et al. , 2002), which may lead to recombinant progeny. must also be handled properly from the point of

While the high turnover of virus provides an oppor- venepuncture or biopsy.

tunity for intervention via antiviral therapy, it also With the increasing use of antiretroviral therapy and

provides opportunities for selection for drug the emergence of drug-resistant HIV strains and

resistance, immune escape and new cell tropisms to substrains, diagnostic methods are required to monitor

evolve.

resistance in managing HIV-infected patients and HIV-infected lymphocytes may be depleted in deciding upon optimal therapy. For the current drugs

several ways (Letvin and Walker, 2003; Pope and targeting RT and protease, certain mutations which

Haase, 2003): (a) by a direct cytopathic eﬀect of HIV, are associated with a high frequency of drug resistance

including cell fusion by SI strains; (b) by cytotoxic T occur in the pol gene. These can either be detected

lymphocytes recognising speciﬁc HIV antigenic pep- phenotypically by testing drug sensitivity of HIV

tides presented on MHC antigens, leading to immune isolates in culture or, in a much more rapid and less

destruction; and (c) by apoptosis, due to lymphocyte laborious test, by genotypic detection of mutations

activation and a changed cytokine and chemokine associated with drug resistance, by PCR or other

milieu.

genomic ampliﬁcation (see later). Although apoptosis correlates with disease progression, it is largely a late measure of activation of the aﬀected cells. Studies on apoptosis in human rapid progressors, non-progressors and chimpanzees

PATHOGENESIS OF HIV INFECTION revealed the extraordinary association between apoptosis, non-speciﬁc activation of the immune system and

HIV Dynamics progression to disease. Indeed, the most important correlates of disease progression after the virus load Primary HIV infection entails symptomatic fever and

and CD4 count are the activation markers, and in this lymphadenopathy in about 50% of infections. Follow-

regard the soluble tumour necrosis factor (TNF) ing seroconversion, there follows an asymptomatic

receptor is as accurate at predicting disease prognosis

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

Figure 25.7 The clinical, virological and immunological phases of HIV/AIDS. The clinical phases of the disease have been staged by the Centers for Disease Control (1985, 1993) to deﬁne primary HIV infection (PHI, CDC stage 1), asymptomatic (CDC

stages 2/3) and symptomatic (CDC stage 4). Viruses are easy to isolate during PHI and are generally a relatively homogenous population of macrophagotropic viruses. During the asymptomatic phase of disease viruses evolve into a heterogenous population but are diﬃcult to isolate; this becomes progressively easier as a population of lymphotropic viruses predominate in the host in symptomatic disease. CD4 lymphocyte counts ( * ) show distinctive changes during disease, with a rapid, short-term decline associated with the viraemia in PHI, a recovery to near-normal levels, a gradual loss of cells during the asymptomatic phase, and

ﬁnally an accelerated decline associated with late symptomatic disease. The reciprocal of these changes is shown by b 2 - microglobulin ( * , top panel), a plasma measure of immune activation. Plasma viral load ( * ) reaches a peak in PHI (mirrored by p24 antigen, & ), declines to a ‘set point’ during early asymptomatic disease, and then gradually increases after about 24 months of

infection at 0.1–0.2 log 10 copies/ml per year. In symptomatic disease viral load and p24 antigen increase progressively. Both viral load and CD4 cell counts in early asymptomatic disease are highly predictive of disease progression. Anti-HIV-1 antibodies ( ~ ) increase to a maximum within 3–6 months of infection and remain detectable throughout the natural course of disease. However, subpopulations of antibodies (i.e. anti-p24 antibodies, ^) may decline in association with, and predict, disease progression. With the introduction of potent antiretroviral therapy and persistently long-term undetectable plasma virus (550 copies/ml), the antigenic stimulus for the immune system is diminished and quantitative declines in plasma anti-HIV-1 antibodies are observed. This model of the natural history of the disease has been shown in practice to exhibit, in a minority of patients, an accelerated course where viral load remains relatively high or rises, p24 antigen remains detectable. CD4 cell counts decline rapidly and patients progress clinically in 55 years (rapid progressors), or a protracted course where the CD4 cell counts remain in the normal range and viral load low or undetectable with asymptomatic disease for 415 years (long-term non-progressors). These diﬀerences probably represent the limits of a normal distribution

731 as is the virus load at the time of infection. There are

HUMAN IMMUNODEFICIENCY VIRUSES

macrophages (Berger et al., 1999; Moore et al., 2004). clear but not absolute correlates between the rate of

Primary, NSI strains of HIV mainly utilise CCR5 (R5 progression to disease and the immunogenetic back-

strains), which is the receptor for the chemokines MIP- ground of the host (Carrington and O’Brien, 2003),

1a, MIP-1b and RANTES, whereas SI strains utilise

e.g. patients with HLA-1 B8 DR3 are unusually rapid CXCR4 (X4 strains), the receptor for stromal derived progressors to disease, whereas in HIV-1 infected

factor 1 (SDF-1). The discovery that chemokine Caucasians, patients with HLA B27 are slow or non-

receptors act as co-receptors to CD4 (Feng et al., progressors to disease.

1996) helped to explain why these chemokines can Chronic activation and progression to disease

inhibit HIV-1 infection in vitro (Cocchi et al., 1995). dependent on the HLA background is seen with

Modiﬁed chemokines and small molecular receptor viruses that encode superantigens. Initial reports that

blockers can potently inhibit HIV-1 entry and may HIV did so have not been conﬁrmed and, although

have promise in therapy (Baba et al., 1999; Simmons superantigen activity with regards to B cell epitopes

et al. , 1996).

have been reported, it is not thought to be suﬃcient to The importance of the CCR5 co-receptor for NSI drive the panactivation seen throughout the immune

viruses in HIV transmission and disease progression is system in HIV-infected patients progressing to AIDS

borne out in people with mutations in the gene for this (Westby et al., 1996). This panactivation is similar to

receptor. In Caucasian populations a deletion in that seen in chronic HLA mismatched transplantation,

CCR5, rendering it non-functional, occurs frequently; which leads to chronic graft-vs.-host disease, and it is

people homozygous for this ‘delta 32’ mutation are notable that HIV incorporates many HLA molecules

resistant to infection by R5 strains of HIV-1, even into its envelope. This property may enhance infectiv-

when exposed, e.g. by regular sexual contact with an ity in vivo and could be one mechanism of activating

HIV carrier (O’Brien and Moore, 2000). Mutations in the immune system resulting in enhancement of

the promoter region of CCR5, which lower the level of infection (Letvin and Walker, 2003; Westby et al.,

CCR5 expression, delay progression to AIDS in 1996). It is notable that sooty mangabey monkeys

infected individuals (An et al., 2000; Carrington and naturally infected with SIVsm have a high virus load

O’Brien, 2003; Kostrikis et al., 1998). without immune activation, and remain healthy

Several host factors that constrain HIV replication (Silvestri et al., 2003).

after virus entry but before provirus integration have also recently been discovered. A human protein, APOBECG3, that blocks HIV-1 infection, is sup-

Cell Tropism and HIV Receptors pressed by the viral Vif protein (Sheehy et al., 2003). A diﬀerent kind of suppression mediated by Lv1 also acts

HIV infects mainly CD4 + cells by binding to CD4 as a at an early step in the HIV replication cycle and receptor (Dalgleish et al., 1984). However, many of

diﬀerentially aﬀects HIV-1, HIV-2 and various strains these cells are not lymphocytes, e.g. monocytes,

of SIV (Hatziioannou et al., 2003). Another type of macrophages, dendritic cells (Langerhans cells) and

early restriction has been observed for HIV-2 some brain cells, such as the microglia, express the

(McKnight et al., 2001).

CD4 receptor, and many are susceptible to infection by Sequence analysis indicates that HIV in diﬀerent HIV. It is likely that infection of these cell types plays a

organs and cell types may represent diﬀerent sub- major role in the pathogenesis of disease (Stevenson,

populations in the various compartments of the body 2003; Weiss, 2003). Importantly, HIV particles bind to

(Atkins et al., 1998). Thus, the dominant HIV the DC-SIGN receptor on dendritic cells without

substrains in the blood and cerebrospinal ﬂuid (CSF) infecting them, and hence may be delivered to CD4

and, within the blood, between CD4 + cells and in lymphocytes in the lymph node (Geijtenbeek et al.,

CD8 + cells may diﬀer in late stage AIDS (Livingstone 2000; Pope and Haase, 2003).

et al. , 1996). Whereas the depletion of CD4 + cells The CD4 cell surface antigen is necessary but not

accounts for the profound immune deﬁciency, and suﬃcient for HIV-1 infection (Dalgleish et al., 1984;

falling CD4 cell counts are a useful marker of disease Maddon et al., 1986). While CD4 is required for high

progression, the wasting disease and central nervous aﬃnity binding of gp120, co-receptors are required for

system (CNS) disease (AIDS dementia) are probably subsequent steps leading to fusion between the viral

more closely related to macrophage infection. This envelope and cell membrane. The co-receptors were

includes the microglial cells of the brain, which are identiﬁed in 1996 to be chemokine receptors and help

derived from monocytes rather than neurectoderm. to explain the diﬀerential tropism for lymphocytes and

Astrocytes may also become latently infected, with

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

Table 25.4 AIDS-deﬁning illnesses in HIV infection changes to local cytokine secretion in the brain leading (deﬁned by Centers for Disease Control in 1985, revised 1993)

to neuronal loss.

Infections

Neoplasms

Pneumocysis carinii pneumonia

Kaposi’s sarcoma

HIV AND AIDS: CLINICAL DISEASE AND

Cytomegalovirus (non-RE

Primary lymphoma (brain)

MANAGEMENT

system)

Non-Hodgkin’s lymphoma

Cryptococcosis (extrapulmonary) HIV infection is characterised by an initial acute viral

Candidiasis (excluding oral)

Invasive cervical cancer a

Mycobacterium avium illness followed by a chronic, asymptomatic phase of (disseminated)

disease associated with active viral replication and M. kansasii (disseminated)

dissemination, and lasting as long as 5–10 years. M. tuberculosis (disseminated) M. tuberculosis (pulmonary) a

Ultimately, immune destruction results in end-stage disease (AIDS) associated with opportunistic infec-

Herpes simplex (41 month, or non-orogenital)

tions, malignancies and neurological disorders. These Cryptosporidiosis (41 month)

clinical events correlate with virological and immuno- Histoplasmosis (extra pulmonary)

logical changes and are shown in Figure 25.7. Toxoplasmosis

Early classiﬁcation of the disease by the Centers for Nocardiosis

Disease Control and Prevention, Atlanta, USA, Recurrent disseminated

deﬁned an asymptomatic phase followed by a list of salmonella

Strongyloidosis (non-intestinal) symptomatic conditions that deﬁned AIDS (Table

Recurrent bacterial pneumonia a 25.4). This was later revised to reﬂect the eﬀects of Coccidioidomycosis

HIV/AIDS infection on clinical status (Table 25.5). In (extrapulmonary)

1993 it was expanded to take account of additional Isosporosis (41 month)

respiratory diseases and recently discovered malignan- Primary HIV-1 dementia

cies, and Table 25.6 outlines the classiﬁcation most Primary HIV-1 wasting disease

widely used to deﬁne entry into contemporary clinical

a Added in revisions of 1993.

trials. The classiﬁcation of paediatric HIV infection evolved independently to reﬂect the unique features of the disease in this patient group (Table 25.7).

Table 25.5 Centers for Disease Control (CDC): classiﬁcation of the eﬀects of HIV-1 infection (CDC, 1985, 1993)

Early Infection

CDC1 Acute infection with seroconversion (PHI)

CDC2 Asymptomatic infection CDC3

Persistent generalised lymphadenopathy Primary HIV infection (PHI) results in a well- CDC4 a

Constitutional disease recognised constellation of clinical, virological and b Neurological disease

immunological responses associated with rapid and c Immunodeﬁciency

widespread dissemination of virus following infection. ci CDC deﬁnitions of AIDS

An initial illness occurs in 50–90% of cases, is cii Infections without deﬁnition

classically described as mononucleosis-like, although d Tumours with CDC deﬁnition

c Others, e.g. carcinomas, interstitial pneumonia in practice <20% have these features. A typical presentation, in order of frequency, may include:

Table 25.6 Centers for Disease Control (CDC) AIDS surveillance case deﬁnition (CDC, 1993) CD4 counts (610 6 /l)

Clinical group

A: asymptomatic PGL or PHI

B: symptomatic but not A or C b C: AIDS conditions a

4 500 A1 B1 C1 200–500

A2 B2 C2 5 200

A3 B3 C3 PGL, persistent generalised lymphadenopathy; PHI, primary HIV infection. a

b Patients in groups C1, C2, C3, and A3 and B3 are reported as AIDS according to Table 25.4 and/or having a CD4 count below 200610 6 Symptomatic but not included in C are those conditions associated with defective immunity. /l.

733 Table 25.7 Centers for Disease Control (CDC) clinical categories for children with HIV-1 infection (CDC, 1994)

HUMAN IMMUNODEFICIENCY VIRUSES

Category Clinical stage

Features

N Asymptomatic

HIV-infected but with no signs or symptoms

A Mildly symptomatic Children with two or more of the following conditions but none in B or C:

Lymphadenopathy Hepatomegaly/splenomegaly Dermatitis Parotitis Persistent urinary tract infections

B Moderately symptomatic Children with conditions other than those in A or C including:

Anaemia Persistent fever (41 month) Cytomegalovirus (onset 51 month of age) Herpes simplex stomatitis (recurrent 5year) Herpes simplex bronchitis/oesophagitis Disseminated varicella Hepatitis Bacterial meningitis, pneumonia or sepsis Toxoplasmosis (51 month) Candidiasis, persistent oral after 6 months age Nocardiosis Diarrhoea, chronic Cardiomyopathy or nephropathy

C Severely symptomatic

Cytomegalovirus (onset 41 month) Herpes simplex oral/internal 41 month Kaposi’s sarcoma Candidiasis, oesophageal/pulmonary Coccidioidomycosis (disseminated) Cryptococcosis (extrapulmonary) Cryptosporidiosis (symptoms 41 month) Histoplasmosis (disseminated) Mycobacterium tuberculosis (disseminated) Mycobacterium avium or M. kansasii Pneumocystis carinii pneumonia Salmonella septicaemia Toxoplasmosis (CNS, 51 month age) Recurrent serious bacterial infections Encephalopathy (developmental delays) Progressive multifocal leukoencephalopathy Wasting syndrome (developmental delays)

acute onset of fever, lethargy, maculopapular rash, The commonest neurological feature is an aseptic myalgia, headache, sore throat, cervical lymphadeno-

meningoencephalitis reﬂecting the neurotropism asso- pathy, arthralgia, oral ulcers, photophobia, oral

ciated with this group of viruses. Less frequently, candida and, rarely, meningoencephalitis. The time

myelopathy, peripheral neuropathy, facial palsy and from exposure to onset of signs and symptoms is

Guillain–Barre´ syndrome are seen. They are usually approximately 10–30 days.

self-limiting. HIV has been detected in the CSF soon Lymphadenopathy is a frequent event during PHI,

after infection, indicating the speed at which the virus usually in the second week of illness and is often

penetrates the blood–brain barrier. Marked changes in associated with a lymphocytosis. The lymph nodes

the lymphocyte count are seen. The CD4:CD8 ratio is tend to decrease in size with time. The architecture

reversed, largely due to an increase in the number of remains relatively normal but HIV can be visualised in

CD8 cells. An early CD4 lymphopenia is characteristic association with both lymphocytes and dendritic

of PHI; it is associated with very high plasma viral load cells. A distinctive feature of PHI is mucocutaneous

levels (in excess of 10 11 copies/l) that occur in early disease involving the buccal mucosa, gingiva, palate,

disease prior to immunomodulation. The mechanism oesophagus, anus and penis.

of this CD4 lymphopenia is explained by a ‘predator– of this CD4 lymphopenia is explained by a ‘predator–

The symptomatic phase of PHI lasts less than 14 days on average and is self-limiting; however, it is now clearly evident that protracted and wide-ranging symptoms, oral and/or oesophageal candida, neurological involvement, and persistently high plasma viral loads are associated with rapid disease progression. The diﬀerential diagnosis of PHI should include: Epstein–Barr virus, cytomegalovirus, herpes simplex, rubella and other acute viral infections; in addition, syphilis, disseminated gonorrhoea, toxoplasmosis and drug reactions should also be considered.

The timing of initiation of therapy in primary infection is controversial. Some investigators have demonstrated that short-term therapy at this time may preserve immune responses, with associated lower levels of proviral DNA and plasma HIV-1 RNA suppression (Kinloch et al., 2003). However, others are more concerned over drug-related reduction in quality of life, with accumulation of adverse events, earlier emergence of resistance and limited future antiretroviral options. Pragmatically, the most important role of therapy at this stage is probably to limit symptoms in a severe seroconversion illness.

The Asymptomatic Phase The period after PHI and prior to symptomatic disease

has been described as the latent or asymptomatic phase. Although it is a clinically latent period, the notion of virological latency is misleading, as virus persistence and active replication and cell turnover clearly occurs throughout the lymphoreticular and other tissues (Perelson, 2002), and continual monitoring of viral load and CD4 cell counts are required. Since these individuals are infectious, advice on sexual health is required. A detailed review of the numerous factors which aﬀect viral latency and persistence is beyond the scope of this chapter (Letvin and Walker, 2003; Levy, 1998; Stevenson, 2003). The duration of the asymptomatic phase may be variable and is related to the severity of PHI, the phenotypic characteristics of the infecting viruses, the status of the host immunity,

the lifestyle of the host and the use of antiretroviral therapies.

Clinically, patients are relatively free of symptoms, although lymphadenopathy may be a complaint in some cases. Patients are oﬀered a clinical review every

3 months and regular plasma viral loads and CD4 lymphocyte counts are used to monitor disease status. Plasma viral loads measured 6–12 months after PHI have been shown to be a powerful predictor of subsequent disease progression (Mellors et al., 1996), superseding CD4 lymphocyte counts taken at the same time; in combination, the two values are the most accurate method for determination of patient prognosis (Loveday and Hill, 1995; Mellors et al., 1996). This phase of clinical care is often supportive, involves surveillance of patients for features of early progression and for the optimum time to commence antiretroviral therapy.

Symptomatic Disease and AIDS

In untreated patients, early features of symptomatic disease were often non-speciﬁc and associated with dermatological manifestations, such as eczema and human papillomavirus eruptions, as well as oral and vaginal candidiasis, recurrent chest infections, night sweats, weight loss and the appearance of lymphadenopathy. Oral lesions become more common with gingivitis, candida, herpes simplex eruptions and aphthous ulcers. ‘Oral hairy leukoplakia’ presents as white, ribbed lesions on the lateral margins of the tongue; it may be asymptomatic or produce soreness within the mouth. It is associated with Epstein–Barr virus infection and may respond to aciclovir therapy. All these features are far less frequent in the era of combined antiretroviral therapy. The progressive immune deﬁciency associated with long-term HIV/ AIDS infection results in the opportunistic infections and malignancies that characterise AIDS, which are often multiple and contribute to the rapid clinical deterioration in patients.

Opportunistic Infections

Opportunistic infections seen in symptomatic HIV-1 disease (Tables 25.4 and 25.7) reﬂect adult and childhood exposure; hence, fungal infections such as histoplasmosis may be seen in persons who come from areas where the organisms are endemic. Infectious agents may be categorised into: (a) those that do not cause disease in the immunocompetent host, e.g.

734

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

735 Pneumocystis carinii ; (b) those that cause mild disease

HUMAN IMMUNODEFICIENCY VIRUSES

Toxoplasma gondii causes space-occupying lesions in in the normal host, e.g. herpes simplex virus (HSV),

the CNS and is treated with combinations of anti- Toxoplasma gondii ; (c) those that are conventional

microbials. These patients present with fever and focal pathogens, e.g. Mycobacterium tuberculosis, but, as a

neurological signs and may have associated chorio- consequence of immunosuppression associated with

retinitis. Serological tests for Toxoplasma are HIV-1 infection, produce widespread debilitating

unreliable, and demonstration of the organism in the disease in the host.

affected tissues by PCR may offer a more definitive Overall, the presenting features of symptomatic HIV

diagnosis. The diﬀerential diagnosis of cerebral lym- disease may be quite distinct, according to the

phoma may be excluded using a highly sensitive and geographical areas in which the hosts are found, e.g.

speciﬁc PCR for Epstein–Barr virus. Pneumocystis carinii pneumonia and Kaposi’s sarcoma

Other CNS-related conditions include progressive are the commonest presenting diseases in the UK,

multifocal leukoencephalopathy (PML), a condition whereas Mycobacterium tuberculosis and gastrointest-

associated with JC virus, producing oligodendritic inal infections are more frequent in Central Africa.

lysis. It is diagnosed by characteristic histological Pneumocystis carinii pneumonia diﬀers from the

changes and conﬁrmed if possible by PCR for JC virus. disease seen in other groups with non-HIV-associated

No speciﬁc treatment is available. In addition, other immunosuppression, in being characterised by sub-

viral encephalites (including herpes simplex) and bacter- acute onset, involving a mild, persistent cough and

ial infections are often implicated in AIDS. Overall, the progressive chest discomfort and fatigue of 2–10 weeks

use of combined antiretroviral therapy has had a duration. It is rare when CD4 cell counts are above

dramatic impact on the frequency and severity of such 200610 6 /l. Subtle bilateral inﬁltrations with a batwing

conditions. Progressive HIV-associated encephalopa- appearance may be seen on a chest radiograph but

thy involves inﬂammatory changes in white and grey 50% of cases are normal at presentation. Minimal

matter and is characterised by foci of inﬂammatory hypoxia is present and diagnosis is made by detection

cells, including microglia, macrophages and multi- of the pneumocystis organism in induced sputum or

nucleate giant cells with actively replicating HIV-1. bronchoalveolar lavage. It is treated with co-trimoxazole

Mycobacterium avium and M. intracellulare (MAI) or pentamidine, and the introduction of prophylaxis in

are two closely related species of ubiquitous environ-

mental organisms which in the past had rarely been markedly reduced the incidence of this infection in

patients with CD4 cell counts <200610 6 /l has

shown to cause disseminated disease. However, MAI is HIV/AIDS, as have antiretroviral drugs.

common in patients with symptomatic HIV infection, Cytomegalovirus is the commonest cause of pro-

the bacteria being ingested or inhaled. MAI produces gressive chorioretinitis in AIDS. The lesions are

disseminated disease with non-speciﬁc symptoms, initially asymptomatic but, as the perivascular exu-

including fatigue, fever, night sweats, weight loss, dates and haemorrhages involve the macula, vision

abdominal pain and diarrhoea, in patients with becomes impaired. Interestingly, CMV pneumonitis is

<50610 6 /l CD4 lymphocytes. It is diagnosed by rare in AIDS patients because it requires active

positive blood cultures using Lowenstein–Jensen solid recruitment of the immune system to cause disease.

medium (3–4 weeks) or a non-speciating broth medium In contrast, CMV retinitis is common in untreated

(1–2 weeks), and conﬁrmation by PCR, in 90% of AIDS patients, as it is an uncontained cytopathic

cases. Treatment and prophylaxis is with speciﬁc anti- disease not requiring immunocompetence. In addition,

tuberculous combination therapy. With the advent of gastrointestinal ulceration, adrenalitis and encephalitis

highly active antiretroviral therapy (HAART), these may occur. Progression of CMV infection can be

therapies need not be lifelong. Both this condition and restricted by parenteral ganciclovir or foscarnet, and

the more classical M. tuberculosis have become the institution of antiretroviral therapies has reduced

increasingly common in HIV-infected patients in the the incidence and/or severity of CMV.

developed as well as the developing world, and Cryptococcus neoformans also causes a diﬀuse

antimicrobial-resistant organisms are evident in this pneumonitis, although more commonly it causes

new tuberculosis epidemic.

meningitis and is widely disseminated. It may also Oral candidiasis is commonly seen, and oesophageal present with fever, granulocytopenia, thrombocyto-

involvement may be present with dysphagia, odyno- penic purpura, maculopapular rashes and ulcerating

phagia and retrosternal burning. Diagnosis is based on gastrointestinal lesions. It may be isolated from many

clinical ﬁndings and simple histology. Treatment is by sites, including throat washings, urine or blood. It is

topical antifungal agents or intravenous therapy in the treated with ﬂuconazole or amphotericin B.

event of refractory and severe disease. Other fungal

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

infections, such as Aspergillus, can produce life- both malabsorption and loss of weight. The clinical threatening respiratory disease, with white plaque-

consequences of this condition may be profound, but like colonies in the bronchi, and cavitation. Infection is

HAART has been found to be of value. diagnosed by culture and treated with itraconazole or

The widespread use of antiretroviral therapy has amphotericin B; without immune restoration following

reduced the incidence of many of the above opportu- antiretroviral therapy the outcome is poor, and time

nistic infections and there is recent evidence to indicate from diagnosis to death is short (months).

that a CD4 cell nadir, prior to restoration with Herpes simplex virus (HSV2>HSV1) infection

HAART, of >200 cells/ml is not predictive of increased commonly presents as a vesicular lesion on an

risk of opportunistic infections (Miller et al., 1999). erythematous base in oral, genital or perianal areas.

However, there has been a concurrent increase in the Attacks are usually more widespread and of longer

morbidity and mortality due to hepatitis B and C duration, with more frequent recurrences in HIV-1

infections. This is not due to an increased prevalence infection; the lesions are associated with secondary

per se , but rather to the longer lifespan of HIV-infected bacterial infection and have been associated with

individuals, enabling the pathogenic processes to widespread epidermal erosion, in some cases requiring

advance. The interplay of host and viral factors in skin grafts. Oesophageal and tracheobronchial invol-

viral hepatitis pathology is complex, and it remains vement is described. Diagnosis is by virus culture and/

unclear whether antiretroviral therapy slows progres- or PCR; treatment is with aggressive use of oral or

sion of these infections. A number of clinical trials are intravenous aciclovir therapy, depending on the

in progress to study the impact of HBV and HCV severity of the condition.

treatment on HIV disease progression and vice versa. Reactivation of varicella zoster virus is common in

Those infected with either hepatitis virus should be HIV-infected patients but is less often seen in advanced

closely monitored by serological and quantitative symptomatic disease. Ophthalmic zoster may threaten

molecular assays.

vision, and any dermatomal presentation may become Many of the opportunistic infections discussed disseminated. Treatment is with high-dose aciclovir,

above manifest disease through an immunopathologi- antibiotics for secondary infection and analgesia.

cal process. It is now becoming apparent that unique Human papillomavirus (HPV-6 and -11)-induced

presentations of these infections can occur consequent warts and molluscum contagiosum (Poxvirus) are

to immune reconstitution induced by retroviral ther- both common skin conditions in up to 25% of patients

apy, e.g. cystoid macula oedema and vitritis due to with HIV infection and may be of abnormal presenta-

cytomegalovirus may be observed in those receiving tion and persistent.

anti-HIV therapy.

Persistent or recurrent diarrhoea, which may be copious in volume and watery in content, is a frequent problem in symptomatic patients. Giardia lamblia,

AIDS-associated Malignancies Entamoeba histolytica , Shigella , Salmonella and

Campylobacter all cause symptomatic disease; how- Malignancy is a common feature of AIDS as a ever, appropriate treatment against these pathogens

consequence of the profound cell-mediated immunosuppression. However, only certain types of cancer are

does not always eliminate the watery diarrhoea. signiﬁcantly increased in AIDS and HIV infection, Cryptosporidium parvum , an enteric coccidium, indicating that the role of immune surveillance in attaches to the epithelial surface of both small and preventing cancer is selective (Boshoﬀ and Weiss, 2002). large intestines and produces protracted and severe It is striking that AIDS-associated tumours mainly have diarrhoea in symptomatic HIV/AIDS patients. The

a viral aetiology. Kaposi’s sarcoma is the commonest diagnosis is by direct modified Ziehl–Neelsen staining AIDS-defining malignancy, with non-Hodgkin’s lym- of stool preparations; treatment is difficult, with a

wide range of antimicrobial agents demonstrating phomas and anogenital squamous carcinomas next in marginal success. CMV, Mycobacterium avium and M.

importance. Tumours present at late stages of infection at a time of diminishing immunocompetence, although

intracellulare , Isospora belli, Microsporidium encepha- and Kaposi’s sarcoma involving the bowel

they may be the ﬁrst symptom of AIDS. lozitozoon

wall may also produce these clinical features. There is growing evidence that HIV-1 itself may cause entero-

Kaposi’s Sarcoma pathic signs and symptoms through infection and

inﬁltration of local target cells, when treatment of Kaposi’s sarcoma (KS) is a rare vascular or lymphatic speciﬁc pathogens is unsuccessful and patients have

tumour (Dupin et al., 1999) that has been simply

737 classiﬁed into diﬀerent epidemiological forms (see

HUMAN IMMUNODEFICIENCY VIRUSES

Histopathologically, a variety of features are seen, Chapter 2F; Boshoﬀ and Weiss, 2002):

which suggests a multipotential mesenchymal cell of origin with markers of lymphatic endothelium in particular (Dupin et al., 1999). Vascular proliferation

1. Classic KS is seen in Jewish, Mediterranean and and spindle-shaped neoplastic cells form a network of Middle Eastern populations. It occurs mainly in reticulin ﬁbres. The lesions start in the mid-dermis elderly people (median 72 years) with a much and extend towards the epidermis. Lesions in the greater prevalence in men than women. Classic KS gastrointestinal tract arise in the submucosa. typically presents in the lower limbs and follows an AIDS-KS has a wide variety of clinical presenta- indolent course. tions. Typically, single or multiple indolent lesions

2. Transplant or iatrogenic KS in immunosuppressed appear as pink macular lesions on the skin; they may patients, often of the same ethnic groups as classic

KS. evolve into reddish-purple maculopapular lesions or

3. Endemic KS in sub-Saharan Africa, presenting in nodules, increasing in size and distribution. They range adults and children. This tumour was already

from benign innocuous lesions to aggressive, invasive commonly seen before the HIV/AIDS era.

and fungating forms over time. The lesions may

4. AIDS-associated KS. This is an aggressive form of occlude or invade lymphatics, producing lymphoedema. tumour, often presenting viscerally and in the lung

A disseminated form occurs, with soft gastrointest- as well as cutaneously.

inal lesions producing dysphagia or gastrointestinal bleeding. Pulmonary KS is associated with space- occupying bronchial lesions, producing wheeze and

All these forms are linked to infection by Kaposi’s cough, dypsnoea, a typical chest X-ray picture, and sarcoma-associated herpesvirus (KSHV or HHV-8)

life-threatening haemoptysis. In both these presenta- and reﬂect the prevalence of this infection. Together

tions, endoscopy and biopsy require considerable with PCP, KS was the sentinel disease that alerted

thought. The prognosis of KS is dependent upon the epidemiologists and physicians to the incipient AIDS

disease stage, the severity of HIV-associated immuno- epidemic in 1981.

suppression and other systemic illnesses (Table 25.8). Early studies of the epidemiology of the AIDS-KS

The treatment of KS in AIDS depends on the deﬁned clusters of cases in homosexual and bisexual

severity of the primary disease and the extent of the men in the developed world, with increasing cases at

KS. Broadly, in otherwise asymptomatic HIV infec- sites associated with the original epidemic. Female

tion, treatment of ‘benign’ skin lesions is only for cases were associated with bisexual male partners. The

psychological or cosmetic reasons, whereas in sympto- sexual practice of oral–anal contact was signiﬁcantly

matic or visceral disease it may be life-saving (Table higher in those with KS and was proposed as a route of

25.9). Before HAART, less than 50% responded to transmission of a potential infectious agent associated

systemic chemotherapy and the response time was with this group. The disease has a lower prevalence in

limited. Intralesional vinblastine will reduce bulk and transfusion-acquired AIDS (4%) and haemophiliacs

number but leave skin pigmentation. Radiotherapy is (<1%), suggesting the blood-borne route as an

used in large skin or oral lesions; responses are good unusual pathway for transmission.

but recurrence common. Immunotherapy using IFN-a, Using representational DNA analysis, a modiﬁed

even in high doses, only produces a 30% response and PCR technique applied to substractive hybridisation,

is complicated by the systemic toxicity of this agent. In Chang et al. (1994) identiﬁed novel DNA sequences in

view of the causative agent, studies using cidofovir and KS that showed homology with gamma-herpesviruses

foscarnet are being explored for therapy and prophy- such as Herpesvirus saimiri and EBV. The new virus

laxis because these anti-herpetic drugs inhibit KSHV was called KSHV or HHV-8. DNA from KSHV was

replication. Above all, the use of HAART for the found in all KS lesions, independent of HIV status

treatment of HIV/AIDS has greatly reduced the (Chapter 2F). PCR and serological assays are available

prevalence of KS in patients with symptomatic disease for testing for KSHV. The presence of KSHV predicts

(Boshoﬀ and Weiss, 2002).

the subsequent development of KS. The increasing frequency of HHV-8 seropositivity with age (Sitas et al. , 1999) suggests that horizontal transmission

Non-Hodgkin’s Lymphoma (NHL) occurs, and paediatric studies conﬁrm vertical transmission, probably via saliva (Boshoﬀ and Weiss,

Most of the lymphoid malignancies are high-grade 2002).

Burkitt’s lymphomas and immunoblastic lymphomas.

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

Table 25.8 TIS staging of AIDS Kaposi’s sarcoma Criteria

Good risk (0)

Poor risk (1)

Tumour bulk (T) Limited to skin, lymph nodes or no oral Tumour-associated oedema or ulceration; involvement (T0)

++ lesions in mouth, GI tract or other organs (T1) Immune status (I)

CD4 cell counts 5200610 6 /l (I1) Systemic disease (S)

CD4 cell counts 4200610 6 /l (I0)

No history of OIs or candidiasis; no B History of OIs, candidiasis; B symptoms; Karnofsky

score 570; other AIDS-related illness (S1) OI, opportunistic infection; Karnofsky score, clinical scale of HIV/AIDS disability in units of 10, from 0=dead to 100=normal, no signs or

symptoms a ; Karnofsky score 470 (S0)

symptoms. a B symptoms—see Table 25.6.

Table 25.9 Choices of treatment for AIDS Kaposi’s sarcoma therapy, a negative thalium uptake scan and a positive EBV PCR result in CSF.

Early disease Advanced disease (T0, I0/1 or S0/1)

(T1, I0/1 or S0/1) Treatment of systemic disease is stratiﬁed according to prognostic factors. Generally, those with major HAART

HAART adverse factors, such as prior AIDS-deﬁning illness, No treatment

Radiotherapy CD4 counts <100610 6 /l, primary cerebral disease or

and low Karnofsky score (<70), receive palliative care Intralesional vinblastine

Systemic chemotherapy and low-toxicity chemotherapy, focusing on quality of (lesions 51 cm 2 )

1st line: bleomycin+vincristine life. The minority, with a better prognosis, are treated or

with conventional ‘curative’ chemotherapy for non- 2nd line: liposomal anthracycline

Hodgkin’s lymphoma. Primary cerebral lymphoma is Radiotherapy

associated with profound immunosuppression and (lesions 41 cm 2 )

+doxil

very low CD4 cell counts; a brief clinical improvement or

is derived from brain radiotherapy and dexametha- Interferon-a sone, but the survival time is only about 2 months.

An increased incidence of Hodgkin’s disease has been reported in patients with HIV/AIDS (Boshoﬀ

The diagnosis is 50 times more common in HIV/AIDS and Weiss, 2002). It is associated with intravenous than in the normal population. Its age distribution is

drug users, and is more aggressive than in the general bimodal, with the former at a peak in 10–20 year-olds

population, with bone marrow or other extra-nodal and the latter in 50–60 year-olds. They are usually

involvement. Patients have mixed cellularity or lym- widespread at presentation and often occur in extra-

phocyte-depleted histology, and a survival time of less nodal sites, particularly the brain. Only about 50% of

than 1 year.

the lymphomas are EBV-related, although higher ﬁgures have been claimed in some populations. This suggests that the pathogenesis is more complicated than the expression of an oncogenic virus in the face of immunosuppression, although it is likely that activa-

Anogenital Squamous Carcinoma tion of virus stimulation has a role, just as in KS. KSHV is causally associated with two rare forms of

Rapidly progressive, squamous intraepithelial carcino- AIDS lymphoma, plasmablastic multicentric Castle-

mas develop in the cervical canals and anorectal man’s disease (Dupin et al., 1999) and primary eﬀusion

junction of women and men, respectively, with HIV/ lymphoma (Chapter 2F). In contrast to KS, NHL has

AIDS. Each site has a squamous-columnar border and become more common in HIV/AIDS, as patients live

there is evidence of viral causation (Mathews, 2003). longer as a consequence of HAART and prophylaxis

These regions are commonly infected with human for opportunistic infections (Boshoﬀ and Weiss, 2002).

papillomavirus (HPV) in these patient groups and, Diagnosis of NHL is essentially by biopsy, but this is

especially as immunosuppression increases in sympto- not routine for suspected primary cerebral disease. In

matic disease, the HPV-encoded oncoproteins (E6 and the latter case, a presumptive diagnosis of a CT lesion

E7) known to promote genetic instability in cells may is made in the absence of response to anti-toxoplasmal

lead to tumour formation.

739 Studies of those with HIV/AIDS conﬁrm an

HUMAN IMMUNODEFICIENCY VIRUSES

children infected with HIV-1 in the developed world. increased risk particularly of in situ cervical cancer

Evidence shows that treatment of the mother with but also of invasive disease (Boshoﬀ and Weiss, 2002),

zidovudine during the last trimester of pregnancy and which follows a more aggressive course in this patient

the infant for the ﬁrst 6 weeks of life produced a 60– group. More frequent (annual) cervical screening is

70% reduction in vertical transmission (Study ACTG oﬀered to patients with HIV/AIDS but anal cytology

076: 23% vs. 8% transmission in recipients of placebo; in men has not yet been systematically evaluated.

Connor et al., 1994). Studies of combination therapies These malignancies are treated conventionally with

are now under way to optimise regimens in relation to chemotherapy and radiotherapy.

drug toxicity and duration of treatment. However, these drugs are not available worldwide and probably less than 10% of HIV-infected mothers are in a

Other Cancers position to beneﬁt at present. Trials of a single dose of nevirapine given to women in labour to prevent

Other tumours that appear to have a high frequency in transmission have shown very good results. However, patients with HIV/AIDS include testicular tumours,

one concern raised by these studies is the emergence of squamous cell carcinoma of the oropharynx, tumours

nevirapine resistance in some of these women—the of the skin, hepatocellular carcinoma, and squamous

rapidity of this process is probably associated with the carcinoma of the conjunctiva in Africa (Allardice et al.,

slow decline in plasma levels following this one dose, 2003; Boshoﬀ and Weiss, 2002). These tumours may be

creating an ideal selective pressure for resistance. associated with immunosuppression and/or viral

Caesarean section gave early inconclusive results, but aetiology, e.g. papillomaviruses, but further evidence

a recent meta-analysis of European and American is awaited. An increase in liver and lung cancers in

studies showed a 55% reduction in risk of vertical HIV/AIDS may be accounted for by increased

transmission.

infection with hepatitis B and C viruses and cigarette Avoiding breast-feeding reduces the rate of trans- smoking, respectively, in HIV-positive persons.

mission and this is strongly advised in the developed world; however, in the developing world the risks to the infant of morbidity and mortality associated with

Clinical HIV/AIDS in Children gastrointestinal infections due to lack of conferred immunity from breast milk, and poor hygiene in

Since 1982, cases of vertically transmitted paediatric preparation of bottle feeds, are greater than those HIV infection have been associated with mothers in

associated with HIV infection. Besides, formula-feed- risk groups (intravenous drug users, sex workers,

ing is often stigmatised as indicating that the mother is partners of haemophiliacs, transfusion recipients and

HIV-positive. Thus, at present the World Health partners of bisexuals). Further large populations of

Organization advises exclusive breast-feeding in Africa infected children were identiﬁed in sub-Saharan Africa,

and avoidance in developed countries. the seat of the epidemic. Rates of vertical transmission

In summary, with the triple strategy of antiretroviral varied between African (30–50%), North American

therapy, Caesarean section and avoidance of breast (20–30%) and European (14%) studies. These diﬀer-

feeding, vertical transmission of HIV can be reduced to ences are probably due to populations at diﬀerent

negligible levels. However, this strategy requires stages of disease being investigated.

knowledge of maternal HIV antibody status in early The virus may be transmitted throughout preg-

pregnancy in order to accurately advise and implement nancy, during delivery and as a consequence of breast-

preventative measures, and at present this is not feeding. HIV has been isolated from ﬁrst/second

universally available. In the developed world the trimester aborted fetuses; twin studies show that the

uptake of HIV antibody testing in pregnancy is only ﬁrst-born has a higher rate of infection, presumed to be

about 50%, and in the developing world only an associated with longer exposure to secretions; and

incomplete infrastructure exists to screen and counsel breast-milk has been demonstrated to carry HIV and

pregnant women.

be infectious as a consequence of ingestion. Contem- porary research evidence indicates that the majority of transmission events occur perinatally and during

Disease Presentation in Children breast-feeding. Strategies for interruption of vertical transmission

Paediatric HIV-1 infection exhibits some distinct have had a dramatic impact on the numbers of

qualities in relation to adult disease; the natural

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

history has not been fully deﬁned but infection of the Wilfert, 1994), although there are increasing numbers host at a stage of immunological and biofunctional

of HIV-positive children surviving through to adoles- immaturity will clearly inﬂuence the expression of

cence and adulthood.

disease. Factors like time of infection, exposure to other pathogens, nutritional status, time of diagnosis and quality of care are all-important. Evidence

Diagnosis and Monitoring of HIV Infection suggests two groups of vertically infected infants:

30% with early clinical problems and life-threatening Serology remains the cornerstone for the diagnosis of illnesses in the ﬁrst year of life; and 70% with few

HIV infection in adults. ELISAs may be used alone to problems in early life but who develop disease after

exclude adult infection, but a positive diagnosis several years. It has been proposed that these two

involves more complex algorithms that are dependent groups represent early transplacental infection in the

upon the mode of transmission (horizontal or vertical) former and late infection in the latter. While this is

and the duration of the infection. The availability of plausible, one must also consider issues such as the

antiretroviral therapies makes rapid diagnosis impera- pathogenicity of infecting viruses and immunological

tive and strategies have therefore been devised to maturation, which may vary widely from case to case.

include PCR in clinical algorithms. The CDC disease classiﬁcation for children is

The algorithm for diagnosis of established adult summarised in Table 25.7. Infections are common in

disease is summarised in Figure 25.8. After the primary HIV-infected children. Herpes simplex, varicella zoster

screen using a sensitive HIV-1+HIV-2 ELISA format, and measles viruses all produce severe forms of the

all negative results are reported once the ‘reactive’ recognised presentations that require prompt and

samples in the run (those with a signal greater than the prolonged therapy to avoid high morbidity and

mean of the negative) are conﬁrmed and the patient mortality. Common paediatric bacterial infections,

identiﬁcation is assured. Reactive samples are entered such as Streptococcus pneumoniae, Haemophilus inﬂu-

into a conﬁrmatory algorithm using three further enzae , Escherichia coli and Salmonella spp., exhibit

ELISAs, with diverse HIV-1 antigens on the solid unusual presentations and severity. Prompt treatment

phase and diﬀerent principles of action (e.g. antiglo- is required until culture results are available, and

bulin, particle agglutination (PAA), competitive, etc.). multiple infections should always be suspected in

A positive report is released if consensus results are unresolving cases. Antimicrobial prophylaxis and

obtained from all three assays (490% of cases), and a immunoglobulin are instituted by some centres.

second blood specimen is requested to conﬁrm patient As in adults, opportunistic infections with Pneumo-

identity.

cystis carinii , Mycobacterium tuberculosis, M. avium If there is non-consensus in the conﬁrmatory tests, and M. intracellulare, CMV, cryptosporidia and non-

the clinical virologist should consider HIV-2 infection, oral candidiasis as a consequence of immunodeﬁciency

seroconversion, non-speciﬁc reactivity or, rarely, a new are common. Failure to thrive is a common feature of

non-reactive HIV subtype. The proposed algorithm for paediatric HIV disease. The reasons are multiple: sick

follow-up of these samples generally resolves 99.9% of mothers; decreased food intake due to oral infections;

cases, but a smaller number remain that often produce decreased absorption due to intestinal disease and

alarming reactivity, which may persist for life or recurrent diarrhoea; and decreased calorie utilisation

disappear over time. These cases are often resolved for growth, due to demands associated with inﬂam-

by repeat negative results with diagnostic PCR of matory responses and tissue repair.

proviral HIV-1 DNA from PBMCs. The Western blot HIV encephalopathy usually occurs in symptomatic

assay is relatively insensitive in relation to modern disease, presenting with developmental delay, motor

ELISAs and is reserved for diﬃcult cases, HIV-2 deﬁcits, and cognitive and behavioural disorders. It

conﬁrmation, seroconverters and research. may be slow or rapid in progression, and cerebral

The adult seroconverter is confounded by initial low atrophy and ventricular enlargement is seen on CT.

or non-detectable levels of anti-HIV-1 antibodies, and Other infectious causes, such as Toxoplasma, Myco-

ELISAs may be negative in the face of clinical signs bacteria , Cryptococcus and JC virus, are unusual but

and symptoms of PHI. If a clinical diagnosis of PHI is should be excluded.

proposed, the sample is tested with all four serological The use of antiretroviral therapies has improved the

assays, a p24 antigen assay and the diagnostic PCR prognosis for all these clinical conditions. We do not

test. In most cases a PCR-positive result is seen prior know the long-term prognosis of acquiring HIV at

to, or associated with, low-level seroreactivity, and in such an early phase of host development (Pizzo and

approximately 50% of cases will show detectable

HUMAN IMMUNODEFICIENCY VIRUSES

Figure 25.8 An algorithm for the diagnosis of HIV-1 infection

plasma p24 antigen. This is repeated at weekly they are PCR-negative and that they have non- intervals and evolving signals in the serological assays

evolving serology. They are followed over time for conﬁrm seroconversion. The Western blot will demon-

reassurance, support if they proceed to any other strate the evolution of a serological response to speciﬁc

medical investigations, repeat testing at 6-monthly HIV antigens but that post-dates the sensitive serolo-

intervals, and in the majority the reactivity will gical assays (Table 25.10).

eventually wane.

Patients with non-speciﬁc reactivity (presumed to The diagnosis of HIV-1 infection in the newborn have cross-reacting antibodies that signal in certain or

infant is confounded by the presence of maternal all assays) show a distinctive virological pattern but

antibodies in the baby’s circulation from approxi- present a diﬃcult clinical problem. They often present

mately 32 weeks of gestation onwards. Early diagnosis through the blood transfusion service as a reactive

is essential for early clinical interventions and con- sample on whom the donation has been withheld. The

siderable eﬀort has been applied to establishing clinical follow-up usually involves establishing that

algorithms. One approach uses Western blot and

Table 25.10 Typical serological and PCR changes associated with early primary HIV-1 infection Time (weeks)

1 2 4 12 Immunometric

7 6 Illness

POS POS HIV-1+2 ELISA

POS POS HIV-1 ELISA

(1/128) (1/512) Western blot

EQUIV POS

(p24/gp160) p24 antigen

(p24 wk b.d.)

NEG NEG ELISA

(510 pg) (510 pg) Diagnostic PCR

POS POS ( pol/gag)

NEG

POS

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

Table 25.11a Typical serological and PCR changes from birth associated with an uninfected baby Time

12 months 18 months PAA

(1/200) (51/16) Western blot

EQUIV NEG

(2 bands) (—) p24 antigen

NEG NEG ELISA

(510 pg) (510 pg) Diagnostic PCR

NEG NEG ( pol/gag)

Table 25.11b Typical serological and PCR changes from birth associated with an infected baby Time

12 months 18 months PAA

(1/200 K) (1/980 K) Western blot

(3 bands) (14 bands) p24 antigen

NEG NEG ELISA

(510 pg) (510 pg) Diagnostic PCR

POS POS ( pol/gag)

titrated particle agglutination assays to measure et al. , 1995) and to demonstrate the rapid initial decline qualitatively and quantitatively antibody concentra-

in plasma HIV-1 that deﬁned the dynamics of virus tions from 3 days of life onwards, serum p24 antigen

replication and allowed the development of new and diagnostic PCR of infant-speciﬁc HIV-1 proviral

theories of pathogenesis (Perelson, 2002). By 1994, DNA (Table 25.11). If available, PBMC culture may

ﬁrst-generation commercial assays were available, also be added to the algorithm. Rapid diagnosis is

based upon plasma HIV-1 nucleic acid capture, reverse conﬁrmed by two positive PCR and one positive p24

transcription and ampliﬁcation and resulting comple- antigen result in samples taken at 3 days, 3 weeks and 3

mentary DNA signalling to quantify plasma viral load. months. Titrated serology in an uninfected baby of an

These genome assays initially detected down to a HIV-infected mother should demonstrate a halving of

level between hundreds and thousands of copies per ml passive antibody concentration every 28 days; if

plasma, but later second-generation assays deﬁned cut- antigen (virus) is present which stimulates an immune

oﬀ values of around 20–50 (Loveday, 1999). These response in an infected baby, this will be reﬂected in a

viral load assays were initially used experimentally to levelling-oﬀ of the exponential decline in antibody

deﬁne virological eﬃcacy in clinical trials (Brun-Vezinet concentrations or an increase over time. It takes up to

et al. , 1997). A large cohort study revealed the

18 months for all passive antibody to disappear in the predictive value of such measures for clinical outcome majority of infants and the analysis and reporting of

(Mellors et al., 1996). Thus, viral load was established these events involves considerable virological experi-

as an essential marker, substituting for clinical end- ence.

points, to define the efficacy of new antiretroviral drug In the early 1990s molecular virologists modified

combinations. In clinical practice, patients have reg- PCR-based technologies to quantify HIV-1 RNA load

ular viral load measures to assist the determination of (viral load) and genotypic resistance in plasma viruses

disease stage, monitor progression, assess responses to (Kaye et al., 1992; Semple et al., 1993). These methods

antiretroviral therapies, and provide evidence of early were useful to monitor the eﬃcacy of antiretroviral

treatment failure.

therapy in trials (Katlama et al., 1996; Schuurman et Early in vitro studies and investigations of patients al. , 1995), to demonstrate their predictive value for

receiving monotherapies revealed that, as viral load disease progression (Loveday and Hill, 1995), to

response failed, drug-resistant viruses evolved in the demonstrate the association between failing virological

plasma. These were detected using both drug-sensitive response and evolving resistance in vivo (Loveday

culture assays (phenotypic) and HIV RT and protease

743 Table 25.12 Comparison of the advantages and disadvantages of genotyping and phenotyping for the determination of

HUMAN IMMUNODEFICIENCY VIRUSES

antiretroviral drug resistance Advantages

Disadvantages

Genotyping Widely available in laboratories

Indirect measure

Rapid PCR-based methods (days) Limit of detection (2–5% of population) Less technically demanding

May not correlate with phenotype Mutations precede phenotype

Complex data generated that needs virological Quantiﬁes the proportion of wild to mutant

interpretation

strains

May be diﬃcult to interpret Expensive (4£200.00/sample) If genetically diverse will not amplify

Phenotyping Direct measure Restricted availability (i.e. no clinical throughput system PCR-based technology gives improved variance

in UK)

Familiar to physicians

Slow (weeks) Technically demanding, needs P3 a Less sensitive than genotyping Very expensive (4£400.00/sample) No analysis of sensitivities of combination of drugs

a P3, high containment laboratory for the culture of certain infectious pathogens like HIV-1.

genome analysis to identify mutations associated with (Torre and Tambini, 2002) concluded that there was resistance (genotypic). These approaches were rapidly

beneﬁt for using genotyping but not phenotyping to adapted for commercial assays, which allowed a wide

support clinical care, and that expert opinion added availabilty of resistance testing in clinical virology

signiﬁcantly to the eﬀect. Recently the MRC ERA trial laboratories to support patient care. The relative

has also demonstrated no additional beneﬁt (in terms advantages and disadvantages of these approaches

of virological and immunological responses) of pheno- are summarised in Table 25.12. Phenotypic testing

typing in addition to genotyping in patients with requires RT/protease genes from patient plasma

limited therapeutic options after 1 year of follow-up viruses to be transfected into recombinant HIV viruses

(Loveday et al., 2003).

and their susceptibility to single antiretroviral drugs to

be evaluated in vitro. Results are expressed as fold change in sensitivity to the drug, relative to a

ANTIRETROVIRAL THERAPY recombinant drug-sensitive laboratory strain of HIV-1.

Clinical cut-oﬀs for susceptibility have been derived for each drug that correlate closely with antiviral activity

Historical Context in humans.

Antiretroviral therapy, where available, has trans- Genotypic testing involves the nucleotide sequence

formed HIV-1 infection into a treatable chronic analysis of RT/protease to identify mutations known

condition rather than a death sentence (Pomerantz to be associated with drug resistance. The results are

and Horn, 2003). With hindsight, many now recognise described according to their codon position (e.g.

the suboptimal policies made during the early period of M184V, the mutation associated with lamivudine

antiretroviral therapy. Zidovudine was the ﬁrst resistance, involves the substitution of a methionine

licenced drug against HIV-1, in the late 1980s, for valine at codon 184 in the RT gene). Tables of

following demonstration of clinical beneﬁt in a mutations are prepared by expert panels based on

placebo-controlled trial, and there was widespread scientiﬁc and clinical experience to provide ‘rules-

demand for monotherapy at this time. Controversy based’ advice of the relative importance of mutational

was fuelled by results of the French–UK Concorde patterns in any given clinical case (www.iasusa.org).

study, which showed little beneﬁt of initiating zidovu- Retrospective data show that antiretroviral resis-

dine therapy early, compared to later in disease. In the tance is associated with poor virological and clinical

early 1990s double therapy with ZDV and didanosine outcomes, but prospective randomised controlled trials

or zalcitabine (or other nucleoside analogues) proved to demonstrate clinical utility of resistance testing vs.

more beneﬁcial than single therapy, which provided standard of care generally involve small patient groups

the underpinning for later combination treatments with short follow-up periods. A recent meta-analysis

using three or more drugs. Monitoring of therapy

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

response was undertaken by CD4 cell count changes

2. Following viral rebound, all components of the during this period, as well as the time to clinical

drug regimen should be changed, if possible, to endpoints, such as AIDS and death.

maximise the beneﬁt of new drugs. The mid-1990s marked a paradigm shift in HIV

3. The advantages of virological suppression should therapy, driven by two independent advances. First,

be balanced against the long-term toxicities of assays for accurate and sensitive quantitation of

therapy, poor adherence and emergence of resis- plasma viraemia became commercialised and therefore

tance, when deciding on the time of initiation of widely available. Second, the results of the ﬁrst clinical

therapy. Currently, clinical cohort data suggest that studies of protease inhibitors (PI) demonstrated the

starting therapy at a CD4 cell count of 200–350 profound potency of this class of drugs when

cells/ml confers similar beneﬁt to patients as starting combined with two nucleoside analogues, deﬁned by

at an earlier stage of infection (Egger et al., 2002). reductions in viral load. Since the application of plasma virus quantitation to natural history cohorts

Therapy of Pregnant Women and Neonates progression (Mellors et al., 1996), a leap of logic was taken to postulate that a therapy-induced fall in viral

showed viral load to be predictive of disease

In general, the indications for treatment of pregnant load would lead to improved prognosis, and this was

women conform to the guidelines for non-pregnant demonstrated subsequently. Since viral replication (as

adults. Those in whom therapy is not indicated, e.g. measured by plasma viral load) became central to

due to a high CD4 cell count, should then initiate theories of pathogenesis, the pendulum swung back to

treatment in the late 2nd/early 3rd trimester, to prevent early initiation of therapy. This approach was exem-

vertical transmission. Many such regimens include pliﬁed by the statement at the time, ‘it’s the virus,

zidovudine in light of the favourable results in ACTG stupid’, and various estimates were made of the

076, and some prefer to use monotherapy if the viral duration of therapy required to eradicate the virus

load is low. Therapy of the neonate should continue from infected individuals.

for 4–6 weeks. Current guidance suggests that oral Unfortunately, this optimism was tempered by two

zidovudine should be given if the mother received this practical manifestations of therapy—adherence pro-

drug. Combination therapy should be considered if the blems and toxicity, as well as the recognition of long-

risk of transmission is high, e.g. the mother starts lasting reservoirs of infection within the body. Long-

therapy late in pregnancy.

term PI therapy generated severe lipid abnormalities, leading to body shape changes and cardiac problems (although some of these are now also known to be

DRUGS AND THEIR RESISTANCE PATTERNS associated with long-term nucleoside analogue treat-

ment; Carr, 2003). The large pill burden of early triple Below, each approved drug is brieﬂy described, with its therapy regimens also caused diﬃculty in drug com-

drug resistance patterns, since clinical virologists are pliance, and rates of therapy failure were signiﬁcant.

increasingly required to advise clinicians on the By the late 1990s, non-nucleoside reverse transcriptase

interpretation of drug resistance test results (Table inhibitors (NNRTIs), such as nevirapine and efavirenz,

25.13). The reader is directed to a more detailed review had started to replace PIs within ﬁrst-line regimens,

for discussion of resistance (D’Aquila et al., 2003) and following encouraging clinical trial data.

also a recent description of recently available drugs More recently, there has been a move towards later

(Gulick, 2003). Further, information on the lipodys- initiation of therapy, in the CD4 count range of 200–

trophic side-eﬀects of anti-HIV-1 drugs can be found 350610 6 /l, based on cohort study data. This is a

in the following reviews (Carr, 2003; John et al., 2001; reﬂection of the remaining inadequacies of therapy,

Nolan et al., 2001).

primarily with respect to adherence, toxicity and resistance issues, rather than representing an ideal approach to a chronic viral infection.

Nucleoside and Nucleotide Analogues The principles currently underpinning antiretroviral

therapy are as follows: Six nucleoside analogues and one nucleotide analogue are currently approved for use. They act to inhibit the

1. Drugs should be used in combination (at least three viral RT. They all represent analogues of one of the drugs), for the purposes of potency and limiting the

four natural nucleosides, thymidine, cytidine, adeno- escape of resistant mutants.

sine and guanosine, and become triphosphorylated

745 Table 25.13 Antiretroviral drugs available to treat HIV-1 infection

HUMAN IMMUNODEFICIENCY VIRUSES

Drug Dose

Viral load a and

Adverse reactions

Comments

resistance b

NRTIs Zidovudine (ZDV)

250–300 mg

Prodrug b.d.

7 0.5 to 0.7 log 10 Nausea, headache, myopathy,

codons 41, 67, 70,

bone marrow suppression

Enters CNS

Combivir-AZT/3TC Didanosine (ddI)

215 and 219, 151

200 mg b.d.

Prodrug 400 mg o.d.

7 0.5 to 0.7 log 10 Nausea, diarrhoea, rarely

4 Eﬃcacy with hydroxyurea (no food)

codons 65, 74, 75,

pancreatitis, peripheral

neuropathy

Zalcitabine (ddC) 0.75 mg t.d.s.

7 0.5 log 10 Oral ulcers and peripheral

Lamivudine (3TC) 150 mg b.d. 7 0.5 to 0.7 log 10 Nausea, bone marrow suppression Prodrug

Well tolerated Stavudine (d4T)

codon 184

Prodrug Abacavir (1592)

40 mg b.d.

0.6 log 10 codon 75

Peripheral neuropathy

300 mg b.d.

7 1.5 log 10 Hypersensitivity: fever, rash, and

Prodrug

codons 65, 74,

fatal rechallenge

Rechallenge contraindicated

Tenofovir 300 mg o.d.

0.3–1.5 log 10 Fanconi’s syndrome—very rare

Nucleotide analogue. Well

codons 65, and

tolerated

41/67, /210/215 together

NNRTIs Nevirapine

200 mg b.d.

Long half-life. initial dose

Rash on induction 415%, 5%

grade 3, induces cytochrome P450 Induction with prednisolone escalation

codons 103, 106,

Delavirdine 400 mg t.d.s.

USA only 600 mg b.d.

Rash 420%, 5% grade 3,

codons 103, 181,

headache, nausea, raised LFTs

Inhibits cytochrome p450

Efavirenz 600 mg nocte

Induces and inhibits (minimise

Headache, dizziness, vivid

cytochrome p450 side-eﬀects)

codons 100, 103,

insomnia, rarely psychoses,

raised LFTs

Protease inhibitors (PIs) Indinivir

800 mg t.d.s.

‘Early eﬃcacy, late toxicity’ fasting/low

7 1 to 2 log 10 Nausea, nephrolithiasis,

x-resistance PIs fat

codons 82, 46,

haematuria, lipodystrophy,

1062nd MTs

hyperlipidaemia

Ritonavir 600 mg b.d.

‘Early toxicity’ (400 in

7 1 to 2 log 10 Nausea, vomiting, taste changes,

x-resistance PIs combo)

codons 82 and

lipid and transaminase elevation

1062nd MTs

Early formulation had poor 1800 mg b.d.

Saquinavir (soft gel) 1200 mg t.d.s. 47 1 log 10 Nausea, diarrhoea, mild

absorption (with food)

codons 48, 90

abdominal pain (self-limiting)

x-resistance PIs Nelﬁnavir

762nd MTs

750 mg t.d.s.

x-resistance PIs 1250 mg b.d.

7 1 to 2 log 10 Diarrhoea: controlled by drug

codon 30

treatment (self-limiting)

(with food)

862nd MTs

Amprenavir 1200 mg b.d.

47 1 log 10 Nausea, vomiting, taste changes,

x-resistance PIs

codon 50

paraesthesia

462nd MTs

LFT, liver function test; MT, mutations; NRTI, nucleoside reverse transcriptase inhibitor; NNRTI, non-NRTI; NR, not recorded; PI, protease a inhibitor.

b Reduction of viral RNA in plasma. Position of mutations conferring resistance in reverse transcriptase (NRTIs, NNRTIs) and protease.

within the cell so that they compete with natural Zidovudine was the ﬁrst licenced drug against HIV-1 nucleotides as substrates for the viral RT. The

(Pomerantz and Horn, 2003). Together with stavudine, nucleotide analogue, tenofovir, is a phosphonate,

it is a thymidine analogue, and these two drugs are structurally similar to a nucleoside monophosphate.

antagonistic, presumably due to competition for the

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

same intracellular phosphorylation pathways. Since it resistance mutations. In addition, the presence of the was the ﬁrst drug to demonstrate eﬃcacy as mono-

T215Y/F/Q mutation predicted a poor short-term therapy, it has remained a key component of

response to a stavudine/lamivudine combination in combination regimens. Beneﬁt was also observed in

zidovudine-experienced patients (Montaner et al., prevention of mother-to-child transmission in the

2000). Although the phenotypic resistance to stavudine landmark ACTG 076 study, in which drug was given

conferred by such mutations is relatively modest, small from the second and third trimester of pregnancy, and

shifts in susceptibility are suﬃcient for this drug to lose to the newborn for 6 weeks following birth. The

eﬃcacy.

relatively high rates of toxicity observed in early Didanosine is an adenosine analogue which requires studies (e.g. anaemia) have been ascribed to the large

deamination prior to phosphorylation. Gastrointest- doses used at that time, and this is less of a problem

inal side-eﬀects have been reduced somewhat by the with current dosing regimens.

introduction of an enteric coated pill, although a major The extensive use of zidovudine has led to a detailed

side-eﬀect remains pancreatitis. The emergence of appreciation of virus resistance. The acquisition of

resistance to didanosine and zalcitabine occurs more high-level zidovudine resistance requires several

slowly than for zidovudine in vivo and in vitro. In changes in the RT, including amino acid positions

addition, those mutations conferring resistance, such

41, 67, 70, 215 and 219. Large surveillance studies of as at positions 65, 69, 74 and 184, lead to only modest nucleoside analogue-experienced patients identiﬁes

increases in IC 50 . Longer experience with these drugs T215Y as the most prevalent drug resistance mutation.

has allowed more subtle identiﬁcation of cross- This is unsurprising in view of the time period over

resistance with other nucleoside analogues. An increas- which zidovudine has been available. It has also

ing resistance to didanosine is observed as nucleoside become apparent that the mutations associated with

analogue mutations are accumulated, and the clinical thymidine analogue resistance cluster according to two

relevance of small changes in fold resistance may have groups, vz. that including mutations at positions 41,

a greater impact than previously recognised.

67, 210 and 215 (Y), and a group including 70, 215 (F) Zalcitabine , a cytidine analogue, is associated with and 219 mutations. The determinants of one or other

peripheral neuropathy. Additionally, potency is only route remain unclear. The key mechanism by which

modest, and therefore this drug is now rarely used in ZDV resistance mutations confer reduced susceptibil-

clinical practice.

ity is thought to be an increase in pyrophosphorolysis, Lamivudine , another cytidine analogue, has potent whereby a ZDV-monophosphate moiety incorporated

activity against HBV as well as HIV, which makes it an into the growing cDNA chain is subsequently cleaved,

important component of therapy for co-infected thus allowing continued reverse transcription rather

individuals. It is well tolerated, and has been combined than chain termination by the drug. It is of interest

within a single pill with zidovudine, as well as with that interactions between mutations are increasingly

zidovudine and abacavir, to improve adherence. High- evident, such as the attenuating eﬀect of M184V

level lamivudine resistance is generated by the M184V (lamivudine resistance), L74V (didanosine resistance)

mutation within RT, and occurs within weeks on and Y181C (nevirapine resistance) on the phenotype of

monotherapy. It is also commonly observed as the viruses containing zidovudine-resistance mutations. At

initial mutation emerging following failure of a least for the M184V mutation, this is due to reversal of

lamivudine-containing triple regimen, suggesting that the pyrophosphorolysis process described above.

the loss of control of this drug drives the evolution of Stavudine , a thymidine analogue, has proven eﬃcacy

resistance against other components of the regimen. within combination regimens and has good bioavail-

Nevertheless, some lines of evidence call into question ability. Concerns about toxicity focus on peripheral

the precise impact of this mutation. Virological neuropathy and lactic acidosis, probably associated

analysis of the NUCA 3001 study in drug-naı¨ve with inhibition of mitochondrial DNA polymerase-g.

patients demonstrated that lamivudine with zidovu- For many years, stavudine resistance was thought to

dine eﬀected greater viral load suppression over 24

be associated with changes at position 75 of RT only, weeks compared to zidovudine alone, despite the which were rarely observed in clinical practice. More

virtually universal emergence of M184V in the recently, long-term stavudine therapy has been asso-

double-therapy arm. A longer-term follow-up on a ciated with the emergence of zidovudine resistance-

similar lamivudine/zidovudine patient cohort showed associated mutations, as well as ‘multi-drug’ resistance

phenotypic resistance to zidovudine, but not lamivu- mutations (see later), and a poor response to stavudine

dine, to be the only independent risk factor for has been associated with the presence of zidovudine

virological failure. Similar results have been presented virological failure. Similar results have been presented

A number of explanations for these observations have been put forward. First, the fitness of the M184V mutant may be reduced, thus contributing to a reduced virological rebound following emergence of lamivudine resistance. Second, this mutation enhances RT fidelity, which in turn would reduce the rate at which new mutants (including drug-resistant mutants) are generated. Third, the presence of M184V partially reverses the zidovudine resistance phenotype in the presence of zidovudine resistance mutations. Finally, pyrophosphorolysis appears to be diminished within a M184V-containing RT, thus enhancing the chain termination effect of the nucleoside analogues. All these mechanisms have been demonstrated within in vitro systems, often with purified RT, and may not be applicable in vivo. Indeed, there is conflicting evidence that reduced fitness or increased fidelity of the M184V mutant is a significant factor with infected individuals. In addition, long-term therapy with zidovudine and lamivudine lead to the emergence of novel mutations, such as at positions 43, 44, R211K, L214F and G333E/

D, suggesting routes to lamivudine resistance that bypass M184V. Abacavir is a guanosine analogue, which is converted to carbovir triphosphate as the active component. A severe hypersensitivity reaction may occur in more than 5% of patients. Since re-challenge has been associated with death, close monitoring is required when initating patients on this drug. In vitro selection experiments lead to a virus with reduced abacavir susceptibility associated with mutations at RT positions 65, 74, 115 and 184. Preliminary data suggest that viral rebound in patients receiving zidovudine/lamivudine/abacavir is initially associated with the appearance of M184V alone. This is likely to reﬂect resistance to lamivudine in these patients, as discussed above, and such viruses appear to be fully susceptible to abacavir. A more extensive analysis of the success of abacavir use within salvage therapy demonstrates that abacavir failure is associated with the presence of three or more zidovudine resistance- associated mutations at baseline. Nevertheless, even in heavily pre-treated patients, the reduced susceptibility to abacavir observed in phenotypic assays is often rather modest (5eight-fold resistant).

Tenofovir is the ﬁrst nucleotide analogue to be approved for HIV treatment, which appears to be unencumbered by the renal toxicity problems of its cousin, adefovir. Like lamivudine, it is a potent inhibitor of HBV as well as HIV-1. As for many other drugs, the RT mutations associated with reduced

activity in the clinic are not necessarily those selected by tenofovir in the laboratory (K65R). This is because the drug has been most widely tested in drug- experienced patients in whom resistant virus already exists and predictors of poor response can be identified. Thus, common nucleoside analogue resistance mutations, such as M41L, L210W (possibly a key marker in this respect) and T215Y, appear to reduce, although not to negate, clinical efficacy. Nevertheless, the widespread use of tenofovir in salvage therapy, and promising first-line treatment trial data, suggest that it represents an important addition to our antiretroviral armoury.

Cross-resistance between Nucleoside Analogues

There is variable cross-resistance between the five licensed drugs discussed above, such that second- or third-line therapy may be compromised following failure of first-line therapy. In addition, combination therapies may select for novel mutations or groups of mutations not observed in monotherapy studies. Some of these have been discussed above. Other examples include the constellation of A62V, V75I, F77L, F116Y and Q151M, which confer cross-resistance to all NRTIs. Of interest is that viruses containing all five of the above mutations appear more replication- competent than wild-type virus within in vitro competition experiments. Multi-nucleoside analogue resistance is also caused by a diverse cluster of amino acid insertions and deletions between positions 67 and

70, commonly 69S-(S-S) or 69S-(S-G), which directly inﬂuence the nucleoside triphosphate binding site of RT. To date, the prevalence of these multi-drug resistance mutations in treated patients appears low; however, this ﬁgure should be expected to rise, and is of major concern.

New Drugs for Use against Nucleoside Analogue-resistant Viruses

Amdoxovir (DAPD) is a new nucleoside analogue prodrug whose oral administration leads to a rapid in

vivo conversion to (7)-b- D -dioxalane guanosine (DXG). Resistance to this drug in the laboratory appears to involve the K65R and L74V mutations, similar to those observed for abacavir (although abacavir failure is rarely associated with these mutations in the clinic). Phase I/II studies demonstrate a reasonable activity of this drug against nucleoside

HUMAN IMMUNODEFICIENCY VIRUSES

747

analogue-resistant viruses, although more data are needed before clarifying its potential role. FTC, a ﬂuorinated derivative of lamivudine, has similar potency and spectrum of action to lamivudine. However, it has a long half-life and can be dosed once daily, which may provide an advantage over lamivudine.

Non-nucleoside Reverse Transcriptase Inhibitors (NNRTIs)

The NNRTIs are a structurally diverse group of compounds, of which nevirapine and efavirenz are approved for use (Table 25.13). Despite this diversity, the current drugs all bind within the same RT pocket, occupied by the side chains of amino acids at positions 181 and 188, and the 103 residue is close to the entry to this site. This pocket is not present in HIV-2, and therefore NNRTIs are ineffective against this virus. The potency of these drugs, and at least equivalence to PI-containing regimens, has led NNRTI-containing regimens to be common as first-line therapy. The most common side-effect of nevirapine is a self-limiting rash; however, a rare but severe (fulminant) hepatitis has been reported, and close monitoring of liver function is required. The major side-effect of efavirenz is insomnia and vivid dreams, especially in the first weeks after starting treatment.

In vitro selection experiments demonstrate the rapid acquisition of high-level resistance to NNRTIs. The mutations associated with resistance may vary between drugs, with variable levels of phenotypic cross- resistance. However, wide cross-resistance appears evident in clinical practice and failure of NNRTIs is often caused by a single mutation. It is likely that these variants therefore pre-exist within the viral population, as for the lamivudine resistance mutation, and can quickly emerge if viral replication is maintained on therapy.

Failure of efavirenz-containing triple regimens is associated with the K103N mutation in up to 90% of cases, producing phenotypic cross-resistance to nevirapine and delavirdine. Other mutations, such as G190S/A/E, Y188L and L100I, may also be observed and they may be acquired sequentially. This suggests that the emergence of high-level resistance with K103N does not preclude further selective pressure. The low genetic barrier to emergence of NNRTI resistance reaﬃrms the importance of maintaining optimal suppression of viral replication in those receiving this class of drugs.

The rapid emergence of resistance to nevirapine in monotherapy studies during the early 1990s led to a halt in further clinical development of this compound. More recently, the eﬃcacy of this drug has been demonstrated within the context of combination regimens. The genetic pathway to resistance is dependent upon co-therapies. Thus, resistance to single therapy is usually caused by the Y181C mutation. By contrast, in the presence of zidovudine, other mutations, such as the K103N, are the preferred route. This may be explained by the in vitro observation that the Y181C mutation suppresses the emergence of zidovudine resistance. Thus, there may be an evolutionary bias against the emergence of the 181 mutation in such co- treated patients. The majority of patients failing a nevirapine-containing therapy will be expected to show NNRTI mutations, the most common combination being K103N and Y181C, but also including changes at codons 101, 106, 108, 179, 188 and 190. This illustrates the variability in pathways to nevirapine resistance, possibly inﬂuenced by the concurrent nucleoside analogues used.

Finally, it is noteworthy that ‘naturally’ occurring resistance to NNRTIs has been observed in group O HIV-1 strains, as well as HIV-2 and SIV. In addition, many non-clade B subtypes of HIV-1 contain polymorphisms at RT positions which may impact on NNRTI susceptibility, such as codons 98, 101 and 179. More work is required to delineate the precise impact of these polymorphisms, and whether they inﬂuence the genetic route to high-level NNRTI resistance.

New NNRTIs The phenomenon of extensive cross-resistance between

NNRTIs is one of the more widely accepted facts of HIV drug resistance, due to the small binding site for this group of drugs within the viral RT. The key mutations in this regard are K103N, T181C and G190A/E, all of which compromise nevirapine, efavirenz and delaviridine responses, and this cross- resistance represents a major limitation of the class as a whole. However, two new compounds, TMC125 and TMC120, appear to have activity against such resistant viruses, both in vitro and in vivo. Another compound (capravirine) demonstrated activity against

a virus bearing the K103N or V106A or L100I single mutation, although high-level resistance to this drug was reported in the presence of mutations at codon 181.

It appears not so much that diﬀerent patterns of resistance mutations are observed with these new

748

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

NNRTIs drugs, but rather that emergence of resistance is much slower than existing NNRTIs—note that single-dose nevirapine in pregnancy is suﬃcient to select for resistant mutants—and that the well-recognised NNRTI mutations have a marginal, and possibly clinically irrelevant, impact on fold susceptibility. It is argued that these properties are a function of the unique structures of these second-generation NNRTIs, in the context of binding to the RT enzyme.

Protease Inhibitors The development of protease inhibitors (PIs) followed

rapidly on from the publication of the crystal structure of HIV-1 protease in 1988 (Pomerantz and Horn, 2003). The enzyme itself is small, comprising a homodimer with 99 amino acids in each strand. Since the functional expression of protease is essential for virus replication, it was initially thought that emergence of drug resistance mutations within such a small gene would be limited. Not only has this proved incorrect but also an extensive polymorphism of this gene has been demonstrated in viruses from PI-naı¨ve patients, such that up to 50% of the amino acids may vary within clade B viruses. This diversity widens when other HIV-1 subtypes are considered. Nevertheless, these variants do not appear to compromise in vitro or in vivo responses to PIs. All PIs are metabolised, at least partially, through the CYP 3A4 isozyme of the cytochrome p450 system. This leads to signiﬁcant interactions with other drugs, and also individual diﬀerences in plasma drug levels achieved. The major limitation of this class of drugs is the syndrome of hyperlipidaemia, insulin resistance and peripheral fat wasting (lipodystrophy), the precise pathogenesis of which is under intense investigation.

Indinavir . The major side-eﬀect is nephrolithiasis, including ﬂank pain with or without haematuria. Early dose ranging studies of indinavir monotherapy generated detailed information on resistance-associated mutations for this drug. Sequential acquisition of mutations was observed, with changes at positions 10,

24, 46, 54, 71, 82, 84 and 90 being significantly correlated with phenotypic resistance. Nevertheless, the V82A/F/T mutation is recognised as the best predictor of reduced indinavir susceptibility occurring early in drug failure, with or without M46I/L/V (although not in themselves leading to significantly reduced susceptibilities). These changes are followed by a series of other more variable changes, which confer increasing resistance and compensate for reduced fitness. A significant advance in our under-

standing of protease inhibitor resistance was made following failure of indinavir therapy with the demonstration of mutations, in conjunction with protease mutations, which map outside the protease gene, but map within the enzyme’s substrate, the gag protease cleavage sites. Changes at the gag p7/p1 site are most commonly observed, and mutagenesis experiments suggest that their major role is to compensate for partial replication deﬁciency caused by the 82 and/or

46 mutations. Similarly, some of the additional mutations acquired within the protease gene itself may also be compensatory.

Saquinavir . The bioavailability of the original hard- gel capsule of this drug was low but it has been increased by the use of the soft-gel formulation. Failure of monotherapy within clinical trials was commonly associated with the L90M mutation and rarely with the G48V, mutations which together severely reduce the catalytic efficiency of the enzyme. Although residue 48 is in an important flap loop of the enzyme, residue 90 appears distant to the active site and may lead to conformational effects on inhibitor binding.

Nelfinavir . Co-adminstration with high-fat meals increases blood levels of nelfinavir significantly, and improves antiviral efficacy. Diarrhoea remains the most important side-effect. Initial monotherapy studies with nelfinavir identified a unique mutation, D30N, as responsible for reduced drug susceptibility, without corresponding cross-resistance to other protease inhibitors. It is now apparent that failure of nelfinavir- containing triple regimens is associated with either the D30N or the L90M mutation. The 30 mutation may be associated with N88D and A71T/V, whereas the 90 mutation emerges together with changes at one or more of positions 10, 20, 46, 60 73 and 74. The constellation of mutations around L90M confers cross-resistance to saquinavir and possibly other PIs, and therefore the route taken for nelfinavir resistance may determine the success of subsequent PI therapies; however, more extensive evidence is required.

Amprenavir . In vitro selection of amprenavir-resistant virus identiﬁes I50V as a key resistance mutation, together with other secondary mutations. However, cross-resistance of isolates from PI-experienced patients to amprenavir can be predicted by the presence of M46I/L, I54L/V, I84V and L90M. Thus, an algorithm of I84V and/or any two of the three mutations 46/54/90, allowed prediction of high-level resistance with a sensitivity of 88% and speciﬁcity of 79%. In view of the poor pharmacokinetics and high pill burden of amprenavir, a prodrug, fos-amprenavir has recently been developed.

HUMAN IMMUNODEFICIENCY VIRUSES

749

Ritonavir . This was the first PI to be licensed in Europe. However, the frequency and severity of side- effects has limited its use as an antiviral drug in its own right. It is one of the most potent p450 inhibitors identified. This has led to it being used primarily at low dose to boost the blood concentrations of other PIs. Such dosing is not thought sufficient to confer antiviral efficacy, or to select for resistance.

Lopinavir . This is co-formulated with ritonavir to achieve drug levels suﬃcient to inhibit viruses with resistance mutations for many other PIs. The ratio of

trough drug concentration divided by the IC 50 value,

termed the inhibitory quotient (IQ), has been introduced in order to quantify the benefit conferred by high drug levels. Such IQ measurements are currently undergoing trials to assess their clinical utility. The major side-effects of lopinavir appear to be lipid abnormalities. In vitro selection experiments identified lopinavir resistance-associated mutations at 84, 10, 46,

71, 32 and 47. However, it appears likely that the clinical benefit of this drug, co-administered with ritonavir, is due to the high plasma levels achieved, which may overcome reduced drug susceptibility, rather than lack of cross-resistance patterns per se. This drug appears to be highly effective when used as first- or second-line PI therapy, and little data are yet available on the mutations which emerge during therapy to lead to drug failure.

New PIs Issues of resistance and cross-resistance are particu-

larly pertinent to the protease inhibitor class of drugs. Many claims have been made on the apparent uniqueness of resistance patterns for specific drugs, based on in vitro data, which do not then translate into clinical benefit for that drug in PI-experienced patients. Two new PIs have now undergone initial clinical evaluation. Atazanavir demonstrated different resistance profiles when used in PI-naı¨ve and PI- experienced patients. In the former group, resistance emerges with the I50L and A71V mutations. This is a unique combination, since amprenavir-resistance mutations include a different amino acid change at position 50 (viz. I50V), although the A71V mutation is

a polymorphism (not infrequently observed in the absence of PI therapy). By contrast, in PI-experienced patients, some level of cross-resistance between atazanavir and other PIs was apparent. Since the I50L– A71V mutation combination does not appear to reduce susceptibility to other PIs, there may be advantage, from a resistance perspective, in using

atazanavir as a first-line PI; however, data are required on the actual efficacy of PI treatment after atazanavir failure to fully assess the importance of the ‘unique resistance pattern’. Clinical data have also been presented for tipranavir, which shows potency against viruses containing a large variety of PI-resistance mutants in vitro. Clinical activity was observed in PI- experienced patients, suggesting that a large number of PI-resistance mutations were required to compromise activity. More work is required to further clarify such ‘clinical cut-offs’, whereby clinicians can be guided on the likely effect of this new drug in a patient with existing PI-resistant virus.

Fusion Inhibitors

Following attachment of the virus to the cell membrane, the helical proteins of the gp41 molecule contract and bring the gp120 and cell receptor into close proximity, thus allowing membrane fusion to occur. The viral genome can then enter the cell. Data are now emerging from the trials of T-20 (enfuvirtide), the first fusion inhibitor to enter the clinic, which acts to inhibit this process (Lalezari et al., 2003). Since the Phase III trials were undertaken in heavily pre-treated patients, it is not surprising that failure rates (lack of full suppression) were relatively high overall. However, this affords the opportunity to characterise the emergence of resistance. Data from Phase II studies demonstrate that the majority of such failure patients had mutations in the gp41 region targeted by the drug, namely between amino acids 36–45, which indeed confirms that activity of the drug is mediated through the proposed mechanism. Since variation in this region is very rare in enfuvirtide-naı¨ve patients, including those infected with non-subtype B viruses, it can be assumed that prior RT inhibitor and PI therapy will not compromise enfuvirtide activity per se. The key issue with use of this drug in salvage therapy will therefore be the choice of other active drugs to combine with it. Of interest, the second-generation fusion inhibitor T-1249 appears to be active against most enfuvirtide-resistant mutants, although so far this is based on in vitro evidence alone. Both compounds have the disadvantage of requiring parenteral administration.

New Drug Targets

Figure 25.9 depicts the many stages of the HIV replication at which inhibition may be possible.

750

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

HUMAN IMMUNODEFICIENCY VIRUSES

Figure 25.9 The life cycle of HIV, indicating the stages at which potential therapeutics might act

Co-receptor antagonists have been developed to inhibit resolved for some years; however, it has taken longer CCR5 and CXCR4 binding to virus. Concern over

to dissect the enzymatic components of the molecule. such compounds has focused on the detrimental eﬀect

Lead compounds have now been developed, and of blockade of important stimulatory molecules, and

clinical studies are due to start (Pomerantz and the ability of the virus to evolve to switch co-receptor

Horn, 2003).

usage following the use of one particular antagonist. Promising data have been presented for the CCR5 inhibitors (Baba et al., 1999; Simmons et al., 1997) and

Transmission of Drug Resistance clinical studies of new, small molecular weight drugs are ongoing at the time of writing.

Epidemiological studies of acute infections or chronic Integrase is a unique enzyme which catalyses the

untreated infections in Europe and North America integration of viral cDNA into the host cell genome.

demonstrate the presence of viruses containing drug As such it is essential, and a promising target for

resistance mutations in up to 20% of cases (UK antiviral drugs. The enzyme structure has been

Collaborative Group on Monitoring the Transmission Collaborative Group on Monitoring the Transmission

Novel Approaches to Management of Multi-resistant Virus Infection

A number of new strategies have been suggested as a means of dealing with multi-resistant HIV-1, some of which have been subject to pilot studies:

1. Treatment interruption. Since wild-type (non-resistant) virus regrows out as the majority species when treatment is stopped, it has been proposed that such

a strategy will allow resensitisation of the virus to treatment (Deeks and Hirschel, 2002). A number of further reasons have been given for the potential advantage of such an approach, such as providing immune stimulation; however, there is little evidence that this provides any lasting beneﬁt in subsequent response to therapy.

2. GIGA-HAART. A pilot study has been undertaken of treatment interruption in patients with low CD4 count, followed by the use of up to eight and nine drugs. It is of interest that this multiple treatment has been shown to provide some beneﬁt (Katlama et al. , 2003).

3. Continuing therapy. Drug-resistant viruses may also have deficiencies in viral replicative capacity (fitness). This has led some to propose that continuing on therapy, to maintain the presence of drug resistance mutations, may be beneficial compared to stopping therapy. An alternative interpretation of these data is that resistance is not all or nothing, and that drugs may maintain some residual activity (Deeks and Hirschel, 2002).

Immunotherapy The fact that HIV induces gradual immunosuppression

over a long period raises the question of reconstitution of the immune system using immunotherapy (Imami and Gotch, 2002). The three major approaches tried to date are:

1. Passive immunisation with plasma selected for high neutralising antibodies.

2. Use of cytokines, such as IL-2 and GM-CSF.

3. Use of therapeutic vaccines. Passive immunisation has been tried by a number of

investigators, using either anti-HIV plasma or selective high-titre anti-HIV V3 anti-loop antibodies and, in the case of children, normal immunoglobulin. Encoura- ging results have been claimed with all these approaches, although a claimed correlation between high-titre anti-V3 loop antibodies and preventive mother-to-child transmission could not be duplicated by others, and no firm evidence for efficacy in vivo exists at present. A limitation of this approach is the availability of high-titre sera containing neutralising antibodies to the relevant HIV strain of the patient being treated. However, the success of human antibodies in the treatment of cancer, such as herceptin for breast cancer and rituximab for lymphoma, raises the possibility that the five available human monoclonal antibodies that have been found to be capable of neutralising a broad range of primary HIV-1 isolates may be better potential candidates for passive immunotherapy.

HAART has made a dramatic impact on patients who are able to receive these drugs frequently. Unfortunately, even though viral load can be reduced to undetectable levels, cessation of the drugs leads to rebound, a rising viral load and falling CD4 count in the majority of patients. Because many patients wish to cease HAART due to side-eﬀects, there is an unmet need to use some other form of treatment to maintain the low viral state when HAART is ceased. Although the immune response to a variety of antigens recovers in patients on HAART, it is clearly not enough to induce the appropriate immune response to HIV.

The concept of enhancing an appropriate immune response and the concept of therapeutic vaccination following the partial immune improvement are actively being pursued in clinical research. Interleukin 2 (IL-2) production in patients with HIV infection is reduced leading to T-helper-1 type deﬁciency, whereas Th-2 type cytokines (IL-4, IL-6) are increased. Replacement by recombinant IL-2 increases the peripheral CD4 counts when given as pulsed therapy or subcutaneous injections. By causing the lymphocyte population to expand, IL-2 could increase viral production by activating cells for HIV propagation. However, a number of studies have shown that IL-2 combined with HAART leads to a substantially greater increase in CD4 count and a larger decrease in viral load than seen with HAART alone. Response to recall antigens is eight-fold higher in IL-2 recipients than in patients treated with HAART alone. However, it is not clear

752

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

753 that this translates into enhanced responses to

HUMAN IMMUNODEFICIENCY VIRUSES

Early in 2003, the ﬁrst results of the randomised trial immunisation in vivo. A large randomised multi-centre

of a lead HIV vaccine candidate were reported, but no study, called ESPRIT, hopes to answer whether IL-2

protective eﬀect was observed in a gp120 vaccine made plus HAART genuinely improves morbidity and

by Vaxgen (Cohen, 2003). This failure has highlighted mortality.

many doubts about the ability of HIV to be success- Other cytokines may also have a role in the

fully contained by prophylactic vaccination. Major management of chronic HIV infection. Chronic

questions with regard to the vaccines involve whether granulocyte macrophage colony stimulating factor

neutralising antibody responses or cell-mediated (GM-CSF) is capable of enhancing monocytes and

responses are the more important and what the macrophages, and can restore the allogenic stimulatory

importance of virus variation is. With regard to the function to accessory cells in patients with AIDS. As

HIV envelope, the dominant antigen, the V3 loop, is dendritic cell function is impaired in HIV patients, a

very variable. Only ﬁve human monoclonal antibodies case for using GM-CSF and IL-2 in the same regimen

have been reported to neutralise a broad range of HIV can be made.

isolates by recognising conserved epitopes across HIV- Therapeutic vaccines are designed to induce the

1 clades, although two of these require CD4 binding of immune response not normally initiated in the HIV

gp120 to expose the epitope. Protection has been patient for a number of diﬀerent reasons (Letvin and

demonstrated in SCID mice reconstituted with human Walker, 2003). With the ability of HAART to reduce

T cells, and with SIV challenge of macaques, although the viral load and IL-2 to increase CD4 and NK

the titres required are high and may be impossible to populations, it is attractive to consider the possibility

achieve by current vaccination methods. of immunising patients with dominant components of

HIV induces a strong CD8 T cell response during HIV. One of these, known as REMUNE, containing

acute viraemia and this can persist in most patients inactivated HIV particles, induced immune responses

(Letvin and Walker, 2003). CD8 T cell responses can to HIV antigens in patients being treated with

select HIV escape mutants. CD8 cells are able to HAART. A therapeutic vaccine based on the recom-

protect against high-dose challenge with several viruses binant glycoprotein 160

in experimental systems and therefore it may be asymptomatic volunteers with CD4 counts above

has been tried in

possible to stimulate CD8 protection in HIV. Unfor- 400. It was concluded that gp160 was safe and

tunately, in SIV and SHIV macaque models single persistently immunogenic, although there was no

amino acid change can escape an eﬀective CD8 evidence that this vaccine had eﬃcacy as a therapeutic

response. Nevertheless, the presence of heavily exposed vaccine in early stage HIV infection, as measured by

but HIV-seronegative people who have CD8 T cell primary endpoints or disease progression.

responses suggest that this may contribute to protec- In conclusion, although it has been shown that

tion as well as IgA neutralising responses. HAART plus IL-2 therapy can greatly improve the

There are many variations on a possible HIV CD4 count and the immune system over time, there are

vaccine, involving a range of vectors and immunogens still deﬁcits in the immune response to HIV. However,

(Table 25.14). A DNA prime, modified vaccinia it took a combination of antiretroviral drugs to achieve significant clinical efficacy over time, and it may well require judicious use of a combination of

Table 25.14 Possible approaches to HIV vaccines cytokines and therapy over the right time interval to

Immunogens

induce a clinically signiﬁcant and beneﬁcial immune

Whole killed virions

response.

Puriﬁed protein subunits Recombinant proteins Synthetic peptides Viral vectors

THE PROSPECTS FOR HIV VACCINES Vaccinia

Canarypox Alpha-virus

Many investigations of many diﬀerent candidate HIV Venezuelan equine encephalitis vaccines are under study at the present time (McMi-

DNA encoding HIV antigens

chael and Hanke, 2003). These agents include envelope

Live attenuated HIV

gp120 or gp140 for humoral immunity, as well as viral

Adjuvants

vectors and DNA-based vaccines encoding Gag, Pol, Those that induce Th1 cellular immune responses Nef, Tat, Rev, and Vpu epitopes.

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

Figure 25.10 (a) Annual AIDS deaths in sub-Saharan Africa (population 640 million) compared with those in USA (population 273 million). (b) Deaths in the USA in more detail, showing the ﬁve leading causes of death in men and women aged 25–44 years. Over the course of 10 years, AIDS came to be the leading cause of death in this generally healthy age group. The sharp decline in mortality followed the introduction of HAART, although the prevalence of HIV infection has not decreased (Weiss, 2003)

Ankara boost immunisation protocol is one of the Although antiretroviral therapy is now being intro- more promising approaches (McMichael and Hanke,

duced into developing countries, and behavioural and 2003). It is clear that any vaccine will need to induce

social changes can help to reduce HIV transmission responses against more than one epitope (because of

(Valdiserri et al., 2003), the most important advance escape) and will probably need to recruit both humoral

for controlling the HIV pandemic will be a safe and and T cell responses. Such a vaccine must either oﬀer

eﬃcacious vaccine to prevent HIV infection. Unfortu- protection across HIV subtypes, or include antigens

nately, HIV has so far defied the best scientific efforts derived from multiple subtypes.

to develop such a vaccine, but it is imperative that this goal continues to be pursued with vigour and innovation.

CONCLUSIONS AND PROSPECTS During the 20 years since HIV-1 was ﬁrst isolated and

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25A

The Human T Cell Lymphotropic Viruses

Graham P. Taylor

Imperial College, London, UK

INTRODUCTION the human T cell leukaemia/lymphoma virus type I. The second was the recognition (1974) and description

The HTLV-BLV viruses are a subfamily of retro- (1977) of a new disease entity, adult T cell leukaemia- viruses. They comprise human T lymphotropic virus

lymphoma (ATLL) by Takatsuki and colleagues, in types I and II (HTLV-I, HTLV-II); bovine leukaemia

Japan (Uchiyama et al., 1997). The clustering of this virus (BLV); and an increasing number of simian or

disease, particularly in south-western Japan, suggested primate T lymphotropic viruses (STLV/PTLVs) closely

an environmental or infectious aetiology. Gallo’s cell related to HTLV-I and -II. HTLV-I infection is usually

line was derived from the lymphocytes of an Afro- asymptomatic but is associated with malignant and

American patient with an aggressive form of cuta- inﬂammatory diseases and mild or selective impair-

neous T cell lymphoma subsequently recognised to be ment of immune function in a minority. Disease

cutaneous ATLL. In 1981 Miyoshi and colleagues associations with HTLV-II are less well established.

produced an immortalised T cell line by the co-culture STLVs have been associated with malignant disease in

of peripheral blood lymphocytes from a woman with non-human primates, but not necessarily in the

ATLL with cord blood cells from a male baby primary host species. In a small proportion of

(Miyoshi et al., 1981). This cell line (MT-2), which naturally infected cattle, BLV is associated with a B

has an XY karyotype, was observed to produce cell leukaemia. However, leukaemia is very common in

numerous extracellular type C retroviral particles experimentally BLV-infected sheep.

and was positive for adult T cell leukaemia antigen by indirect immunoﬂuorescence (Hinuma et al., 1981). Using viral antigens from MT-2 cells to develop a

HISTORY serological test, they demonstrated that nearly all ATLL patients and a high proportion of their

The discovery of HTLV-I as an important human relatives had antibodies to this virus (Yoshida et al., pathogen was the result of two distinct lines of

1982). Known initially in Japan as adult T cell research. One, the long search for cancer-causing

leukaemia virus (ATLV), sequence analysis showed retroviruses in humans, was dependent on the earlier

that ATLV was almost identical with HTLV-I. discovery and reﬁnement of tests for reverse tran-

Seroprevalence studies revealed that ATLV/HTLV-I scriptase, together with the identiﬁcation and use in

was endemic in south-western Japan, with 15% of the cell culture of T cell growth factor, now known as

population seropositive, rising to 30% in some interleukin 2 (IL-2). In 1980, Gallo’s team found one

villages. In Central Japan only 1% of the population of their many transformed T cell lines derived from

were seropositive, with higher rates again in the north. leukaemia/lymphoma patients to contain a retrovirus

The inhabitants of central Japan are believed to have (Poiesz et al., 1980). This virus, now known as the

come from mainland Asia circa BC 300, displacing the human T lymphotropic virus type I, is also known as

‘older’ population to the north and south-west. This

Principles and Practice of Clinical Virology , Fifth Edition. Edited by A. J. Zuckerman, J. E. Banatvala, J. R. Pattison, P. D. Griﬃths and B. D. Schoub & 2004 John Wiley & Sons Ltd ISBN 0 470 84338 1 Principles and Practice of Clinical Virology , Fifth Edition. Edited by A. J. Zuckerman, J. E. Banatvala, J. R. Pattison, P. D. Griﬃths and B. D. Schoub & 2004 John Wiley & Sons Ltd ISBN 0 470 84338 1

associated myelopathy (HAM),

was

described in Japan the following year (Osame et al., 1986). This disease is commonly referred to as HAM/ TSP but here the acronym HAM will be used, as the data presented refer only to HTLV-I associated myelopathy. HTLV-I-seronegative TSP occurs.

A related virus, HTLV-II was isolated from the cells of a patient with an atypical hairy cell leukaemia (HCL) in 1982 (Kalyanaraman et al., 1982). Hairy cell leukaemias are usually of B cell origin; however, the infected cell line expressed T cell markers. Although HTLV-II was isolated from a second patient with HCL, subsequent extensive investigation has failed to conﬁrm any association between HCL and HTLV-II infection. HTLV-II shares 60–70% sequence homology with HTLV-I and anti-HTLV-II antibodies are detected by HTLV-I lysate or whole virus-based assays.

Simian T lymphotropic viruses have been found in macaques and other Old World monkeys, and STLV-I appears to be more closely related to HTLV-I than to other STLV/PTLVs. HTLV-I/STLV-I strains cluster geographically rather than by host species. During the last few years a number of STLVs closely related to but discrete from HTLV-I have been characterised. These include relatives of HTLV-II (Giri et al., 1994) and viruses, which cluster with neither HTLV/STLV-I nor -II. The ﬁrst of these, found in an Eritrean baboon (Papio hamadryas), was designated PTLV-L after Leuven, where the baboon was residing and the virus was isolated (Goubau et al., 1994), HTLV-III having already been used as a former name for human immunodeﬁciency virus type 1. STLVs isolated from wild-caught red-capped mangabeys (Cercocebus torquatus ) from Cameroon are distinct from, but cluster with, PTLV-L in the newly designated PTLV-3 type (Meertens et al., 2002).

THE VIRUS Morphologically, HTLV-I and -II resemble C-type

retroviruses (Figure 25A.1). They can be grown in vitro in immortalised lines obtained by culturing patients’ cells with phytohaemagglutinin (PHA) and IL-2. De novo infection of T cells and cell lines requires co- cultivation using irradiated or mitomycin-C-treated HTLV producer cell lines. The HTLVs are not readily

transmissible in cell-free form and this is reﬂected in the observation that only whole fresh blood transfusion and not plasma or other cell-free fractions results in transmission (Okochi et al., 1984).

Susceptible cell lines can be identified by the formation of syncytia (giant multinucleated cells) upon contact with virus-producing cells. This interaction requires the presence of the HTLV-I envelope and specific cell membrane ligands (receptors). The syncytial assay has proved useful in studying HTLV-I, particularly for the detection of neutralising antibodies and cellular receptors (Clapham et al., 1984). In vitro, many cell types from a broad range of species, including astrocytes, can be infected. In vivo, HTLV-I primarily infects CD4 + lymphocytes whilst HTLV-II infects CD8 + lymphocytes. However HTLV-I infection of CD8 + lymphocytes does occur and is more common among HTLV-I specific cytotoxic CD8 + lymphocytes than other CD8 + cells (Hanon et al., 2000). Richardson et al. (1990) found no evidence of HTLV-I infection of B cells, monocytes or natural killer (NK) cells in vivo but others have reported infection in B cells, monocytes (Koyanagi et al., 1993) and NK cells (Igakura et al., 2003). Whether HTLV-I infection of neural cells occurs in vivo remains controversial. Although the cellular receptor has not been identified the coding gene has been reportedly localised to chromosome 17. Various receptors have been proposed with neuropilin-1 (Ghez et al., 2003) and GLUT-1 (Manel et al., 2003) the most recent. The envelope proteins of HTLV-I and HTLV-II are the target for neutralising antibodies. Sera from British or American patients and asymptomatic carriers of HTLV-I equally neutralise viruses from other countries including Japan (Clapham et al., 1984). This suggests that there is a single worldwide serotype for HTLV-I.

The 9 kb genome of HTLV-I contains the three major open reading frames (ORFs), gag, pol and env, of all retroviruses flanked by two long terminal repeats (LTR) with an additional regulatory region, pX (Figure 25A.2). The names, product size and functions of the genes of HTLV-I are summarised in Table 25A.1. The HTLV-II genome is very similar except that five ORFs have been identified in pX.

The LTR containing the viral promoter and other regulatory elements are divided into three regions, U3, R and U5. The U3 region contains elements that control proviral transcription, messenger RNA termination and polyadenylation.

The ﬁrst major reading frame (gag) encodes a 429 amino acid precursor polyprotein Pr53 Gag . A second major reading frame within the gag–pol complex

760

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

HUMAN T CELL LYMPHOTROPIC VIRUSES

Figure 25A.1 Electron micrograph of HTLV-I particles produced by a T cell line transformed in vitro. Note the variable size of the particles and the diﬀuse spherical core

Figure 25A.2 Schematic representation of HTLV-I and HTLV-II genomes. Reproduced from Franchini (1995)

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

Table 25A.1 Products of HTLV-I genes 5’ LTR

Contains regulatory elements essential for viral replication gag

G roup antigen nucleocapsid proteins

pol Poly merase

Reverse transcriptase

Transcription of DNA from RNA

Proteinase

Cleavage of protein precursors

RNaseH

Digestion of RNA template

Integration of proviral DNA into host genome env

Integrase

Env elope

Surface glycoprotein

p12 I (Rof) Infectivity

ORF II p13 II Mitochondrial protein p30 II (Tof) Modulation of cellular gene transcription

ORF III

p27

Rex Regulatory gene of ‘X’ region

Cytoplasmic protein unknown function ORF IV

p21 rexIII

Tax Trans activating gene of ‘X’ region 3’ LTR

p40

Contains regulatory elements essential for viral replication

encodes a second precursor, Pr76 Gag–Pro , which gives reading frame and are cleaved from a 99 kDa precursor rise to the viral protease. This frame slightly overlaps

protein. RT transcribes the single viral RNA strand, with gag and is generated by a frameshift suppression

generating ﬁrst the complementary DNA strand and of the gag terminator codon. During viral assembly

then using that as the template for the viral DNA the gag precursors accumulate at the inner face of the

version of the original RNA, which is digested by plasma membrane and are anchored there by the

RNaseH. This double-stranded DNA is integrated into matrix domain. In the absence of protease, immature

a host cell chromosome. Having no proof-reading viral particles are released. During and early after viral

mechanism, RT is error-prone and a high frequency of budding, cleavage of Pr53 Gag into the three major

mutation would be expected to result in HTLV/STLV structural proteins of the core of the virus, 19 kDa

sequence diversity.

(matrix protein), 24 kDa (capsid protein) and 15 kDa Env encodes a 481 amino acid protein that after (nucleocapsid protein), results in the formation of the

glycosylation has a molecular weight of 62 kDa and is mature virion. The viral genome is transported to the

processed into an outer surface protein of 46 kDa and cell envelope bound to the gag precursor. The matrix

a transmembrane protein of 21 kDa. The envelope domain of the gag precursor is involved in targeting of

precursor is translated from a 4.2 kb mRNA from the gag precursor–genome complex to the plasma

which gag and pol have been spliced out. membrane (this also requires myristoylation of the

The ﬁfth genomic region, referred to as pX, is polyprotein) and in eﬃcient budding of the viral

located between env and the 3’ LTR. The region codes particle from the cell. The mature (cleaved) matrix

for tax and rex, which are translated from a double- protein also plays a role early in the replication cycle,

spliced 2.1 kb mRNA as well as three additional in viral entry, as virions with defective matrix proteins

proteins encoded by open reading frames I, II and have reduced infectivity even when properly released

III, whose functions are slowly being revealed. Tax, a (Le Blanc et al., 1999). The capsid proteins assemble

40 kDa protein that is expressed early, drives viral during viral maturation to form the shell around the

transcription from Tax-responsive elements (21 bp core of the nucleus. The N-terminal domain is

repeats) in the LTR U3 and is an important essential for viral particle formation (Rayne et al.,

upregulator of viral replication. In addition, tax 2001). In the viral particle the nucleocapsid protein is

transactivates a range of host cellular genes, through also a key component of the capsid and is in close

which it is thought to aﬀect cell replication and play an contact with the genome. In the cell the nucleocapsid

important role in the pathogenesis of ATLL. The domain of the Gag precursor polyprotein is important

immunodominant peptides for the host cytotoxic T- for recognition and packaging of the viral genome.

lymphocyte response are also usually found in the Tax Retroviral Gag function is reviewed in Wills and

protein. This may be important both for the control of Craven (1991).

infection and in the pathogenesis of inﬂammatory The remaining products of pol, reverse transcriptase

disease associated with HTLV-I, of which the arche- (RT) and integrase are encoded by a diﬀerent open

type is HTLV-I associated myelopathy. Rex is a

HUMAN T CELL LYMPHOTROPIC VIRUSES

27 kDa phosphoprotein which determines the export of binant peptides are highly sensitive and speciﬁc for unspliced gag–pol mRNA and singly-spliced env

HTLV-I and HTLV-II. Immunoﬂuorescence assays mRNA from the nucleus, thereby controlling the

using ﬁxed infected cells with uninfected cells as production of viral proteins and infectious virus (Rex

controls can be used to conﬁrm and type infections, production results in increased export of env mRNA

but are subjective. Peptide-based assays that discrimi- and unspliced viral RNA for the Gag/Pol proteins

nate between HTLV-I and HTLV-II have been essential for the production of new virions and aids a

developed. HTLV-I infection is usually conﬁrmed by shift from doubly-spliced pX RNA to gag–pol and env

the detection of antibodies to Gag (p19 and p24) and RNA). Thus, the overall eﬀect of Rex may be virion

Env (gp21 and gp46) by Western blot (Figure 25A.3), production soon after infection, followed by down-

although radio-immune precipitation assays (RIPA), regulation of viral expression. This may protect the

radio-immune binding assays (RIBAs), line immuno- infected cell from death due to the cytolytic eﬀect of

precipitation assays (LIPAs) and competitive ELISAs virion production and protect both the infected cell

can also be used. p19 Antibody is often absent in, and and the virus from the host immune response (Hidaka

not required to conﬁrm, HTLV-II infection. Recom- et al ., 1998).

binant Env peptides have improved the sensitivity and The Tax (p37) and Rex (p26) proteins of HTLV-II

speciﬁcity of Western blots and type-speciﬁc antigens are slightly smaller than those of HTLV-I. HTLV-II

rgp46-I and rgp-46-II enable the infections to be Tax shares 72–74% amino acid homology with

discriminated without resort to molecular methods. HTLV-I Tax. However, the C termini of HTLV-II

Minimal criteria for the diagnosis of HTLV infection Tax (both HTLV-IIa and HTLV-IIb) are diﬀerent

are the detection of antibodies to one Gag and one from HTLV-I Tax. Tax IIa has a 22 amino acid

Env protein. Western blots which reveal some virus- truncation at the C terminus, whilst the C terminus of

speciﬁc bands but which are not suﬃcient to make a Tax IIb has 25 amino acids, which are totally

positive diagnosis are termed ‘indeterminate’. Further diﬀerent from Tax I (Lewis et al., 2000). In vitro,

investigation is then warranted, including DNA HTLV-II Tax proteins activate transcription of NF-

ampliﬁcation using generic or type-speciﬁc primers k

B and the CREB pathways as well as HTLV-I Tax, and repeat serology after a few weeks. Viral culture although Tax from some HTLV-IIa isolates had

may be required to conﬁrm an infection but is costly, much less transcriptional activity, possibly due to

time consuming and must be conducted in a Category low levels of expression of Tax (Lewis et al., 2002).

3 laboratory (HTLV European Research Network, However both Tax IIa and Tax IIb are less eﬃcient at

1996). Although HTLV-I viral DNA load is fre- transforming rat ﬁbroblast cell lines than Tax I (Endo

quently high in asymptomatic carriers very low levels, et al ., 2002).

5 1 HTLV DNA copy per 100 000 PBMCs, can be The additional proteins are p13 and p30 (ORF-II)

found and a negative PCR does not exclude the

and p12 from ORF I. p30 II Localises to the nucleus

diagnosis of HTLV infection. In European studies, and may modulate transcription of cellular genes. p13 II HTLV-I/II infection was not conﬁrmed in the

Localises to mitochondria and in vitro disrupts the majority of low risk subjects with WB indeterminate mitochondrial inner membrane potential. Although

sera, whereas among HIV-infected injecting drug users not essential for viral replication, these proteins are

(IDU) HTLV-II infection might be found by DNA

ampliﬁcation. Indeterminate WB patterns are com- oncogenic activity and is important in infectivity

important for viral infectivity. p12 I Exhibits weak

mon in the tropics, and may be more common than (reviewed by Ciminale et al., 1996; Johnson et al.,

conﬁrmed HTLV-I infection, even in endemic areas. 2001).

In this setting antibodies to Gag proteins only have been associated with a low risk of HTLV infection (Rouet et al., 2001).

DIAGNOSIS The presence of the recombinant surface membrane glycoprotein (RD21 in the HTLV 2.4 WB, Genelabs, Detection of HTLV-I and -II infection is primarily by

Singapore) alone may reﬂect recent infection and a serology. A particle agglutination assay based on

follow-up sample should be obtained. Antibodies to whole viral lysate is sensitive for both viruses but

the Gag proteins p19 and p24 usually appear early limited by poor speciﬁcity. First-generation commer-

following seroconversion, whereas antibodies to the cial enzyme immunoassays (EIAs) were more speciﬁc

transmembrane glycoprotein gp46 appear later. Anti- than the agglutination assays but less sensitive

Tax antibodies occur late or not at all (Manns et al., particularly for HTLV-II. EIAs incorporating recom-

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

Figure 25A.3 Western blot with recombinant peptides which discriminate between HTLV-I and HTLV-II infection (Genelabs HTLV 2.4, Singapore). Courtesy of John Parry, Health Protection Agency, London, UK

VIRAL VARIATION HTLV-I by geography rather than by species (Figure 25A.4). This suggests that there have been a

HTLV-I has a highly conserved genome with only number of simian–human transmissions (Koralnik et about 4% sequence variation between isolates from

al ., 1994). HTLV-II shares 60–70% sequence homol- around the world. The only exceptions are isolates

ogy with HTLV-I. HTLV-II isolates generally belong from Australian aborigines and Melanesians, which

to genotype a or b. HTLV-IIc (Eiraku et al., 1996) has form a distinct genotype or clade, HTLV-I MEL and

to date only been described in Brazil, whilst HTLV-IId which have up to 8% diversity in LTR and/or env.

was described in Congolese pygmies (Vandamme et al., Most other isolates from Africa, Japan or the

1998). A number of primate T lymphotropic virus Caribbean belong to the Cosmopolitan clade, HTLV-

isolates fall outside the recognised HTLV/STLV clades

I COS . However, three other clades have been recog-

as described above.

nised, from Central Africa, from West Africa and from The highly conserved nature of HTLV-I, HTLV-II Japan (Ureta-Vidal et al., 1994). In phylogenetic

and their simian counterparts is unexpected for a analyses simian T lymphotropic virus type I (STLV-

retrovirus (see discussion of reverse transcriptase,

I) which is almost identical to HTLV-I, clusters with above) and quite distinct from the rapid evolution of

HUMAN T CELL LYMPHOTROPIC VIRUSES

Figure 25A.4 Phylogenetic analysis showing the relationship between HTLV-I/STLV-I subtypes. Reproduced from Meertens et al. 2001

the human immunodeﬁciency viruses. RNA viruses infection has been found to be relatively common in usually evolve at 1/100–1/10 000 nucleotide substitu-

southern Africa, particularly Natal. Up to 0.5% of tions/site/year, whereas HTLV-II is mutating at a rate

blood donors in Brazil are HTLV-I seropositive, with of 1.7–7.31/10 000 000 nucleotide substitutions/site/

considerable interstate variation. HTLV-I infection is year in the LTR. From family studies involving two

recognised among many native South American or three generations it appears that HTLV-I only

indigenous peoples as well as among black and undergoes a few replication cycles, perhaps early

Japanese immigrants. However, population mixing following infection, in each generation (Van Dooren

has been extensive and in Brazil the proportion of et al ., 2001). It has been shown that HTLV-II is

neurology patients with HAM/TSP is equal among the evolving faster in IDU than in endemic populations:

main racial groups. Northern Iran is another recently

2.7 nucleotide substitutions per 10 000 sites per year in described area of HTLV-I infection with cases IDU, which would be consistent with the virus being

described in neighbouring Middle East and central transmitted from one host to another after a shorter

Asian countries. The prevalence of HTLV-I among period than in the endemic populations in whom

European Union (EU) blood donors is low (2–7/ mother-to-child transmission plays an important role

100 000) but remarkably similar across the length and in maintaining the virus in the population (Salemi et

breadth of the EU. Seroprevalence rates 50–100 times al ., 1999).

higher have been found among women attending antenatal clinics and among men and women attending STD clinics. However, the numbers tested are much

EPIDEMIOLOGY smaller and in many countries such studies have not been conducted (Figure 25A.6). In central and eastern

It has been estimated that 20 million persons world- Europe very few studies have been conducted, but wide are infected with HTLV-I, including 1.2 million in

cases of HTLV-I-related pathology have been diag- Japan. Other endemic areas are the Caribbean, parts of

nosed in patients from Bulgaria, Romania and in a the south-eastern states of the USA, Melanesia and

Georgian family. HTLV-I/II infection would appear to parts of sub-Saharan Africa, especially west and

be rare in Hungary, although there are occasional central Africa (Figure 25A.5). Recently, HTLV-I

reports of detection of deleted HTLV gene sequences

766 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

Figure 25A.5 Global seroprevalence of (a) HTLV-I and (b) HTLV-II

HUMAN T CELL LYMPHOTROPIC VIRUSES

Figure 25A.6 Comparison between HTLV-I/II seroprevalence rates in diﬀerent risk groups in Europe. ^, Blood donors; &, STD clinic attendees; s, pregnant women; l, IDU. Rates of HTLV-I/II among European blood donors are low but relatively uniform. Rates among the three other groups tend to have much wider 95% conﬁdence intervals due to the smaller sample size. Compared to blood donors, the seroprevalence rate of (predominantly) HTLV-II in IDU populations is approximately 1000-fold higher; of HTLV-I among men and women attending clinics for sexually transmitted infections are approximately 100-fold higher; and of (predominantly) HTLV-I among pregnant women 10-fold higher. Data from Taylor (2002)

in seronegative subjects. Despite the importance of oped’ world by IDU. In Europe HTLV-II is common IDU in the spread of HIV-1 infection in former

among IDU in Eire, Spain, Italy and Scandinavia, but member states of the USSR, HTLV-II has not yet been

rare in Germany and France. The ﬁrst isolations of detected in these high-risk populations (reviewed in

HTLV-II in Africa were from pygmy tribes both from Taylor, 1996). The most important recent develop-

Ethiopia and from West Africa (Goubau et al., 1992; ments in European epidemiology of HTLV infection

Gessain et al., 1995). Although infection in West have been: (a) the conﬁrmation, in a large multicentre

African prostitutes need not imply a local origin, study, that HTLV-I/II infection in the EU is 10-fold

HTLV-II has also been described in the Gabon in a more common among pregnant women than among

male with no history to suggest exposure to an blood donors (Taylor et al., 2001); and (b) the

imported virus. This virus is genetically close both to emergence of data showing HTLV-I infection and

the Cameroonian pygmy isolate and to a North disease (ATLL) to be relatively common in Romania.

American isolate, emphasising the conserved nature Romania can be considered to be the most recently

of these viruses (Letournier et al., 1998). These described endemic area for HTLV-I infection and the

findings, and the identification of an HTLV-II-like first to be described in a Caucasian population.

primate virus in Central Africa, suggest that HTLV-II, HTLV-II infection is found among native American

like HTLV-I, may have originated in Africa. However, Indians in North, Central and South America and was

a number of questions remain as HTLV-II is widely until recently considered a New World virus. At some

spread throughout the Americas but has been found in time prior to the AIDS epidemic, HTLV-II infection

few populations in Africa. To date PTLVs have not moved into, and has been spread around the ‘devel-

been identiﬁed in New World primates and, with the been identiﬁed in New World primates and, with the

TRANSMISSION For both viruses transmission may be by three routes:

from mother to child; through sexual intercourse; and through blood–blood contact. Family studies in Japan suggested that HTLV-I was mainly transmitted from mother to child (Kajiyama et al., 1986). HTLV-I was identified in lymphocytes in breast milk and transmission through breast-feeding was demonstrated in marmosets and rabbits. The mother-to-child transmission rate in Japan was 25% if babies were breast-fed, but only 5% if babies were bottle-fed. It may be that maternal anti-HTLV antibodies protect breast-feeding infants until their titre starts to decline, as in one study in Japan short-term breast-fed babies were no more likely to be infected than bottle-fed babies (Takahashi et al ., 1991). Breast-feeding for 6 months is associated with higher rates of transmission, which continue to increase if weaning is further delayed. Although in one study HTLV-I was detected in cord blood, none of the cord-blood-positive babies became infected. HTLV-I has been detected but not quantified in cervico-vaginal secretions and the contribution of in utero and perinatal infections to the total infection rate is uncertain. In the Gabon the mother-to-child transmission rate was 9.7% with transmission associated with higher maternal viral load (Ureta-Vidal et al., 1999). There are conflicting data on mother-to-child transmission of HTLV-II but among the Kayapo Indians in Brazil the risk of transmission from an HTLV-II infected mother to her child was 30–50% (Ishak et al., 1995).

The aforementioned family studies also indicated that infection was likely to pass from husband to wife. In a Japanese cohort study of 100 discordant couples practising unprotected sexual intercourse, there were seven seroconversions during 5 years of observation. Uninfected females were 3.9 times more likely to become infected than uninfected males (Stuver et al., 1993). Higher rates of transmission have been reported: 60% of wives of seropositive husbands over

10 years in one study, but only 0.4% of husbands of seropositive wives; 50% of wives during 1–4 years of marriage in another. In a study of HTLV-I- and HTLV-II-infected US blood donors, those with higher HTLV-I proviral loads were more likely to have an infected partner. A similar association of transmission

with high proviral load was seen in HTLV-II, although the viral burden is generally much less than in HTLV-I (Kaplan et al., 1996). Both HTLV-I and HTLV-II are more common in females. It is reasonable to suggest that condoms will eﬃciently protect against transmission, but no data are available.

HTLV-I is transmitted by cell-containing blood products but not by plasma or plasma-derived products. This has been demonstrated in the rabbit model and through clinical observation. Fresh blood is more infectious than older blood, due to the short life of stored lymphocytes. Infection has occurred following transfusion of 41 ml blood. Following transfusion with HTLV-I-infected blood the median time to seroconversion was 51 days (Manns et al., 1991). The mean incubation period of HAM following infection by transfusion is 3 years, shorter than by other routes. In a post mortem study the average time to ﬁrst symptoms was 8.5 months but the duration of symptoms was not diﬀerent from other patients with HAM (Iwasaki, 1990). The risk of HAM was 7.7-fold higher than expected among transfusion recipients in Japan and following the introduction of blood donor screening in 1986 the incidence of HAM fell by 16%. HTLV-I is also transmitted through injecting drug use but this route is more commonly associated with HTLV-II. Indeed, outside of the endemic areas of the Americas, HTLV-II is primarily transmitted through re-use of injection paraphernalia. It seems likely that HTLV-II was introduced into the IDU population of the USA during the 1970s and into Europe slightly later.

HTLV-ASSOCIATED DISEASE

HTLV-I is recognised as a carcinogen by the Interna- tional Agency for Research on Cancer (IARC Monographs on the Evaluation of Carcinogenic Risk to Humans, 1996). HTLV-I is clearly associated with ATLL and serological documentation of HTLV-I infection is an essential part of conﬁrming this diagnosis, whereas the demonstration of clonality is only required in unusual cases. The search for seroepidemiological evidence of an association between HTLV-I and other malignancies has been complicated by the frequent history of previous blood transfusion in patients with malignant disease included in such studies. However, two studies have demonstrated an increased rate of cervical carcinoma in patients with HTLV-I and in one of these studies patients with HTLV-I were also found to have more advanced disease. An interaction between HTLV-I and HPV

768

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

769 directly or through an eﬀect of HTLV-I on cell-

HUMAN T CELL LYMPHOTROPIC VIRUSES

excluded (Table 25A.2). The female:male ratio is 2:1 mediated immunity is biologically plausible. However,

and the onset is most common in the 3rd and 4th since both HPV and HTLV-I are sexually transmitted,

decades, resulting in decades of morbidity. The the association may reﬂect sexual activity rather than a

condition is occasionally rapidly progressive and fatal biological interaction between two oncogenic viruses

within 2 years of onset.

(IARC Monographs on the Evaluation of Carcino- Uveitis is more common in HTLV-I patients than in genic Risk to Humans, 1996).

the general population. HTLV-I-associated uveitis Although originally described in two patients with

usually responds to treatment with topical steroids; atypical hairy cell leukaemia, extensive studies have

less often systemic steroids are required. The condition failed to associate HTLV-II infection with HCL.

is generally mild but recurs in 25% of cases (Mochi- HTLV-II infection was also reported in two patients

zuki et al., 1992). In a prospective study of 200 patients with large granular lymphocytosis and one with large

with HTLV-I in Martinique, 14.5% had uveitis, but granular lymphocytic leukaemia but the virus was

37% had keratoconjunctivitis sicca and interstitial found in the T lymphocytes and not the abnormal

keratitis was also common. (Merle et al., 2002). cells. Rare cases of CD8 + lymphoproliferation in

Polymyositis, alveolitis, arthritis and thyroiditis patients with HIV-1/HTLV-II co-infection have been

have been reported in subjects with HTLV-I, often in reported, including one in which clonal expansion of

patients with HAM. Whilst these associations have not HTLV-II-infected cells was demonstrated. In sum-

been conﬁrmed epidemiologically, the histology/cytol- mary, there are as yet inadequate data to aﬀord

ogy characterised by lymphocytic inﬂammatory HTLV-II carcinogenic status, although it immortalises

inﬁltration is consistent with an HTLV-I aetiology. T cells in vitro (reviewed in Araujo et al., 2002).

Infective dermatitis is an eczematous condition of ATLL may present as acute leukaemia, chronic or

children that only responds to long-term antibiotic smouldering leukaemia, or as a lymphoma, including a

treatment against Streptococcus and Staphylococcus cutaneous presentation. ATLL cells are CD3 + CD4 +

spp., which are otherwise not usually considered and CD25 + (IL-2 receptor) and in the leukaemic form

pathogenic. Skin biopsies reveal an inﬂammatory characteristic polylobed ‘ﬂower’ cells (Figure 25A.7)

inﬁltrate of CD8 + lymphocytes. HTLV-associated can be easily identiﬁed. Although the cells are

infective dermatitis is rare outside the tropics. morphologically mature, the malignancy is aggressive

Studies in Japan have repeatedly shown reduced and survival measured in months. The most common

delayed hypersensitivity to tuberculin in HTLV-I presentation is acute leukaemia, with lymphoma the

infected persons. Recently an increased adjusted odds next most common. Chronic and smouldering forms

risk of previous TB was reported in HTLV-II-positive are associated with longer survival. Hypercalcaemia

blood donors in the USA (Murphy et al., 1997b) but due to expression of PTHr peptide is a common and

conversely there was no abnormality in delayed characteristic ﬁnding. Patients may present with acute

hypersensitivity skin testing to mumps virus or renal failure and lytic bone lesions. Atypical presenta-

Candida albicans antigens in the same population tion with opportunistic infections should also be

(Murphy et al., 2001). Encrusted (Norwegian) scabies considered.

has been reported in patients with HTLV-I and HTLV-I is associated with a number of inﬂamma-

suggested as a marker for the development of ATLL.

Failure to clear Strongyloides stercoralis despite inﬁltration of the target organ (Figure 25A.8). The

tory conditions characterised by a lymphocytic

appropriate treatment is also recognised with HTLV- lifetime risk of HAM in HTLV-I-infected persons is 2–

I infection and investigation for HTLV-I is considered 7%, except in Japan where it has been estimated at

an essential part of the management of patients with 0.25%. HAM has been diagnosed in about 150

proven strongyloidiasis. Increased rates of infection patients in the UK, mostly of Caribbean origin, but

and treatment failure have also been reported for also in Caucasians. This includes at least one case

Schistosoma mansoni in Brazil (Porto et al., 2002). following transfusion with HTLV-I-infected blood in

A myelopathy similar to that seen with HTLV-I has the UK. The condition is a chronic progressive spastic

been reported in several subjects with HTLV-II paraparesis without the relapsing/remitting character

infection in the absence of HIV infection (Jacobson of multiple sclerosis. Chronic backache, hyperactive

et al ., 1993; Black et al., 1996; Murphy et al., 1997a), bladder, constipation and impotence are common.

although some patients also have ataxia (Harrington et Sensory signs and upper limb disease are unusual but

al ., 1993). Although the frequency of this condition is can be found in long-standing disease. Other causes of

unknown and the causative role of HTLV-II unpro- myelopathy, including cord compression, should be

ven, many specialists accept that there is a growing

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

Figure 25A.7 Flower cells. A blood smear showing characteristic convoluted T cells in ATLL. Courtesy of Dr E. Matutes and Professor D. Catovsky

body of evidence to suggest that HTLV-II-associated and Sjo¨gren’s syndrome in endemic areas but this has myelopathy is a real entity.

not yet been conﬁrmed in properly aged-matched In a cohort study of US blood donors, respiratory

studies. HTLV-I-defective provirus has been ampliﬁed and urinary infections were more common among the

from the salivary glands of seronegative patients with HTLV-II infected donors than in matched controls

Sjo¨gren’s syndrome. Rare cases of seronegative but (Murphy et al., 1999).

PCR positive HAM/TSP are reported with defective An association between mycosis fungoides and the

provirus. Tax sequences are present in all these presence of HTLV-I tax DNA has now been refuted.

conditions. The absence of both humoral and cellular There appears to be an association between HTLV-I

immune responses in these exceptional cases makes an understanding of the pathogenesis of these inﬂamma- tory conditions even more elusive.

Table 25A.2 Diﬀerential diagnosis of HAM Compressive myelopathy

PATHOGENESIS

Syringomyelia

Familial spastic paraparesis Primary lateral sclerosis

Our understanding of the pathogenesis of ATLL is Multiple sclerosis

incomplete, based to a large extent on in vitro studies Transverse myelitis

of HTLV-I-infected cells and Tax constructs, and has Devic’s disease

to take into account a number of apparent discrepan- Motor neuron disease

cies. HTLV-I can immortalise T lymphocytes in vitro Vitamin B 12 deﬁciency

Folate deﬁciency and these cells express HTLV-I proteins, but there

Syphilis appears to be little expression of HTLV-I by ATL cells Human immunodeﬁciency virus

in vivo . Only a small proportion (2–4%) of those Schistosomiasis

infected with HTLV-I develop ATLL and then only Sarcoidosis

after many decades of infection. Indeed, the observa- Neurological lupus

tion that only a proportion of the mothers of patients Behc¸et’s disease

with HAM but all the mothers of ATLL patients are Sjo¨gren’s syndrome

Carcinomatous meningitis carriers of HTLV-I suggests that infection during Paraneoplastic syndrome

infancy is important for the development of ATLL. In common with other malignancies, it is likely that

771 transformation is a multi-step phenomenon, with the

HUMAN T CELL LYMPHOTROPIC VIRUSES

Table 25A.3 Cellular genes transactivated by HTLV-I Tax infection of a lymphocyte by HTLV-I only one of several events leading to ATLL. Since HTLV-I does

IL-1, IL-2, IL-3,

Interleukins

not contain an oncogene and malignant transforma- IL-6, IL-8

Interleukin 2 receptor a chain (CD25) tion is not related to the integration disturbing cellular

IL-2Ra

House-keeping gene genes important in oncogenesis, the transactivating

Vimentin

Human leukocyte antigens characteristics of Tax assume importance. Although

Class I MHC

Granulocyte-macrophage colony adult T cell leukaemic cells contain randomly inte-

GM-CSF

stimulating factor grated (the integration site varies between subjects but

NGF

Nerve growth factor

is constant within a subject) HTLV-I that may be Transforming growth factor

TGFb1

Parathyroid hormone related protein defective, tax is preserved.

PTHr-P

Tumour necrosis factor (cachexin) In vitro , HTLV-I induces T cell activation and

TNF-a, TNF-b

Cellular oncogenes (proteins localise to proliferation and immortalises primary human lym-

c-fos, c-myc

nucleus)

Growth factor related to platelet-derived these cells, as in other tumour virus models, Jak/Stat

phocytes, which can become IL-2-independent. In

c-sis

growth factor

proteins are constitutively activated (Johnson et al., Proliferating cell nuclear antigen (cyclin)

PCNA

Multidrug resistance gene 2001). Tax has been shown to stabilise and inactivate

MDR1

Early response cellular genes the tumour suppressor p53. Tax also interacts with cell

egr-1, egr-2

Nuclear factor cycle genes, inducing phosphorylation of cyclin D1-

NF-kB

c-AMP response element cdk4/6 and cyclin D3, which may contribute to the

CRE

Serum-responsive element shift from G 1 to S phase in the cycle. Tax inactivates a cdk-inhibitor and the human mitotic arrest deﬁciency-

SRE

1 protein. where the NF-kB p50 is able to activate transcription Other examples of cellular genes transactivated by

of IL-2Ra. A secondary eﬀect of Tax is that the Tax are listed in Table 25A.3. Tax has an inhibitory

dissociation of p50 from the heterodimer releases the eﬀect on b polymerase, a DNA repair gene. This may

p65 protein, which is then free to dimerise with the increase the likelihood of mutagenesis. Tax does not

product of the c-Rel oncogene. This p65-Rel hetero- act directly on the cellular genes, or on the promoter

dimer is also able to activate the NF-kB motif. Thus, sequences in the viral LTR, but binds to a number of

Tax may further enhance the activation of the many speciﬁc transcription factors, with resulting enhance-

genes under the inﬂuence of NF-kB. ment of their interaction with the target genes.

One theory of T cell transformation by HTLV-I has Although others may exist, the three recognised target

been that, by promoting activity of both IL-2R and IL- sequences are the cyclic AMP-response element

2 genes, Tax gives rise to continuous proliferation of (CRE), the NF-kB binding site and the serum-

the cells by positive feed-back—the autocrine loop responsive element (SRE). Tax can bind to one or

theory. However, stimulation of Tax-transduced T several members of a family of cellular transcription

cells can occur in an IL-2-independent manner, Tax- factors, which in turn bind to the CRE promoters.

transfected cells continue to grow after anti-CD3 Similarly, Tax can bind to various members of the NF-

stimulation in the absence of IL-2, and not all Tax- k

B family of transcription activators. transformed T cells express IL-2, although they all The interaction between the IL-2Ra gene and Tax

express IL-2R.

has been of particular interest because of the high Whilst the oncogenic potential of HTLV-I has been expression of IL-2R (CD25) by T lymphocytes in

recognised from the time of its discovery, a similar role ATLL. IL-2Ra expression normally only occurs after

has not been proven for HTLV-II. This could merely antigenic stimulation by the T cell receptor. However,

be a function of their diﬀerent epidemiology; the it appears that Tax is able to induce constitutive

HTLV-II endemic populations are small and often expression of the IL-2Ra gene. There is a 12 bp

remote and therefore rare malignancies might be sequence in the 5’ IL-2Ra gene promoter required for

missed, whilst in industrialised states HTLV-II is tax transactivation, which shows homology with the

mostly spread among adults who may not incubate NF-kB binding site. The NF-kB precursor, a p105

the infection for long enough. With the exception of a heterodimer, is usually found in the cytoplasm because

few isolates of HTLV-IIa, in which Tax protein its nuclear localisation signal is masked. Tax causes

expression is low, the transactivating function of NF-kB to dissociate from its inhibitor, I-kB, which

HTLV-II Tax via NF-kB does not diﬀer signiﬁcantly results in increased transport of NF-kB to the nucleus,

from HTLV-I in vitro (Lewis et al., 2002).

The pathogenesis of HAM/TSP is also incompletely understood. It is rare to obtain histopathology at the time of the initial symptoms but a perivascular lymphocytic inﬁltration of the spinal cord, which is at ﬁrst CD4 + and later predominantly CD8 + is found, followed by demyelination and atrophy. HTLV-I is rarely found in the lesions and when detected is probably in circulating CD4 + lymphocytes. The peripheral lymphocyte proviral load is approximately 10-fold higher in HAM/TSP than in asymptomatic carriers although there is considerable overlap between the ranges. There appears to be a threshold of about 1 HTLV DNA copy/100 PBMCs, above which the risk of HAM increases exponentially (Nagai et al., 1998).

HTLV-I Tax-speciﬁc CTL are found in the majority of HAM/TSP patients (Jacobson et al., 1990) but also in asymptomatic carriers (Parker et al., 1992) and, by limiting dilution assays, have been found in high frequency (Daenke et al., 1996). Using MHC-peptide tetramers, as many as 10% of circulating CD8 lymphocytes have been found to recognise a single HTLV-I epitope (Bieganowska et al., 1999), although tetramer assays do not identify cytotoxic activity.

T helper responses to HTLV-I have been diﬃcult to study but, using a short incubation ELIspot assay, higher frequencies of CD4 cells secreting interferon-g in response to HTLV-I Env and Tax peptides have been found in patients with HAM (median 2/1000 CD4 cells) compared with asymptomatic carriers (Goon et al., 2002).

Polyclonal expansion of HTLV-I-infected lymphocytes has been reported in patients (without malignancy) with high proviral load, particularly patients with HAM/TSP. As with the oligo/monoclonal proliferation of T lymphocytes in ATLL, viral replication in these cells occurs through cell division, without the need for reverse transcription and integration. Thus, HTLV-I appears to be able to replicate through two quite distinct mechanisms, cell division and virion production. The relative contribution of ‘cellular’ and ‘viral’ replication to HTLV-I proviral load in patients with HAM/TSP and in asymptomatic carriers is uncertain. The continuous presence of a strong anti-Tax CTL response suggests continuing expression of Tax by some cells. It is possible that this may drive the proliferation of the clonally expanding cell populations. Despite the low rate of detection of HTLV-I proteins in vivo, there is now good evidence that HTLV-I-infected CD4 and CD8 cells are capable of expressing viral proteins and that such cells are lysed by HTLV-I-speciﬁc CTL. It has been proposed that the eﬃciency of these CTL in vivo at identifying and lysing virus protein expressing cells contributes sig-

niﬁcantly to the apparent absence of viral expression described. Recently direct cell-to-cell spread of HTLV-

I through a virus-induced synapse has been demonstrated in primary cells ex vivo (Figure 25A.9) (Igakura et al ., 2003). This, if it occurs to any degree in vivo, may help explain the absence of free virions in plasma.

There are three main theories concerning HAM/TSP pathogenesis: (a) the inflammatory response is directed against virus in the CNS; (b) the inflammatory response against HTLV-I targets similar host CNS peptides; (c) CNS tissue is an innocent bystander, damaged when CTL recognise migrating, HTLV-I- expressing, CD4 + cells. The latter has been considered most probable, although the potential role of HTLV- specific CD4 cells and NK cells might also be considered. Molecular mimicry between HTLV-I and the human neural ribosomal nucleoprotein A1 has, however, been reported (Levin et al., 2002). The effect of Tax on matrix metalloproteinases and on tissue inhibitors of metalloproteinases disturbing the neuronal milieu which has been reported in vitro could be implicated in the degeneration of neurons.

Although HAM/TSP is associated with high proviral load, differences in the rates of HAM/TSP per HTLV-I infected subjects in different populations suggest that other, possibly host factors, are important. Of the candidate factors, HLA types have attracted considerable interest. HLA types are known to be associated with other inflammatory diseases but could also influence outcome by controlling viral replication. In Japanese ATLL patients, HLA-A26, B61 and DR9 were found at an increased frequency, whilst HLA0A24 and HLA-Cw1 were less frequently found than in controls. Conversely, in patients with HAM/TSP HLA-Cw7, B7 and DR1 were found more commonly than in controls and patients with ATLL. It has been suggested that in Japan the A26Cw3B61DR9DQ3 haplotype is representative of ATLL and is associated with a low immune response, whilst A24Cw7B7DR1DQ1 is the representative haplotype of HAM/TSP and is associated with a high immune response. Amongst different ethnic groups HLA class I haplotypes were variable but examination of class II suggested that some haplotypes associated with disease are panethnic, whilst others are ethnospecific (Sonoda et al., 1996). In

a study of Japanese blood donors and patients with HAM, possession of the HLA-A*02 and HLA-Cw*08 were found to protect against HAM through an association with lower HTLV-I viral burden, whereas HLA-B*5401was associated with an increased risk of disease (Jeﬀery et al., 1999, 2000). Further studies of single nucleotide polymorphisms in the same population

772

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

773 have revealed that TNF-963A predisposes to HAM

HUMAN T CELL LYMPHOTROPIC VIRUSES

HAM/TSP is also diﬃcult to treat, and there have to (OR=9.7), SDF-1+801A 3’UTR reduced the risk of

date been only two randomised controlled studies. HAM by half, whilst the IL-15 191C allele also

Early reports of the use of pulsed high-dose steroids, conferred protection against HAM by being associated

steroid-sparing cytotoxics and plasmapheresis, all with lower proviral loads (Vine et al., 2002).

targeting the immune response, were encouraging (Osame et al., 1990) but the improvements have not been long term (Matsuo et al., 1989). High-dose vitamin C (Kataoka et al., 1993) and oxpentifylline

TREATMENT have also been promoted (Shirabe et al., 1997). Extensive research into the treatment of HIV-1 has

Few instances of curative treatment of ATLL have resulted in the licensing of four classes of antiretroviral been reported and there have been no randomised

therapy. Unfortunately, the current protease inhibitors controlled clinical trials. Until recently the overall

and non-nucleoside reverse transcriptase inhibitors management has been to treat the disease as for other

have no activity against HTLV-I. The nucleoside malignancies and the regimens for non-Hodgkin’s

analogue reverse transcription inhibitors (NRTIs) lymphoma, such as CHOP (cyclophosphamide, adria-

zidovudine and zalcitabine have been shown to be mycin, vincristine and prednisolone), are favoured.

active against HTLV-I in vitro and in animal models. Although successive improvements in chemotherapy

Unfortunately, zalcitabine and two other NRTIs, have increased remission rates to 42%, ATLL is

didanosine and stavudine, are neurotoxic. Zidovudine essentially a highly drug-resistant malignancy and

was reported to improve mobility in ambulant patients survival remains less than 12 months for the acute

in one study (Sheremata et al., 1993) but not in another leukaemia and lymphoma presentations. Enhanced

(Gout et al., 1991); neither reported proviral load transcription of the multi-drug resistance gene

measurements. In the management of HAM/TSP it is (MDR1) with signiﬁcant P-glycoprotein-mediated

important to consider that in long-standing disease drug eﬄux in the T cells of HTLV-I-infected indivi-

demyelination and atrophy may prevent improvement, duals, with and without malignancy, has been

even if viral replication or the production of damaging demonstrated, as has the ability of the HTLV-I tax

cytokines has been reduced. Clinical improvement with protein to activate the MDR1 promoter. Other

a reduction in HTLV-I proviral load was reported with treatments, such as deoxycoformycin, interferons b

lamivudine (Taylor et al., 1999), but in a randomised and g and topoisomerase II have been tried with

placebo-controlled study zidovudine plus lamivudine limited success. Since CD25 is expressed by all ATLL

was not eﬀective (Taylor et al., 2003). cells, anti-Tac (CD25) antibodies have been tried,

The earliest studies of interferon-a were of short although with limited success. Improved remission

duration but temporary beneﬁt was observed (Shi-

bayama et al., 1991; Kuroda et al., 1992). In a anti-Tac antibodies (Waldmann et al., 1995). Success-

rates (56%) were obtained with 99 Yttrium-labelled

randomised study of interferon-a given for 4 weeks ful eradication, not only of ATLL cells but also of

with 2 months follow-up, a dose-related eﬀect was HTLV-I infection, has been reported following bone

observed. Thus, the 16 patients prescribed 0.3610 6 IU marrow transplantation (BMT) but patients often fail

did not beneﬁt, but a clinical response was reported in to survive to or through BMT. Improved remission

3/17 and 6/16 patients randomised to 1610 6 and rates and survival have been reported, in uncontrolled

3610 6 IU daily, respectively (Izumo et al., 1996). studies, by three groups using the combination of

Interferon-b1A for 6 months did not alter the clinical interferon-a and zidovudine (Gill et al., 1995; Hermine

status of 12 patients in an observational study and et al ., 1995; Matutes et al., 2001). The mechanism of

although a reduction in CTL activity was documented activity of these compounds in ATLL is not clear; they

in three patients, this was not speciﬁc to HTLV-I are not eﬀective when given independently and do not

(Mora et al., 2003).

appear to have a cytotoxic eﬀect. Interest in this Symptomatic management remains the mainstay of treatment followed anecdotal improvement in a patient

therapy. Bladder spasticity with urgency, frequency, with ATLL and HIV. The immune suppression seen in

nocturia and incontinence is often distressing. Some patients with ATLL is more severe than in other

patients respond well to oxybutinin. The anabolic malignancies, and prophylaxis against Pneumocystis

steroid danazol has been reported to improve these pneumonia is recommended. Where appropriate,

symptoms. Intravesical instillation of capsaicin has infection with Strongyloides stercoralis should be

been shown to reduce the bladder spasticity in these sought and treated.

patients, with symptomatic improvement lasting

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

months (Dasgupta et al., 1996). Intranasal antidiuretic rather than HAM/TSP, although the latter causes hormone, cautiously used, may reduce nocturia in

more morbidity and is a much more common refractory cases. In a minority of patients the bladder

consequence.

is hypotonic and catheterisation, intermittent or HTLV-I, and probably HTLV-II, can also be permanent, is necessary to both relieve symptoms

transmitted through organ donation. In endemic and protect renal function. A titrated dose of baclofen

areas it has been suggested that donors and recipients may reduce limb spasticity—tizanidine may be tried if

should be matched for HTLV-I antibody status. baclofen is not tolerated. The combination of urinary

Elsewhere the arguments that apply to blood donor frequency with impaired mobility is particularly

screening should apply to donated organs. frustrating for patients with HAM. Lumbar pain

Eighty per cent of mother-to-child transmission can which has a radicular pattern is common in some,

be prevented by avoidance of breast feeding. In Japan, but not all, patient series. Management can be complex

carrier mothers are identiﬁed through ante-natal and long-acting local anaesthesia can be useful in more

screening programmes. In the UK the prevalence of severe cases.

HTLV-I/II infection in women attending metropolitan HTLV-I associated uveitis usually responds to non-

ante-natal clinics is approximately 1:200 (cf. HIV-1) speciﬁc therapy with corticosteroids. Patients with

but HTLV-I antibody testing has never been oﬀered as HTLV-I-infection and strongyloidiasis should be care-

a routine part of ante-natal care. Although breast- fully followed after therapy with thiabendazole and/or

feeding is less common in the UK, there is now ivermectin, as initial treatment is not always curative.

increasing concern to prevent the transmission of other viral infections by this route and recognition that when oﬀered there is a high uptake, by the mothers, of

PREVENTION OF DISEASE interventions to reduce transmission. Reduction of HTLV-I transmission from mothers to their infants is

There is no vaccine for HTLV-I or HTLV-II infection. likely to prevent most cases of HTLV-associated Although the association of HAM/TSP with high

disease in the UK—ATLL by preventing infection of proviral load suggests that antiretroviral therapy may

the infant, and HAM/TSP by both preventing early in the future be part of the treatment or prevention of

infection and reducing the pool of infected persons this disease, current strategies must be directed at

who will transmit HTLV-I sexually in adult life. Any preventing infection. Two transmission routes can be

eﬀect on the incidence of disease would not be seen for targeted relatively easily. Many blood transfusion

several decades.

services now include HTLV-I/II antibody screening of all or of new donors. This was ﬁrst introduced in 1986 in Japan, where blood transfusion accounted for

HIV AND HTLV CO-INFECTION up to 60% of seroconversions in the Kyushu region of

South West Japan (Kamihira et al., 1987). Blood Co-infections with HTLV-I plus HIV-1 or HTLV-II donor screening has been standard practice in the USA

plus HIV-1 occur where the two families of viruses and Australia for many years and more recently was

occur in the same risk population. Thus, HTLV-I and introduced in South American countries. With the

HIV-1 co-infections are likely to be most common in commercial production of suitably sensitive and

Latin America, the Caribbean and West/Central speciﬁc screening assays, HTLV-I/II blood donor

Africa, whereas HTLV-II co-infection with HIV-1 is testing has been introduced in the majority of EU

common among IDUs in Europe and North America. countries. In France more than 300 HTLV-I-positive

HTLV-I and HIV-1 share tropism for CD4 + blood donors have been detected since screening began

lymphocytes but CD4 + lymphocyte counts and in 1991. In the UK donor screening was introduced in

CD4:CD8 ratios are normal in HTLV-I infection August 2002. Prior to that, the risk of transmission

alone. Studies in Brazil and French Guiana have was almost certainly reduced by leukodepletion,

reported decreased survival in patients co-infected with introduced in 1999 to protect recipients from the

HTLV-I and HIV-1 and more advanced HIV disease transmission of prions. Screening of blood donors

than anticipated from the CD4 counts. Thus, the undoubtedly prevents infection in recipients, but cost-

initiation of prophylaxis against Pneumocystis pneu- eﬀectiveness in terms of preventing HTLV-I-associated

monia and/or antiretroviral therapy at CD4 levels disease in them has been questioned (Tynell et al.,

higher than currently recommended should be con- 1998). In such analyses more weight has been given to

sidered. No eﬀect of HTLV-II on HIV-1 outcome was the ‘cost’ of developing ATLL following transfusion

found in a study of North American and Italian IDUs,

775 whereas increased risk of sensory motor polyneuro-

HUMAN T CELL LYMPHOTROPIC VIRUSES

Ghez D, Lepelletier Y, Amulf B et al. (2003) Identiﬁcation of pathy has been observed with this co-infection. HTLV-

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HTLV-I and HTLV-2. AIDS Res Hum Retrovirol, 19 (suppl), O28–S15.

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adult T-cell leukaemia–lymphoma with a combination of association is causal is uncertain. HTLV-II infection

interferon a and zidovudine. N Engl J Med, 332, 1744–1748. may be more diﬃcult to detect serologically and

Giri A, Markham P, Digilio L et al. (1994) Isolation of a indeterminate Western blot results warrant further

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NH 2 -terminal domain of the human T-cell leukaemia virus Uchiyama T, Yodoi J, Sagawa K et al. (1997) Adult T-cell type 1 capsid protein is involved in particle formation. J

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African origin of human T-lymphotropic virus type 2 Sheremata WA, Benedict BS, Squilacote DC et al. (1993)

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26 Human Prion Diseases

John Collinge

University College London, London, UK INTRODUCTION TO PRIONS AND HISTORICAL PERSPECTIVE

The prion diseases, or transmissible spongiform encephalopathies, are a closely related group of neurodegenerative conditions that aﬀect both humans and animals. They have previously been called ‘the sub-acute spongiform encephalopathies’, ‘slow virus diseases’ and ‘transmissible dementias’. The prototypic disease is scrapie, a naturally occurring disease of sheep and goats, which has been recognised in Europe for over 200 years and is present in many countries worldwide. Other animal prion diseases, described over the last few decades, include transmissible mink encephalopathy and chronic wasting disease of mule deer and elk, both principally in the USA, and since the 1980s bovine spongiform encephalopathy (BSE), ﬁrst described in the UK and now seen in most European Union and some other countries. The more recently described feline spongiform encephalopathy of domestic cats and spongiform encephalopathies of an increasing number of species of zoo animals (Kirkwood et al., 1990) are now also recognised as animal prion diseases.

The human prion diseases have been traditionally classified into Creutzfeldt–Jakob disease (CJD), Gerstmann–Straüssler syndrome (GSS) (also known as Gerstmann–Straüssler–Scheinker disease) and kuru. Although these are rare neurodegenerative disorders, affecting about 1 person per million worldwide per annum, remarkable attention has been focused on these diseases in recent years. This is because of the unique biology of the transmissible agent or prion, and also because of the fears that the epizootic BSE could

pose a threat to public health through dietary exposure to infected tissues.

Scrapie was demonstrated to be transmissible by inoculation between sheep (and goats) following prolonged incubation periods in 1936. It was assumed that some type of virus must be the causative agent and Sigurdsson coined the term ‘slow virus infection’ in 1954. There was considerable interest in the 1950s in an epidemic of a neurodegenerative disease, kuru, characterised principally by a progressive ataxia, amongst the Fore linguistic group of the Eastern Highlands of Papua New Guinea. Subsequent field work, by a number of investigators, suggested that kuru was transmitted during cannibalistic feasts. In 1959, Hadlow drew attention to the similarities between kuru and scrapie at the neuropathological, clinical and epidemiological levels, leading to the suggestion that these diseases may also be transmissible. A landmark in the field was the transmission, by intracerebral inoculation with brain homogenates into chimpanzees, of first kuru and then CJD by Gajdusek and colleagues in 1966 and 1968 respectively (Gibbs et al., 1968). Transmission of GSS followed in 1981. This work led to the concept of the ‘transmissible dementias’. The term ‘Creutzfeldt–Jakob disease (CJD)’ was introduced by Spielmeyer in 1922, drawing from the case reports of Creutzfeldt (1920) and Jakob (1921), and was used in subsequent years to describe a range of neurodegenerative conditions, many of which would not meet modern diagnostic criteria for CJD. The criterion of transmissibility allowed diagnostic criteria for CJD to be assessed and refined; atypical cases could be classified as CJD on the basis of their transmissibility. All the animal and human conditions

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

share common histopathological features. The classical Many of the key advances in understanding the diagnostic triad of spongiform vacuolation (aﬀecting

pathogenesis of the prion diseases have come from any part of the cerebral grey matter), neuronal loss and

study of the various forms of human prion disease. In astrocytic proliferation may be accompanied by

particular, the recognition that the familial forms of amyloid plaques.

the human diseases are autosomal dominant inherited The nature of the transmissible agent in these

conditions, associated with PRNP coding mutations diseases has been a subject of intense and often heated

(Hsiao et al., 1989; Owen et al., 1989), as well as being debate for many years. The understandable initial

transmissible to laboratory animals by inoculation, assumption that the agent must be some form of virus

strongly supported the contention that the transmis- was challenged, however, both by the failure to directly

sible agent, or prion, was composed principally of an demonstrate such a virus (or any immunological

abnormal isoform of prion protein. response to it) and by evidence indicating that the transmissible agent showed remarkable resistance to treatment expected to inactivate nucleic acids (such as ultraviolet radiation or treatment with nucleases). Such

STRUCTURAL BIOLOGY OF PRIONS ﬁndings had led to suggestions as early as 1966 by Alper and others that the transmissible agent might be

A wide body of data now supports the idea that prions devoid of nucleic acid (Alper et al., 1966, 1967) and to

consist principally or entirely of an abnormal isoform Griﬃth’s (1967) suggestion that the transmissible agent

of a host-encoded protein, the prion protein (PrP), might be a protein and his proposal of several

designated PrP Sc (for review see Prusiner, 1991). PrP Sc hypothetical mechanisms for replication. Progressive

is derived from PrP C by a post-translational mechan- enrichment of brain homogenates for infectivity

ism (Borchelt et al., 1990) and no covalent diﬀerences resulted in the isolation of a protease-resistant

(Caughey and Raymond 1991) between PrP C and PrP Sc sialoglycoprotein, designated the prion protein (PrP),

have been demonstrated. It is proposed that PrP Sc acts by Prusiner and co-workers in 1982. This protein was

as a template which promotes the conversion of PrP C the major constituent of infective fractions and was

to PrP Sc and that this conversion involves only found to accumulate in aﬀected brains and sometimes

conformational change.

to form amyloid deposits. The term ‘prion’ (from The conformation of the cellular isoform was ﬁrst pro teinaceous infectious particle) was proposed by

established by NMR measurements of recombinant Prusiner (1982) to distinguish the infectious pathogen

mouse PrP (Riek et al., 1996). Since then NMR from viruses or viroids. Prions were deﬁned as ‘small

measurements on recombinant hamster, human and proteinaceous infectious particles that resist inactiva-

other mammalian PrPs have shown that they have tion by procedures which modify nucleic acids’.

essentially the same conformation; however, despite The protease-resistant PrP extracted from aﬀected

strenuous eﬀorts, no group has yet determined the brains was of 27–30 kDa and became known as

three-dimensional structure of PrP C by crystallo- PrP 27–30 . N-terminal sequencing of PrP 27–30 enabled

graphic methods.

production of isocoding mixtures of oligonucleotides Following cleavage of an N-terminal signal peptide that were used to screen cDNA libraries prepared from

and removal of a C-terminal peptide on addition of a scrapie-infected hamsters. These studies led to recovery

glycosylphosphatidylinositol (GPI) anchor, the mature of cognate cDNA clones by Weissmann and colleagues

PrP C species consists of an N-terminal region of about in 1985. Remarkably, PrP 27–30 was encoded by a single

100 amino acids, which is unstructured in the isolated copy host chromosomal gene rather than by a putative

molecule in solution, and a C-terminal segment, also nucleic acid in fractions enriched for scrapie infectivity.

around 100 amino acids in length. The C-terminal PrP 27–30 was found to be derived from a larger molecule

domain is folded into a largely a-helical conformation of 33–35 kDa designated PrP Sc (denoting the scrapie

(three a-helices and a short anti-parallel b-sheet) and is isoform of the protein; Oesch et al., 1985). The normal

stabilised by a single disulphide bond linking helices 2 product of the PrP gene, however, is protease-sensitive

and 3. There are two asparagine-linked glycosylation and was designated PrP C (denoting the cellular isoform

sites (see Figure 26.1).

of the protein). No differences in amino acid sequence The N-terminal region contains a segment of five between PrP Sc and PrP C have been identified. PrP Sc is

repeats of an eight-amino acid sequence (the octapep-

tide-repeat region), whose expansion by insertional process (Borchelt et al., 1990; Caughey and Raymond

known to be derived from PrP C by a post-translational

mutation leads to inherited prion disease. While 1991).

unstructured in the isolated molecule, this highly

HUMAN PRION DISEASES

781 hydrogen/deuterium exchange measurements on the

human protein, which show that the overall equilibrium constant describing the distribution of folded and unfolded states is the same as the protection factor (Hosszu et al., 1999). This shows that there are no partially unfolded forms or intermediates that have a population greater than the unfolded state. The data suggest that PrP Sc is unlikely to be formed from a kinetic folding intermediate, as has been hypothesised in the case of amyloid formation in other systems. In fact, on the basis of population it would be more likely that PrP Sc were formed from the unfolded state of the molecule.

Inherited prion diseases may produce disease by destabilising PrP C , which would predispose the molecule to aggregate. Alternatively, a mutation could facilitate the interaction between PrP C and PrP Sc or affect the binding of a ligand or co-protein. In order to relate the folding stability of PrP C to its propensity for forming PrP Sc , several of the human mutations have been copied into the recombinant mouse protein (Liemann and Glockshuber, 1999). Although this work broadly concluded that there is no absolute correlation between stability and disease, all of the fully penetrant pathogenic mutations show significant destabilisation, while non-pathogenic polymorphisms have little effect.

PrP Sc is extracted from aﬀected brains as highly Figure 26.1 Model of glycosylated human prion protein

aggregated, detergent insoluble material that is not indicating positions of N-linked glycans, the single disulphide

bond joined helixes 2 and 3 and GPI anchor to outer surface amenable to high-resolution structural techniques.

of cell membrane However, Fourier transform infrared (FTIR) spectro- scopic methods show that PrP Sc , in sharp contrast to

PrP C , has a high b-sheet content (Pan et al., 1993). PrP Sc is covalently indistinguishable from PrP C (Pan et conserved region contains a tight binding site for a

al. , 1993; Stahl et al., 1993).

single Cu 2+ ion with a dissociation constant (K d ) of

The underlying molecular events during infection

which lead to the conversion of PrP C to the scrapie present upstream of the octa-repeat region but before

10 7 14 M. A second tight copper site (K d =10 7 13 M) is

agent remain ill-deﬁned. The most coherent and the structured C-domain (Jackson et al., 2001;

general model thus far proposed is that the protein, Hornshaw et al., 1995; Sto¨ckel et al., 1998). Clearly,

PrP, ﬂuctuates between a dominant native state, PrP C , it is possible that the unstructured N-terminal region

and a series of minor conformations, one or a set of may acquire structure following copper binding. A role

which can self-associate in an ordered manner to for PrP in copper metabolism or transport is possible

produce a stable supramolecular structure, PrP Sc , and disturbance of this function by the conformational

composed of misfolded PrP monomers. Once a stable transitions between isoforms of PrP could be involved

‘seed’ structure is formed, PrP can then be recruited, in prion-related neurotoxicity.

leading to an explosive, auto-catalytic formation of The structured C-domain folds and unfolds rever-

PrP Sc . Such a system would be extremely sensitive to sibly in response to chaotropic denaturants, and recent

three factors: (a) overall PrP C concentration; (b) the

equilibrium distribution between the native conforma- et al. , 1999) demonstrates that there are no populated

work on the folding kinetics of mouse PrP C (Wildegger

tion and the self-associating conformation; and (c) intermediates in the folding reaction and that the

complimentarity between surfaces which come protein displays unusually rapid rates of folding and

together in the aggregation step. All three of these unfolding. These ﬁndings have been reinforced by

predictions from this minimal model are manifest in

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

the aetiology of prion disease: an inversely propor- degree of periodic order (i.e. extensive regions of

secondary structure). However, such alternative states incubation period in transgenic mice (Bueler et al.,

tional relationship between PrP C expression and prion

do not have precisely and tightly packed side-chains, 1993; Collinge et al., 1995b; Prusiner et al., 1990;

which are the hallmark of the native state of orthodox Telling et al., 1995); predisposition by relatively subtle

globular proteins.

mutations in the protein sequence (Collinge, 1997); Studies on a large fragment of the human prion and a requirement for molecular homogeneity for

protein (PrP 91–231 ) have shown that at acidic pH PrP eﬃcient prion propagation (Palmer et al., 1991;

can fold to a soluble monomer comprised almost Prusiner et al., 1990).

entirely of b-sheet in the absence of denaturants Little is known for certain about the molecular state

(Jackson et al., 1999). Reduction of the native of the protein that constitutes the self-propagating,

disulphide bond was a prerequisite for b-sheet forma- infectious particle itself. There are examples of

tion and these observations of alternative folding infectivity in the absence of detectable PrP Sc (Collinge

pathways, dependent upon solvent pH and redox et al. , 1995a; Lasme´zas et al., 1997; Shaked et al., 1999;

potential, could have important implications for the Wille et al., 1996) and diﬀerent strains of prions (see

mechanism of conversion to PrP Sc . Indeed, this below) are known to diﬀer in their degree of protease

monomeric b-sheet state was prone to aggregation resistance. A single infectious unit corresponds to

into ﬁbrils with partial resistance to proteinase K around 10 5 PrP molecules (Bolton et al., 1982). It is

digestion, characteristic markers of PrP Sc . Unusually unclear whether this indicates that a large aggregate is

for a protein with a predominantly helical fold, the necessary for infectivity or, at the other extreme,

majority of residues in PrP 91–231 have a preference for whether only a single one of these PrP Sc molecules is

b -conformation (55% of non-glycine/proline residues). actually infectious. This relationship of PrP Sc mole-

In view of this property, it is possible that the PrP cules to infectivity could, however, simply relate to the

molecule is delicately balanced between radically rapid clearance of prions from the brain known to

diﬀerent folds with a high-energy barrier between occur on intracerebral challenge.

them; one dictated by local structural propensity (the Direct in vitro mixing experiments (Bessen et al.,

b -conformation) and one requiring the precise docking 1995; Kocisko et al., 1994, 1995) have been performed

of side-chains (the native a-conformation). Such a in an attempt to produce PrP Sc . In such experiments an

balance would be inﬂuenced by mutations causing excess of PrP Sc is used as a seed to convert recombinant

inherited human prion diseases. It is also worthy of PrP C to a protease-resistant form (designated PrP RES ).

note that individuals homozygous for valine at However, the relative ineﬃciency of these reactions has

polymorphic residue 129 of human PrP (where either precluded determining whether new infectivity has

methionine or valine can be encoded) are more been generated. An artiﬁcial species barrier has,

susceptible to iatrogenic CJD (Collinge et al., 1991a), however, been exploited to address this issue, and

and valine has a much higher b-propensity than does such conversion products, expected to have a diﬀerent

methionine.

host speciﬁcity (and so can be bioassayed in the The precise subcellular localisation of PrP Sc propa- presence of an excess of starting material), have not

gation remains controversial. However, there is shown any detectable infectivity (Hill et al., 1999).

considerable evidence implicating either late-endo- These results argue that acquisition of protease

some-like organelles or lysosomes (Arnold et al., resistance by PrP C is not suﬃcient for the propagation

1995; Laszlo et al., 1992; Mayer et al., 1992; of infectivity. Despite the obvious limitations of such

Taraboulos et al., 1992). The environments of these experiments, they may represent an initial step in the

organelles are evolved to facilitate protein unfolding at generation of the infectious isoform of PrP, which

low pH prior to degradation by acid-activated requires additional, as yet unknown, co-factors for the

proteases. It is possible that the a-PrP to b-PrP acquisition of infectivity.

conversion, caused by reduction and mild acidiﬁcation, The diﬃculty in performing structural studies on

is relevant to the conditions that PrP C would encounter native PrP Sc has led to attempts to produce soluble b-

within the cell, following its internalisation during sheet-rich forms of PrP, which may be amenable to

recycling. Such a mechanism could underlie prion NMR or crystallographic structure determination. It is

propagation and account for the transmitted, sporadic now recognised that the adage ‘one sequence, one

and inherited aetiologies of prion disease (see Figure conformation’ is not strictly true. Depending on

26.2). Initiation of a pathogenic self-propagating solvent conditions, probably any protein chain can

conversion reaction, with accumulation of aggregated adopt a variety of conformations in which there is a

b -PrP, may be induced by exposure to a ‘seed’ of

HUMAN PRION DISEASES

783 et al. , 1992), although they were completely resistant to

prion disease following inoculation and did not replicate prions (Bueler et al., 1993). However, these mice were then shown to have abnormalities in synaptic physiology (Collinge et al., 1994) and in circadian rhythms and sleep (Tobler et al., 1996). While none of these observations deﬁne a molecular

role for PrP C , it has been argued that PrP may act as a receptor for an as-yet unidentiﬁed extracellular ligand. Newly synthesised PrP C is transported to the cell surface and then cycles rapidly via a clathrin-mediated mechanism, with a transit time of around an hour, between the surface and early endosomes (Shyng et al., 1994).

Figure 26.2 Possible mechanism for prion propagation. It is possible that Prnp 0/0 mice may be viable and Largely a-helical PrP C proceeds via an unfolded state (A) to

healthy due to secondary compensatory mechanisms re-fold into a largely b-sheet form, b-PrP (B). b-PrP is prone

during neurodevelopment, as has been documented in to aggregation in physiological salt concentrations. Prion

other models of targeted gene knockout. The most replication may require a critical ‘seed’ size. Further

recruitment of b-PrP monomers (C) or unfolded PrP (D) direct approach to answering this question was by then occurs as an essentially irreversible process

knocking out neuronal PrP expression in a developed nervous system, avoiding potential compensatory mechanisms activated during neurodevelopment, and thus to directly observe the eﬀects of acute depletion of

aggregated b-PrP following prion inoculation, or as a PrP, revealing any critical function. This has now been rare stochastic conformational change, or as an

achieved and excludes PrP loss of function as a cause inevitable consequence of expression of a pathogenic

of prion neurodegeneration (Mallucci et al., 2002). mutant that is predisposed to form b-PrP. It

PrP C

remains to be demonstrated whether such alternative conformational states of the protein are suﬃcient to cause prion disease in an experimental host or whether

PRION STRAINS other cellular co-factors are also required.

A major problem for the ‘protein-only’ hypothesis of prion propagation has been how to explain the NORMAL CELLULAR FUNCTION OF P R P

existence of multiple isolates, or strains, of prions. Dickinson, Fraser and colleagues isolated multiple

While PrP plays a central role in pathogenesis of prion distinct strains of naturally occurring sheep scrapie in

mice. Such strains are distinguished by their biological scrapie and do not accumulate PrP Sc or propagate

diseases, and mice devoid of PrP C are resistant to

properties: they produce distinct incubation periods infectivity (Bueler et al., 1992), its normal biological

and patterns of neuropathological targeting (so-called function remains unclear. PrP is highly conserved

lesion profiles) in defined inbred mouse lines (for amongst mammals, has been identified in marsupials,

review, see Bruce et al., 1992). As they can be serially amphibians and birds, and may be present in all

propagated in inbred mice with the same Prnp vertebrates. It is expressed during early embryogenesis

genotype, they cannot be encoded by diﬀerences in and is found in most tissues in the adult (Manson et al.,

PrP primary structure. Furthermore, strains can be re- 1992). However, highest levels of expression are seen in

isolated in mice after passage in intermediate species the central nervous system. The protein is found

with diﬀerent PrP primary structures (Bruce et al., predominantly in neurons, particularly at synapses in

1994). Conventionally, distinct strains of conventional cholesterol-rich microdomains or caveolae, known to

pathogen are explained by diﬀerences in their nucleic play a central role in neuronal signalling events (for

acid genome. However, in the absence of such a scrapie review, see Anderson, 1993). PrP is also widely

genome, alternative possibilities must be considered. expressed in cells of the immune system (Dodelet and

Weissmann proposed a ‘uniﬁed hypothesis’ where, Cashman, 1998). Mice lacking PrP as a result of gene

although the protein alone was argued to be suﬃcient knockout (Prnp o/o ) showed no gross phenotype (Bueler

to account for infectivity, it was proposed that strain

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

characteristics could be encoded by a small cellular only’ hypothesis of infectivity and suggest that strain nucleic acid, or ‘co-prion’ (Weissmann, 1991).

variation could be encoded by a combination of PrP Although this hypothesis leads to the testable predic-

conformation and glycosylation. Furthermore, poly- tion that strain characteristics, unlike infectivity,

morphism in PrP sequence can inﬂuence the generation would be sensitive to UV irradiation, no such test

of particular PrP Sc conformers (Collinge et al., 1996b). has been reported. At the other extreme, the protein-

Transmission of PrP Sc fragment sizes from two only hypothesis (Griﬃth, 1967) would have to explain

diﬀerent subtypes of inherited prion disease to trans- how a single polypeptide chain could encode multiple

genic mice expressing a chimaeric human mouse PrP disease phenotypes. Clearly, understanding how a

has also been reported (Telling et al., 1996). As PrP protein-only infectious agent could encode such

glycosylation occurs before conversion to PrP Sc , the phenotypic information is of considerable biological

diﬀerent glycoform ratios may represent selection of interest.

particular PrP C glycoforms by PrP Sc of diﬀerent Support for the idea that strain speciﬁcity may be

conformations. According to such a hypothesis, PrP encoded by PrP itself was provided by study of two

conformation would be the primary determinant of distinct strains of transmissible mink encephalopathy

strain type, with glycosylation being involved as a prions which can be serially propagated in hamsters,

secondary process. However, since it is known that designated hyper (HY) and drowsy (DY). These

different cell types may glycosylate proteins differently, strains can be distinguished by differing physiochem-

PrP Sc glycosylation patterns may provide a substrate ical properties of the accumulated PrP Sc in the brains

for the neuropathological targeting that distinguishes of aﬀected hamsters (Bessen and Marsh, 1992).

diﬀerent prion strains (Collinge et al., 1996b). Parti- Following limited proteolysis, strain-speciﬁc migration

cular PrP Sc glycoforms may replicate most favourably patterns of PrP Sc on polyacrylamide gels were seen

in neuronal populations with a similar PrP glycoform which related to diﬀerent N-terminal ends of HY and

expressed on the cell surface. Such targeting could also DY PrP Sc following protease treatment and implying

explain the diﬀerent incubation periods which also diﬀering conformations of HY and DY PrP Sc (Bessen

discriminate strains, targeting of more critical brain and Marsh, 1994).

regions, or regions with higher levels of PrP expression, Distinct human PrP Sc types have been identiﬁed

producing shorter incubation periods. which are associated with diﬀerent phenotypes of CJD

Recent work has shown strain-speciﬁc protein (Collinge et al., 1996b; Parchi et al., 1996). The

conformation to be inﬂuenced by metal binding to diﬀerent fragment sizes seen on Western blots follow-

PrP Sc (Wadsworth et al., 1999). Two diﬀerent human ing treatment with proteinase K suggests that there are

PrP Sc types, seen in clinically distinct subtypes of several diﬀerent human PrP Sc conformations. How-

classical Creutzfeldt–Jakob disease, can be intercon- ever, while such biochemical modiﬁcations of PrP are

verted in vitro by altering the metal-ion occupancy. clearly candidates for the molecular substrate of prion

The dependence of PrP Sc conformation on the binding strain diversity, it is necessary to be able to demon-

of copper and zinc represents a novel mechanism for strate that these properties fulﬁl the biological

post-translational modiﬁcation of PrP and for the properties of strains. In particular, that they are

generation of multiple prion strains. transmissible to the PrP in a host of both the same

Molecular strain typing of prion isolates can now be and diﬀerent species. This has been demonstrated in

applied to molecular diagnosis of vCJD (Collinge et studies with CJD isolates, with both PrP Sc fragment

al. , 1996b; Hill et al., 1997a) and to produce a new sizes and the ratios of the three PrP glycoforms

classiﬁcation of human prion diseases with implica- (diglycosylated, monoglycosylated and unglycosylated

tions for epidemiological studies investigating the PrP) maintained on passage in transgenic mice

aetiology of sporadic CJD (Figure 26.3). Such methods expressing human PrP (Collinge et al., 1996b).

allow strain typing to be performed in days rather than Furthermore, transmission of human prions and

the 1–2 years required for classical biological strain bovine prions to wild-type mice results in murine

typing. This technique may also be applicable to PrP Sc , with fragment sizes and glycoform ratios which

determining whether BSE has been transmitted to correspond to the original inoculum (Collinge et al.,

other species (Collinge et al., 1996b) and thereby poses 1996b). Variant CJD is associated with PrP Sc glyco-

a threat to human health, e.g. to sheep (Hill et al., form ratios, which are distinct from those seen in

1998; Hope et al., 1999; Kuczius et al., 1998). classical CJD. Similar ratios are seen in BSE and BSE

Such ability of a single polypeptide chain to encode when transmitted to several other species (Collinge et

information specifying distinct phenotypes of disease al. , 1996b). These data strongly support the ‘protein

raises intriguing evolutionary questions. Do other

HUMAN PRION DISEASES

Figure 26.3 Molecular strain typing of human prions

proteins behave in this way? The novel pathogenic (Williams et al., 1997). Although PrP C expression is mechanisms involved in prion propagation may be of

required for susceptibility to the disease, a number of far wider signiﬁcance and relevant to other neurologi-

observations argue that PrP Sc , and indeed prions cal and non-neurological illnesses; indeed, other prion-

(whether or not they are identical), may not themselves like mechanisms have now been described and the ﬁeld

be highly neurotoxic. Prion diseases in which PrP Sc is of yeast and fungal prions has emerged (Wickner,

barely or not detectable have been described (Medori 1997; Wickner and Masison, 1996).

et al. , 1992a; Collinge et al., 1995a; Hsiao et al., 1990; Lasme´zas et al., 1997). Mice with reduced levels of

PrP C expression have extremely high levels of PrP Sc NEURONAL CELL DEATH IN PRION DISEASE

and prions in the brain and yet remain well for several months after their wild-type counterparts succumb

The precise molecular nature of the infectious agent (Bueler et al., 1993). Conversely, Tg20 mice, with high and the cause of neuronal cell death remains unclear.

levels of PrP C , have short incubation periods and yet The current working hypothesis is that an abnormal

produce low levels of PrP Sc after inoculation with isoform of PrP is the infectious agent and, to date, the

mouse prions (Fischer et al., 1996). In addition, brain most highly enriched preparations contain one infec-

grafts producing high levels of PrP Sc do not damage tious unit per 10 5 PrP monomers (Bolton et al., 1982).

adjacent tissue in PrP knockout (Prnp o/o ) mice Various hypotheses have been proposed to explain the

(Brandner et al., 1996). The cause of neurodegenera- mechanism of spongiform change and neuronal cell

tion in prion diseases remains unclear. It remains loss. These have included direct neurotoxic eﬀects from

possible that prion neurodegeneration is related, at

a region of the prion protein encompassing residues least in part, to loss of function of PrP C . That Prnp o/o 106–126 (Brown et al., 1994; Forloni et al., 1993;

mice (other than those associated with overexpression Tagliavini et al., 1993b) to increased oxidative stress in

of the Prnp-like gene Dpl (Moore et al., 1999) do not

develop neurodegeneration could be due to compen- proposed to function as an antioxidant molecule

neurons as a result of PrP C depletion, which has been

during neurodevelopment. (Brown et al., 1997). Neurotoxicity of PrP 106–126

satory

adaptations

Complete or near-complete ablation of PrP expression is, however, controversial (Kunz et al., 1999). It has

in an adult mouse using conditional gene expression

methods has not yet been achieved. A recent study has apoptosis with disturbance of normal cellular levels of

also been suggested that PrP C plays a role in regulating

demonstrated that mice inoculated with Sc237 hamster PrP during infection leading to cell death (Kurschner

prions replicate prions to high levels in their brains, and Morgan, 1995, 1996). Certainly there have been

but do not develop clinical signs of prion disease numerous recent reports of apoptotic cells being

during their normal lifespan, arguing that PrP Sc and identiﬁed in the neuronal tissue of prion disease brains

indeed prions (whether or not they are identical) may

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

not themselves be highly neurotoxic (Hill et al., 2000). most eﬃciently when the interacting PrP Sc and PrP C An alternative hypothesis for prion-related neurode-

were of identical primary structure (Collinge et al., generation is that a toxic, possibly infectious,

1991; Palmer et al., 1991a). However, it has been long intermediate is produced in the process of conversion

recognised that prion strain type aﬀects ease of of PrP C to PrP Sc , with PrP Sc present as highly

transmission to another species. Interestingly, with aggregated material, being a relatively inert end-

BSE prions the strain component to the barrier seems product. The steady-state level of such a toxic

to predominate, with BSE not only transmitting monomeric or oligomeric PrP intermediate could

eﬃciently to a range of species, but maintaining its then determine the rate of neurodegeneration. One

transmission characteristics even when passaged possibility is that Sc237-inoculated mice propagate

through an intermediate species with a distinct PrP prions very slowly and that such a toxic intermediate is

gene (Bruce et al., 1994). For instance, transmission of generated at extremely low levels that are tolerated by

CJD prions to conventional mice is diﬃcult, with few if the mouse (Hill et al., 2000).

any inoculated mice succumbing after prolonged incubation periods, consistent with a substantial species barrier (Collinge et al., 1995b; Hill et al.,

THE ‘SPECIES BARRIER’ 1997a). In sharp contrast, transgenic mice expressing only human PrP are highly susceptible to CJD prions,

Transmission of prion diseases between diﬀerent with 100% attack rate and consistent short incubation mammalian species is restricted by a ‘species barrier’

periods which are unaltered by second passage, (Pattison, 1965). On primary passage of prions from

consistent with a complete lack of species barrier species A to species B, usually not all inoculated

(Collinge et al., 1995b). However, vCJD prions (again animals of species B develop disease and those that do

comprising human PrP of identical primary structure) have much longer and more variable incubation

transmit much more readily to wild-type mice than do periods than those that are seen with transmission of

classical CJD prions, while transmission to transgenic prions within the same species, where typically all

mice is relatively less eﬃcient than with classical CJD inoculated animals would succumb with a relatively

(Hill et al., 1997a). The term ‘species barrier’ does not short, and remarkably consistent, incubation period.

seem appropriate to describe such eﬀects and ‘species– On second passage of infectivity to further animals of

strain barrier’ or simply ‘transmission barrier’ may be the species B, transmission parameters resemble

preferable (Collinge, 1999). Both PrP amino acid within-species transmissions, with most if not all

sequence and strain type aﬀect the 3D structure of animals developing the disease with short and con-

glycosylated PrP, which will presumably, in turn, aﬀect sistent incubation periods. Species barriers can

the eﬃciency of the protein–protein interactions therefore be quantitated by measuring the fall in

thought to determine prion propagation. mean incubation period on primary and second

Mammalian PrP genes are highly conserved. Pre- passage, or, perhaps more rigorously, by a compara-

sumably only a restricted number of diﬀerent PrP Sc tive titration study. The latter involves inoculating

conformations (which are highly stable and can serial dilutions of an inoculum in both the donor and

therefore be serially propagated) will be permissible

thermodynamically and will constitute the range of eﬀect of a very substantial species barrier (e.g. that

host species and comparing the LD 50 s obtained. The

prion strains seen. PrP glycosylation may be important between hamsters and mice) is that few, if any, animals

in stabilising particular PrP Sc conformations. While a succumb to disease at all on primary passage, and then

signiﬁcant number of diﬀerent such PrP Sc conforma- at incubation periods approaching the natural lifespan

tions may be possible amongst the range of of the species concerned.

mammalian PrPs, only a subset of these would be Early studies of the molecular basis of the species

allowable for a given single mammalian PrP. Sub- barrier argued that it principally resided in diﬀerences

stantial overlap between the favoured conformations in PrP primary structure between the species from

for PrP Sc derived from species A and species B might which the inoculum was derived and the inoculated

therefore result in relatively easy transmission of prion host. Transgenic mice expressing hamster PrP were,

diseases between these two species, while two species unlike wild-type mice, highly susceptible to infection

with no preferred PrP Sc conformations in common with Sc237 hamster prions (Prusiner et al., 1990). That

would have a large barrier to transmission (and, most sporadic and acquired CJD occurred in indivi-

indeed, transmission would necessitate a change of duals homozygous at PRNP polymorphic codon 129

strain type). According to such a model of a prion supported the view that prion propagation proceeded

transmission barrier, BSE may represent a thermo-

787 dynamically highly favoured PrP Sc conformation that

HUMAN PRION DISEASES

and other lymphoreticular tissues (for review, see is permissive for PrP expressed in a wide range of

Fraser et al., 1992). Spleen titres rise to a plateau diﬀerent species, accounting for the remarkable pro-

early in the incubation period, a considerable period miscuity of this strain in mammals. A contribution of

before neuroinvasion is detectable. CNS prion replica- other components to the species barrier is possible and

tion then rises to high levels and the clinical phase may involve interacting co-factors which mediate the

occurs.

eﬃciency of prion propagation, although no such The route of entry of prions following oral exposure factors have yet been identiﬁed.

may follow invasion of Peyer’s patches and other gut Recent data has further challenged our understand-

lymphoid tissues; the relative protease-resistance of ing of transmission barriers (Hill et al., 2000). The

prions presumably allows a signiﬁcant proportion of assessment of species barriers has relied on the

infectivity to survive the digestive tract. It is unclear development of a clinical disease in inoculated animals.

how prions transit the intestinal mucosa, although M On this basis there is a highly eﬃcient barrier limiting

cells may be involved (Heppner et al., 2001). It has transmission of hamster Sc237 prions to mice. Indeed,

been suggested that myeloid dendritic cells mediate the hamster scrapie strain Sc237 (which is similar to

transport within the lymphoreticular system (Aucou- the strain classiﬁed as 263K; Kimberlin and Walker,

turier et al., 2001). While mature B cells are required 1978), is regarded as non-pathogenic for mice (with no

for peripheral prion propagation, this appears to be clinical disease in mice observed for up to 735 days

because they are required for maturation of follicular post-inoculation; Kimberlin and Walker, 1978) and

dendritic cells (FDCs). PrP Sc accumulates in FDCs, was used in studies of species barriers in transgenic

which are a long-lived cell type, and it is thought that mice (Kimberlin and Walker, 1979; Prusiner et al.,

they are the site of prion propagation in the spleen 1990; Scott et al., 1989). It was demonstrated that

(Mabbott et al., 2000, 2003; Montrasio et al., 2000). transgenic mice expressing hamster PrP (in addition to

However, neuroinvasion is possible without FDCs, endogenous mouse PrP), in sharp contrast to conven-

indicating that other peripheral cell types can replicate tional mice, were highly susceptible to Sc237 hamster

prions (Oldstone et al., 2002; Prinz et al., 2002). prions, with consistent short incubation periods which

Neuroinvasion involves the autonomic nervous system were inversely correlated to hamster PrP expression

innervating lymphoid tissue with retrograde spread to levels (Scott et al., 1989; Prusiner et al., 1990).

the spinal cord or via the vagus to the brainstem Importantly, however, these studies deﬁned transmis-

(Beekes et al., 1998; Bencsik et al., 2001). Prions have sion using clinical criteria and did not report PrP Sc

been detected in the blood at low levels in some rodent levels and types, or prion titres in the brains of

models and experimental BSE-infected primates (Bons clinically unaﬀected animals. However, while not

et al. , 2002; Brown et al., 1999; Holada et al., 2002) developing a clinical disease, and indeed living as

and transmission of BSE prions between sheep by long as mock-inoculated mice, Sc237-inoculated mice

transfusion has been reported (Hunter et al., 2002). may accumulate high levels of prions in their brains

Several reports of infrequent transmission from human (Hill et al., 2000). Previous studies on the species

blood to rodents have been reported (for review, see barrier between hamsters and mice (using the Sc237 or

Brown, 1995).

263K strain) did not report whether PrP Sc and/or While prominent lymphoreticular involvement is infectivity were present in clinically unaﬀected animals

seen in some experimental models or natural prion (Prusiner et al., 1990; Scott et al., 1989) or have

diseases, it is undetectable in others (for review, see attempted passage from mice only up to 280 days post-

Fraser et al., 1992). Both host and prion strain eﬀects inoculation (Kimberlin and Walker, 1978). The barrier

are relevant, e.g. infection of sheep with BSE prions to primary passage appears in this case to be to the

results in a wide tissue distribution of infectivity but development of rapid neurodegeneration and the

infection of cattle with this strain does not, infectivity resulting clinical syndrome, rather than a barrier to

being largely conﬁned to the CNS. In humans infected prion propagation itself.

with sporadic CJD prions, infectivity is largely conﬁned to the CNS, while in variant CJD there is prominent involvement of lymphoreticular tissues (Hill

PATHOGENESIS et al. , 1997b, 1999; Wadsworth et al., 2001). It is possible that species-barrier eﬀects are also relevant,

In some experimental rodent scrapie models in which and it has been suggested that, on passage of prions in prions are inoculated outside the CNS, and in natural

a new species, there is an obligate lymphoreticular sheep scrapie, infectivity is ﬁrst detectable in the spleen

phase. This would be of considerable importance with phase. This would be of considerable importance with

ANIMAL PRION DISEASES An increasing number of animal prion diseases are

recognised. Scrapie, a naturally occurring disease of sheep and goats, has been recognised in Europe for over 200 years and is present endemically in many countries. Accurate epidemiology is lacking, although scrapie appears to be relatively common in some countries. Remarkably little is known about its natural routes of transmission. Transmissible mink encephalopathy (TME) and chronic wasting disease of mule deer and elk were described from the 1940s onwards, principally in the USA. It has more recently become apparent that chronic wasting disease is a common condition in wild deer and elk in Colorado. Again the routes of transmission are unclear. TME has occurred as infrequent epidemics amongst ranched mink and may result from food-borne prion exposure.

The appearance of BSE in UK cattle from 1986 onwards, which rapidly evolved to a major epidemic (Anderson et al., 1996; Wilesmith et al., 1988), was widely attributed to transmission of sheep scrapie, endemic in the UK and many other countries, to cattle via contaminated feed prepared from rendered carcasses (Wilesmith et al., 1988). However, an alternative hypothesis is that epidemic BSE resulted from recycling of rare sporadic BSE cases, as cattle were also rendered to produce cattle feed. Whether or not BSE originated from sheep scrapie, however, it was clear from 1990 onwards, with the occurrence of novel spongiform encephalopathies amongst domestic and captive wild cats, that its host range was different to that of scrapie. Many new species have developed spongiform encephalopathies coincident with or following the arrival of BSE, including greater kudu, nyala, Arabian oryx, Scimitar-horned oryx, eland, gemsbok, bison, ankole, tiger, cheetah, ocelot, puma and domestic cats. Several of these have been confirmed to be caused by a BSE-like prion strain (Bruce et al., 1994; Collinge et al., 1996b), and it is likely that most or all are BSE-related. More than 180 000 BSE cases have been confirmed in cattle in the UK, although the total number of infected animals has been estimated to be around 2 million. BSE has since been reported in many European countries, with

signiﬁcant epidemics reported in Switzerland and Portugal, and a number of other countries including Canada and Japan.

AETIOLOGY AND EPIDEMIOLOGY AND HUMAN PRION DISEASE

The human prion diseases have been traditionally classiﬁed into Creutzfeldt–Jakob disease (CJD), Gerst- mann–Stra¨ussler–Scheinker disease and kuru, and they can be further divided into three aetiological categories: sporadic, acquired and inherited.

Sporadic CJD makes up around 85% of all recognised human prion disease. It occurs in all countries with a random case distribution and an annual incidence of 1 per million. Hypothesised causes of sporadic CJD include spontaneous production of PrP Sc via rare stochastic events, somatic mutation of PRNP or unidentified environmental prion exposure. An association with sheep scrapie is not supported by epidemiological studies, which have found a fairly uniform worldwide incidence of sporadic CJD, irrespective of scrapie prevalence (Brown et al., 1987). Although spatiotemporal groupings of sporadic CJD have been reported previously (Adikari and Farmer, 2001; Farmer et al., 1978), no direct evidence for exposure to a common source of infectious prions has been provided. Indeed, such apparent clustering of cases, while appearing to reach levels of significance when viewed in isolation, can be deemed to be expected by chance alone when analysed within the population as a whole (Collins et al., 2002). However, the lack of such evidence does not exclude the possibility that a fraction of sporadic CJD, is caused by environmental exposure to animal or human prions. There is marked genetic susceptibility in sporadic CJD in that most cases occur in homozygotes at codon 129 of PRNP, where either methionine or valine may be encoded. Heterozygotes appear significantly protected against developing sporadic CJD (Collinge et al., 1991b; Palmer et al., 1991; Windl et al. , 1996). Additionally, a PRNP susceptibility haplotype has been identified indicating additional genetic susceptibility to sporadic CJD at or near to the PRNP locus (Mead et al., 2001).

The acquired prion diseases include iatrogenic CJD and kuru, and arise from accidental exposure to human prions through medical or surgical procedures or participation in cannibalistic feasts. The two most frequent causes of iatrogenic CJD occurring through medical procedures have arisen as a result of implanta-

788

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

789 tion of dura mater grafts and treatment with human

HUMAN PRION DISEASES

diseases, iatrogenic CJD and kuru, occur in all codon growth hormone derived from the pituitary glands of

129 genotypes as the epidemic evolves, with codon 129 human cadavers (Brown et al., 1992, 2000). Less

heterozygotes having the longest mean incubation frequent incidences of human prion disease have

periods (Collinge, 1999; Lloyd et al., 2002; Poulter et resulted from iatrogenic transmission of CJD during

al. , 1992). Human BSE infection of other PRNP corneal transplantation, contaminated electroencepha-

genotypes may simply have a longer latency (Collinge, lographic (EEG) electrode implantation and surgical

1999) and may also have a diﬀerent phenotype (Hill et operations using contaminated instruments or appa-

al. , 1997a).

ratus (Brown et al., 1992, 2000). PRNP codon 129 Estimates of the mean incubation period of human- genotype is also relevant to susceptibility and incuba-

to-human prion transmission come from study of tion period (see below).

growth hormone-related iatrogenic CJD and kuru. The Around 15% of human prion disease is inherited

mean incubation period has been estimated in both and all cases to date have been associated with coding

examples to be around 12 years; in kuru, incubation mutations in the prion protein gene (PRNP), of which

periods can exceed 40 years (for review, see Collinge, over 30 distinct types are recognised (Figure 26.3). The

1999). The eﬀect of a species barrier is to considerably inherited prion diseases can be diagnosed by PRNP

increase mean incubation periods and the range of analysis and the use of these deﬁnitive genetic

incubation periods, which may approach the usual diagnostic markers has allowed the recognition of a

lifespan of the species concerned. The cattle to mouse wider phenotypic spectrum of human prion disease to

barrier for the BSE strain results typically in a three- to include a range of atypical dementias and fatal

four-fold increase in mean incubation period. Mean familial insomnia (Collinge et al., 1990, 1992; Medori

incubation periods of human BSE infection of 30 years et al. , 1992b). The protective eﬀect of PRNP codon

or more should be considered (Collinge, 1999). 129 heterozygosity is also seen in some of the

Furthermore, prion disease in mice follows a well- inherited prion diseases, with a later age at disease

deﬁned course with a highly distinctive and repeatable onset in heterozygotes (Baker et al., 1991; Hsiao et al.,

incubation time for a given prion strain in a deﬁned 1992).

inbred mouse line. In addition to the prion protein The occurrence of cases of apparently sporadic CJD

gene, a small number of additional genetic loci with a in unusually young people in 1995 (Bateman et al.,

major eﬀect on incubation period have been mapped. 1995; Britton et al., 1995; Tabrizi et al., 1996) led to

It can be anticipated that the human homologues of concerns that BSE transmission to humans may have

such loci may play a key role in human susceptibility to occurred. Arrival of further cases in 1996 led to the

prion disease, following both accidental human prion recognition of a novel clinicopathological type of

exposure and exposure to the BSE agent. By deﬁnition, human prion disease, new variant CJD (vCJD) (Will

the patients identiﬁed to date with vCJD are those with et al. , 1996), indicating the arrival of a new risk factor

the shortest incubation periods for BSE. These, in for CJD in the UK (Collinge and Rossor, 1996). A link

turn, given that no unusual history of dietary, with BSE seemed highly likely on epidemiological

occupational or other exposure to BSE has been grounds and this was strongly supported by experi-

identiﬁed, would be expected to be predominantly mental data, ﬁrst from molecular strain typing studies

those individuals with short incubation time alleles at (Collinge et al., 1996b) and later by transmission

these multiple genetic loci in addition to having the studies into both transgenic and conventional mice

codon 129 methionine homozygous PRNP genotype. (Hill et al., 1997a; Bruce et al., 1997). PRNP mutations

The vCJD cases reported to date may therefore are absent in vCJD, and all cases studied to date have

represent a distinct genetic subpopulation with un- been methionine homozygotes at codon 129 (Collinge

usually short incubation periods to BSE prions. It is et al. , 1996a; Zeidler et al., 1997b and unpublished).

possible, therefore, that a human BSE epidemic will be That vCJD is caused by the same prion strain as that

multiphasic, and that recent estimates of the size of the causing BSE in cattle raised the possibility that a major

vCJD epidemic based on uniform genetic susceptibility epidemic of vCJD will occur in the UK and other

may substantially underestimate the eventual size countries as a result of dietary or other exposure to

(D’Aignaux et al., 2001; Ghani et al., 2000). Genes BSE prions, and also (Ghani et al., 1999) concerns of

involved in species barrier eﬀects, which would further potential iatrogenic transmission of preclinical vCJD

increase both the mean and range of human BSE via medical and surgical procedures. That only PRNP

incubation periods, are also likely to be relevant. In 129MM individuals are susceptible to BSE infection is

this context, it will be very diﬃcult to accurately questionable, since the other acquired human prion

predict a human epidemic until such loci are identiﬁed

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

and their gene frequencies in the population can be reported which are not only clinically atypical but determined (Lloyd et al., 2001).

which lack the classical histological features entirely (Collinge et al., 1990). Signiﬁcant clinical overlap exists with familial Alzheimer’s disease, Pick’s disease,

CLINICAL FEATURES AND DIAGNOSIS frontal lobe degeneration of non-Alzheimer type and amyotrophic lateral sclerosis with dementia. Although

With the advances in our understanding of their classical GSS is described below, it now seems more aetiology, it now seems more appropriate to divide

sensible to designate the familial illnesses as inherited the human prion diseases into inherited, sporadic and

prion diseases and then to sub-classify these according acquired forms, with CJD, GSS and kuru being

to mutation. Acquired prion diseases include iatro- clinicopathological syndromes within a wider spectrum

genic CJD, kuru and now vCJD. Sporadic prion of disease. Kindreds with inherited prion disease have

diseases at present consist of CJD and atypical variants been described, with phenotypes of classical CJD and

of CJD. Cases lacking the characteristic histological GSS and also with other neurodegenerative syndromes

features of CJD have been transmitted. As there are at including fatal familial insomnia (Medori et al.,

present no equivalent aetiological diagnostic markers 1992b). Some kindreds show remarkable phenotypic

for sporadic prion diseases to those for the inherited variability which can encompass both CJD- and GSS-

diseases, it cannot yet be excluded that more diverse like cases, as well as other cases which do not conform

phenotypic variants of sporadic prion disease exist. to either CJD or GSS phenotypes (Collinge et al.,

The key clinical features and investigations for the 1992). Cases diagnosed by PrP gene analysis have been

diagnosis of prion disease are given in Table 26.1.

Table 26.1 Diagnosis of human prion disease Sporadic (classical) CJD

. Rapidly progressive dementia with two or

. Most cases aged 45–75

more of myoclonus, cortical bindness,

Serial EEG shows pseudoperiodic

pyramidal signs, cerebellar signs, extra-

complexes in most cases

pyramidal signs, akinetic mutism

. CSF 14–3–3 protein usually positive

. PRNP analysis: no pathogenic mutations

. CT and MRI normal, or atrophy, or

. If brain biopsy performed:

abnormal signal basal ganglia

PrP immunocytochemistry or Western

. PRNP analysis: most are

129 homozygotes Iatrogenic CJD

blot for PrP Sc types 1–3

. Progressive cerebellar syndrome and

. May be young

behavioural disturbance, or classical

. EEG, CSF and MRI generally less helpful

CJD-like syndrome, with history of

than in sporadic cases

iatrogenic exposure to human prions

. PRNP analysis: most are

(pituitary-derived hormones, tissue

129 homozygotes

grafting or neurosurgery) . PRNP analysis: no pathogenic mutations . If brain biopsy performed: PrP

immunocytochemistry or Western blot for PrP Sc types 1–3

Variant CJD

. Early features: depression, anxiety, social

. Cerebellar ataxia, chorea or athetosis

withdrawal, peripheral sensory symptoms

often precedes dementia, advanced

. EEG: non-speciﬁc slow waves

disease as sporadic CJD

. PRNP analysis: no pathogenic mutations

. Most in young adults

. If tonsil biopsy performed: characteristic

. CSF 14–3–3 may be elevated

PrP immunostaining and PrP Sc on

. MRI: may be high T2-weighted signal

in posterior thalamus bilaterally . PRNP analysis: all 129MM to date Inherited prion disease

Western blot (type 4t)

. PRNP analysis: pathogenic mutations

. Varied clinical syndromes between and within kindreds: should consider in all pre-senile dementias and ataxias irrespective of family history

. PRNP analysis: codon 129 genotype may predict age at onset in pre-symptomatic testing

791 Sporadic Prion Disease

HUMAN PRION DISEASES

the cerebrospinal ﬂuid is normal, although raised neuronal-speciﬁc enolase (NSE), S-100 and 14–3–3 protein have been proposed as useful markers,

Creutzfeldt–Jakob Disease although it is clear that they are not speciﬁc for CJD The core clinical syndrome of classic CJD is a rapidly

and represent markers of neuronal injury or astrocyte progressive multifocal dementia, usually with promi-

activation (Jimi et al., 1992; Otto et al., 1997; Zerr et nent myoclonus. Onset is usually in the 45–75 year age

al. , 1995). A positive 14–3–3 appears to be a useful group, with peak onset between 60–65. The clinical

adjunct to diagnosis in the appropriate clinical context progression is typically over weeks and may progress

(Collinge, 1996); it is also positive in recent cerebral to akinetic mutism and death in 2–3 months. Around

infarction or haemorrhage and in viral encephalitis, 70% of cases die in under 6 months. Prodromal

although these conditions do not usually present features, present in around one-third of cases, include

diagnostic confusion with CJD. Neuroimaging with fatigue, insomnia, depression, weight loss, headaches,

CT or MRI is essential to exclude other causes of general malaise and ill-deﬁned pain sensations. In

subacute neurological illness and may also show addition to mental deterioration and myoclonus,

cerebral and cerebellar atrophy. MRI scanning may frequent additional neurological features include extra-

demonstrate signal changes in the basal ganglia that, pyramidal signs, cerebellar ataxia, pyramidal signs and

although not speciﬁc, can be diagnostically helpful cortical blindness. About 10% of cases present initially

(Schroter et al., 2000). The electroencephalogram with cerebellar ataxia.

(EEG) may show characteristic pseudoperiodic sharp Routine haematological and biochemical investiga-

wave activity (Figure 26.4), which is useful in diagnosis tions are normal, although occasional cases have been

but present only in around 70% of cases. To some noted to have raised serum transaminases or alkaline

extent, demonstration of a typical EEG is dependent phosphatase. There are no immunological markers and

on the number of EEGs performed and serial EEG is acute phase proteins are not elevated. Examination of

indicated to try to demonstrate this appearance.

Figure 26.4 Electroencephalogram in sporadic CJD

Prospective epidemiological studies have demonstrated that cases with a progressive dementia, and two or more of the following—myoclonus; cortical blindness; pyramidal, cerebellar or extrapyramidal signs; or akinetic mutism in the setting of a typical EEG— nearly always turn out to be confirmed as histologically definite CJD if neuropathological examination is performed. Brain biopsy may be considered in selected cases to exclude alternative, potentially treatable diagnoses; inherited prion disease should be excluded prior to biopsy by gene analysis. Neuropathological confirmation of CJD is by demonstration of spongiform change, neuronal loss and astrocytosis. PrP amyloid plaques are usually not present in CJD, although PrP immunohistochemistry, using appropriate pre-treatments (Bell et al., 1997; Budka et al., 1995), will nearly always be positive (Figure 26.5). PrP Sc , seen in all the currently recognised prion diseases, can be demonstrated by immunoblotting of brain homogenates. Genetic susceptibility to CJD has been demonstrated in that most cases of classical CJD are homozygous with respect to the common 129 polymorphism of PrP.

Atypical Forms of Creutzfeldt–Jakob Disease Atypical forms of Creutzfeldt–Jakob disease are well

recognised. Around 10% of cases of CJD have a much more prolonged clinical course, with a disease duration of over 2 years (Brown et al., 1984). These cases may represent the occasional occurrence of CJD in individuals heterozygous for PrP polymorphisms. Around 10% of CJD cases present with cerebellar ataxia rather than cognitive impairment, so-called ‘ataxic CJD’ (Gomori et al., 1973). Heidenhain’s variant of CJD refers to cases in which cortical blindness predominates with severe involvement of the occipital lobes. The panencephalopathic type of CJD refers to cases with extensive degeneration of the cerebral white matter in addition to spongiform vacuolation of the grey matter, and has been predominately reported from Japan (Gomori et al., 1973).

Amyotrophic variants of CJD have been described with prominent early muscle wasting. However, most cases of dementia with amyotrophy are not experimentally transmissible (Salazar et al., 1983) and their relationship with CJD is unclear. Most cases are probably variants of motor neuron disease with associated dementia. Amyotrophic features in CJD are usually seen in late disease when other features are well established.

Molecular Classification of Sporadic CJD

The marked clinical heterogeneity observed in human prion diseases has yet to be explained. However, it has been clear for many years that distinct isolates, or strains, of prions can be propagated in the same host and these are biologically recognised by distinctive clinical and pathological features (Collinge, 2001; Hill and Collinge, 2001). It is therefore likely that a proportion of clinicopathological heterogeneity seen in sporadic CJD and other human prion diseases relates to the propagation of distinct human prion strains. The identification of strain-specific PrP Sc structural properties would thus allow an aetiology- based classification of CJD by typing of the infectious agent itself.

Four types of human PrP Sc have now been reliably identiﬁed using molecular strain typing (Collinge et al., 1996b; Hill et al., 1997a, 2003; Wadsworth et al., 1999) (Figure 26.3). Sporadic and iatrogenic CJD are associated with PrP Sc types 1–3, while type 4 human PrP Sc is uniquely associated with vCJD and is characterised by a fragment size and glycoform ratio that is distinct from PrP Sc types 1–3 observed in classical CJD (Collinge et al., 1996b; Hill et al., 1997a, 2003; Wadsworth et al., 1999). The methionine/valine polymorphism at codon 129 of PRNP is associated with diﬀerent PrP Sc types. PrP Sc types 1 and 4 have so far only been detected in methionine homozygotes, type 3 cases are predominantly associated with at least one valine allele, while type 2 is seen in any PRNP codon 129 genotype (Collinge et al., 1996a, 1996b; Hill et al. , 1999, 2003; Wadsworth et al., 1999). PrP Sc types

1 and 2 are associated with two clinically distinct subtypes of sporadic CJD and have N-terminal structures determined by the coordination of metal ions (Hill et al., 2003; Wadsworth et al., 1999). Importantly, the identification of strain-specific PrP Sc structural properties has enabled investigation of the influence of human PrP primary structure, in particular polymorphic residue 129, in determining PrP Sc structure. Transgenic mice expressing human PrP with either valine or methionine at residue 129 have revealed that this polymorphism constrains both the propagation of distinct human PrP Sc conformers and the occurrence of associated patterns of neuropathology (Asante et al., 2002; Collinge et al., 1996b; Hill et al. , 1997a; and unpublished data). These data strongly support the biological relevance of molecular strain typing, which can now be applied to rapid molecular diagnosis of classical CJD or vCJD and to produce a new classification of human prion diseases.

792

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

793 Molecular strain typing has major implications for

HUMAN PRION DISEASES

communities to engage in consumption of dead epidemiological surveillance of sporadic CJD, whose

relatives as a mark of respect and mourning. Women aetiology remains obscure. While spontaneous conver-

and children predominantly ate the brain and internal sion of PrP C to PrP Sc as a rare stochastic event, or

organs, which is thought to explain the diﬀerential age somatic mutation of the PrP gene resulting in

and sex incidence. Preparation of the cadaver for expression of a pathogenic PrP mutant, are plausible

consumption was performed by the women and explanations for sporadic CJD (Collinge, 1997), other

children, such that other routes of exposure may also causes for at least some cases, including environmental

have been relevant. It is thought that the epidemic exposure to human or animal prions, has not been

related to a single sporadic CJD case occurring in the ruled out by existing epidemiological studies (Collins et

region some decades earlier. Epidemiological studies al. , 1999). Sub-classiﬁcation of sporadic CJD based

provided no evidence for vertical transmission, since upon PrP Sc type immediately allows a more precise

most of the children born after 1956 (when cannibal- molecular classiﬁcation of human prion disease, and

ism had eﬀectively ceased) and all of those born after re-analysis of epidemiological data using these mole-

1959 of mothers aﬀected with or incubating kuru, were cular subtypes may reveal important risk factors

unaﬀected (Alpers, 1987). From the age of the young- obscured when sporadic CJD is analysed as a single

est aﬀected patient, the shortest incubation period is entity. For example, it will be important to review the

estimated as 4.5 years, although it may have been incidence of sporadic CJD associated with PrP Sc type 2

shorter, since the time of infection was usually and other molecular subtypes, in both BSE-aﬀected

unknown. Currently, two or three cases are occurring and unaﬀected countries, in the light of recent ﬁndings

annually, all in individuals aged 40 or more, consistent suggesting that human BSE prion infection may result

with exposure prior to 1956 and indicating that in propagation of either type 4 PrP Sc or type 2

incubation periods can be 40 years or more (Whitﬁeld, PrP Sc (Asante et al., 2002). Individuals that propagate

Alpers and Collinge, unpublished). PRNP codon 129 type 2 PrP Sc as a result of BSE exposure may present

genotype has a signiﬁcant eﬀect on kuru susceptibility with prion disease that would be indistinguishable on

and most elderly survivors of the kuru epidemic are clinical, pathological and molecular criteria from that

heterozygotes (Mead et al., 2003). The marked survival found in classical CJD.

advantage for codon 129 heterozygotes provides a powerful basis for selection pressure in the Fore. Remarkably, an analysis of worldwide haplotype

Acquired Prion Diseases diversity and allele frequency of PRNP coding and non-coding polymorphisms suggests that balancing selection at this locus is much older and more

While human prion diseases can be transmitted to geographically widespread. Evidence for balancing experimental animals by inoculation, they are not selection (where there is more variation than expected contagious in humans. Documented case-to-case in a gene due to heterozygote advantage) has been spread has only occurred during ritual cannibalistic demonstrated in only a few human genes. Given recent practices (kuru) or following accidental inoculation

with prions during medical or surgical procedures biochemical and physical evidence of cannibalism on (iatrogenic CJD).

ﬁve continents, one explanation is that ancient and worldwide cannibalism resulted in a series of prion disease epidemics in human prehistory, thus imposing balancing selection on PRNP (Mead et al., 2003).

Kuru Kuru aﬀects both sexes and the onset of disease has ranged from age 5 to over 60. The mean clinical Kuru reached epidemic proportions amongst a deﬁned

duration of illness is 12 months, with a range of 3 population living in the Eastern Highlands of Papua

months to 3 years; the course tends to be shorter in New Guinea. The earliest cases are thought to date

children. The central clinical feature is progressive back to the early part of the century. Kuru aﬀected the

cerebellar ataxia. In contrast to classical CJD, demen- people of the Fore linguistic group and their neigh-

tia is much less prominent, although in the later stages bours, with

many patients have their faculties obtunded (Alpers, predominantly aﬀected women and children (of both

whom they

intermarried. Kuru

1987). The occasional case in which gross dementia sexes), with only 2% of cases in adult males (Alpers,

occurs is in contrast to the clinical norm. Detailed 1987), and was the commonest cause of death amongst

clinical descriptions have been given by a number of women in aﬀected villages. It was the practice in these

observers and the disease does not appear to have observers and the disease does not appear to have

A prodrome and three clinical stages are recognised: . Prodromal stage. Kuru typically begins with

prodromal symptoms consisting of headache, aching of limbs and joint pains, which can last for several months.

. Ambulatory stage. Kuru was frequently self- diagnosed by patients at the earliest onset of unsteadiness in standing or walking, or of dysarthria or diplopia. At this stage there may be no objective signs of disease. However, gait ataxia worsens and patients develop a broad-based gait, truncal instability and titubation. A coarse postural tremor is usually present and accentuated by movement; patients characteristically hold their hands together in the midline to suppress this. Standing with feet together reveals clawing of toes to maintain posture. This marked clawing response is regarded as pathognomonic of kuru. Patients often become withdrawn at this stage and occasionally develop a severe reactive depression. Prodromal symptoms tend to disappear. Astasia and gait ataxia worsen and the patient requires a stick for walking. Intention tremor, dysmetria, hypotonia

and dysdiadochokinesis develop. Although eye movements are ataxic and jerky, nystagmus is rarely seen. Strabismus, usually convergent, may occur, particularly in children. This strabismus does not appear to be concomitant or paralytic and may fluctuate in both extent and type, sometimes disappearing later in the clinical course. Photophobia is common and there may be an abnormal cold sensitivity with shivering and piloerection even in a warm environment. Tendon reflexes are reduced or normal and plantar responses are flexor. Dysarthria usually occurs. As ataxia progresses, the patient passes from the first (ambulatory) stage to the second (sedentary) stage. The mean clinical duration of the first stage is around 8 months and correlates closely with total duration (Alpers, 1964).

. Sedentary stage. At this stage patients are able to sit unsupported but cannot walk. Attempted walking with support leads to a high steppage, wide-based gait with reeling instability and flinging arm movements in an attempt to maintain posture. Hyperreflexia is seen, although plantar responses usually remain flexor with intact abdominal reflexes. Clonus is characteristically short-lived. Athetoid and choreiform movements and Parkin- sonian tremors may occur. There is no paralysis, although muscle power is reduced. Obesity is

common at this stage but may be present in early disease associated with bulimia. Characteristically, there is emotional lability and bizarre uncontrollable laughter, which has led to the disease being referred to as ‘laughing death’. There is no sensory impairment. In sharp contrast to CJD, myoclonic jerking is rarely seen. EEG is usually normal or may show non-speciﬁc changes (Cobb et al., 1973). This stage lasts around 2–3 months. When truncal ataxia reaches the point where the patient is unable to sit unsupported, the third or tertiary stage is reached.

. Tertiary stage . Hypotonia and hyporeflexia develop and the terminal state is marked by flaccid muscle weakness. Plantar responses remain flexor and abdominal reflexes intact. Progressive dysphagia occurs and patients become incontinent of urine and faeces. Inanition and emaciation develop. Transient conjugate eye signs and dementia may occur. Primitive reflexes develop in occasional cases. Brainstem involvement and both bulbar and pseudobulbar signs occur. Respiratory failure and bronchopneumonia eventually lead to death. The tertiary stage lasts 1–2 months.

Iatrogenic Creutzfeldt–Jakob Disease Iatrogenic transmission of CJD has occurred by

accidental inoculation with human prions as a result of medical procedures. Such iatrogenic routes include the use of inadequately sterilised neurosurgical instruments, dura mater and corneal grafting, and use of human cadaveric pituitary-derived growth hormone or gonadotrophin. It is of considerable interest that cases arising from intracerebral or optic inoculation manifest clinically as classical CJD, with a rapidly progressive dementia, while those resulting from peripheral inoculation, most notably following pituitary-derived growth hormone exposure, typically present with a progressive cerebellar syndrome, and are in that respect somewhat reminiscent of kuru. Unsurprisingly, the incubation period in intracerebral cases is short (19–46 months for dura mater grafts) as compared to peripheral cases (typically 15 years or more). There is evidence for genetic susceptibility to iatrogenic CJD, with an excess of PRNP codon 129 homozygotes (Collinge et al., 1991b).

Epidemiological studies have not shown increased risks of particular occupations that may be exposed to human or animal prions, although individual CJD cases in two histopathology technicians, a neuro- pathologist

and

a neurosurgeon have been

794

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

documented. While there have been concerns that CJD may be transmissible by blood transfusion, extensive epidemiological analysis in the UK has found that the frequency of blood transfusion and donation was no different in over 200 cases of CJD and a matched control population (Esmonde et al., 1993). Recipients of blood transfusions who developed CJD had clinical presentations similar to those of sporadic CJD patients and not to the more kuru-like iatrogenic cases arising from peripheral exposure to human prions. Further- more, experimental transmission studies have shown only weak evidence for infectivity in blood (Brown, 1995), even when inoculated via the most efficient (intracerebral) route. It cannot be assumed that the same picture will hold for vCJD, as this is caused by a distinct prion strain (Collinge et al., 1996b) from those causing classical CJD and has a distinct pathogenesis. It is also possible that many individuals in the UK and other countries with significant BSE exposure are sub- or pre-clinically infected with vCJD prions.

Variant CJD In late 1995, two cases of sporadic CJD were reported

in the UK in teenagers (Britton et al., 1995). Only four cases of sporadic CJD had previously been recorded in teenagers, and none of these cases occurred in the UK. In addition, both cases were unusual in having kuru- type plaques, a ﬁnding seen in only around 5% of CJD cases. Soon afterwards a third very young sporadic CJD case occurred (Tabrizi et al., 1996). These cases caused considerable concern and the possibility was raised that they might suggest a link with BSE. It was clearly of some importance to see whether any further such extraordinarily rare cases occurred in the UK. By March 1996, further extremely young onset cases were apparent and review of the histology of these cases showed a remarkably consistent and unique pattern. These cases were named ‘new variant’ CJD, although it was clear that they were also rather atypical in their clinical presentation; in fact, most cases did not meet

HUMAN PRION DISEASES

795

Figure 26.5 Mutations and polymorphisms in the human prion protein gene (PRNP)

the accepted clinical diagnostic criteria for probable CJD. Extensive studies of archival cases of CJD or other prion diseases failed to show this picture and it seemed that it did represent the arrival of a new form of prion disease in the UK. The statistical probability of such cases occurring by chance was vanishingly small and ascertainment bias seemed most unlikely as an explanation. It was clear that a new risk factor for CJD had emerged and appeared to be specific to the UK. The UK Government advisory committee on spongiform encephalopathy (SEAC) concluded that, while there was no direct evidence for a link with BSE, exposure to specified bovine offal (SBO), prior to the ban on its inclusion in human foodstuffs in 1989, was the most likely explanation. A case of vCJD was soon afterwards reported in France (Chazot et al., 1996). Direct experimental evidence that vCJD is caused by BSE was provided by molecular analysis of human prion strains and transmission studies in transgenic and wild-type mice. While it is now clear that vCJD is human BSE, it is unclear why this particular age group should be affected and why none of these cases had a pattern of unusual occupational or dietary exposure to BSE. However, very little is known about which foodstuffs contained high-titre bovine offal. It is possible that certain foods containing particularly high titres were eaten predominately by younger people. An alternative possibility is that young people are more susceptible to BSE following dietary exposure, or that they have shorter incubation periods. It is important to appreciate that BSE-contaminated feed was fed to sheep, pigs and poultry, and that although there is no evidence of natural transmission to these species, it would be prudent to remain open- minded about other dietary exposure to novel animal prions.

The clinical presentation is often, although not always, with behavioural and psychiatric disturbances, and in some cases with sensory disturbance (Will et al., 1996). Initial presentation may be to a psychiatrist and the most prominent feature is depression, but anxiety, withdrawal and behavioural change is also frequent (Zeidler et al., 1997a). Suicidal ideation is infrequent and response to antidepressants poor. Delusions, which are complex and unsustained, are common. Other features include emotional lability, aggression, insomnia and auditory and visual hallucinations. A prominent early feature in some patients is dysaesthe- siae or pain in the limbs or face, or pain that is persistent rather than intermittent and unrelated to anxiety levels. A few cases have been noted to have forgetfulness or mild gait ataxia from an early stage but in most overt neurological features are not

apparent until some months into the clinical course (Zeidler et al., 1997b). In the majority of patients a progressive cerebellar syndrome develops with gait and limb ataxia. Dementia usually develops later in the clinical course with progression to akinetic mutism. Myoclonus is seen in most patients, and chorea, sometimes severe, is seen in some cases. Cortical blindness develops in a minority of patients in the late stages of disease. Upgaze paresis, an uncommon feature of classical CJD, has been noted in some patients (Zeidler et al., 1997b). The age at onset, although initially in young adults, has since broa- dened, with a range of 12–74 years (mean 29 years) and the clinical course is relatively prolonged (9–35 months, median 14 months) compared to sporadic CJD. The EEG is abnormal, most frequently showing generalised slow wave activity, but without the pseudoperiodic pattern seen in most sporadic CJD cases. Neuroimaging by CT is either normal or shows only mild atrophy. MRI scanning has proved helpful in diagnosis of vCJD, with high signal in the pulvinar (the ‘pulvinar sign’; Zeidler et al., 2000) (Figure 26.6). Although clearly of value, and reported to be present on scrutiny of scans from a high proportion

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

Figure 26.6 MRI signs in human prion disease: pulvinar sign in vCJD

HUMAN PRION DISEASES

Figure 26.7 Neuropathology of sporadic and variant CJD. Mild spongiform vacuolation of the cortical grey matter is a hallmark of Creutzfeldt–Jakob disease (A). In this case there was no deposition of kuru-like plaques but widespread deposition of abnormal prion protein in a synaptic pattern (B). In contrast, variant CJD is characterised by frequent so-called ﬂorid plaque, consisting of a round amyloid core, surrounded by a daisy-like vacuolation (C). This hallmark of variant CJD can be found in several cortical areas and in the cerebellum and may be associated with vacuolar degeneration. Immunostaining for the pathological prion protein (using monoclonal antibody KG9) highlights the ﬂorid plaques but also reveals synaptic staining (D). Scale bar: 50 mm

of patients meeting clinical or neuropathological amyloid plaques in cerebral and cerebellar cortex. criteria for diagnosis of vCJD, its speciﬁcity and

These consisted of kuru-like, ‘ﬂorid’ (surrounded by sensitivity as a diagnostic sign have not been

spongiform vacuoles) and multicentric plaque types. formally evaluated in unselected series. CSF 14–3–

The ‘ﬂorid’ plaques, seen previously only in scrapie,

3 protein may be elevated, but much less frequently were a particularly unusual but highly consistent than in sporadic CJD. PRNP analysis to date has

feature. There was also abundant pericellular PrP demonstrated that all cases studied were homozy-

deposition in the cerebral and cerebellar cortex. A gous for methionine at codon 129. No known or

further highly unusual feature was the extensive PrP novel pathogenic mutations are found in the coding

deposition in the molecular layer of the cerebellum. sequence (Collinge et al., 1996a).

The ﬂorid plaque pathology of vCJD has been The neuropathological appearances of vCJD are

reproduced in an animal model of vCJD: transgenic striking and consistent (Figure 26.7). While there is

mice expressing human PrP methionine 129, but not widespread spongiform change, gliosis and neuronal

mice expressing valine 129, develop strikingly similar loss, most severe in the basal ganglia and thalamus, the

neuropathology when infected with either BSE or most remarkable feature was the abundant PrP

vCJD prions (Asante et al., 2002).

Importantly, a clear tissue diagnosis of vCJD can now be made by tonsil biopsy with detection of characteristic PrP immunostaining and PrP Sc (Collinge et al. , 1997; Wadsworth et al., 2001). It has long been recognised that prion replication, in experimentally infected animals, is ﬁrst detectable in the lymphoreticular system, considerably earlier than the onset of neurological symptoms. Importantly, PrP Sc is only detectable in vCJD, and not other forms of human prion disease studied. The PrP Sc type detected on Western blot in vCJD tonsil has a characteristic pattern designated type 4t(45) (Figure 26.8). A positive tonsil biopsy obviates the need for brain biopsy, which may otherwise be considered in such a clinical context to exclude alternative, potentially treatable diagnoses.

It is of considerable interest that some of the features of vCJD are reminiscent of kuru, in which behavioural changes and progressive ataxia predominate. In addition, peripheral sensory disturbances are well recognised in the kuru prodrome. Kuru plaques are seen in around 70% of cases and are especially abundant in younger kuru cases. The observation that iatrogenic prion disease related to peripheral exposure to human prions has a more kuru-like than CJD-like clinical picture may well be relevant and would be consistent with a peripheral prion exposure.

Other Phenotypes of BSE Infection in Humans? The relatively stereotyped clinical presentation and

neuropathology of vCJD contrasts sharply with sporadic CJD. This may be because vCJD is caused by a single prion strain and may also suggest that a relatively homogeneous genetically susceptible subgroup of the population with short incubation periods to BSE has been selected to date (Collinge, 1999). A widening of the recognised phenotypic range of vCJD from that based on the earliest patients can be anticipated and indeed is already emerging. Other phenotypic presentations of BSE prion infection in humans, particularly involving other PRNP genotypes, are to be anticipated (Hill et al., 1997a). It will be important to remain open-minded about such phenotypes. Recent studies in transgenic mice expressing human PrP have shown that BSE infection can result in two distinct phenotypes, one with the neuropathological and molecular phenotype of vCJD and a second with the molecular phenotype of the commonest subtype of sporadic CJD (associated with PrP Sc type 2) (Asante et al., 2002). This raises the possibility that BSE infection of humans could also cause some cases of apparently sporadic CJD.

Inherited Prion Diseases

Gerstmann–Stra¨ussler–Scheinker Disease The ﬁrst case was described by Gerstmann in 1928 and

was followed by a more detailed report on seven other aﬀected members of the same family in 1936 (Gerstmann et al., 1936). The classical presentation of GSS is with a chronic cerebellar ataxia accompanied by pyramidal features, with dementia occurring later in

a much more prolonged clinical course than that seen in CJD. The mean duration is around 5 years, with onset usually in either the third or fourth decades. Histologically, the hallmark is the presence of multicentric amyloid plaques. Spongiform change, neuronal loss, astrocytosis and white matter loss are also usually present. Numerous GSS kindreds from several countries (including the original Austrian family described by Gerstmann et al., 1936) have now been demonstrated to have mutations in the PrP gene. GSS is an autosomal dominant disorder, which can now be classiﬁed within the spectrum of inherited prion disease.

Other Inherited Prion Diseases The identiﬁcation of one of the pathogenic PrP gene

mutations in a case with neurodegenerative disease allows not only molecular diagnosis of an inherited prion disease but also its sub-classiﬁcation according to mutation (see Figure 26.8). Pathogenic mutations reported to date in the human PrP gene consist of two groups: (a) point mutations within the coding sequence, resulting in amino acid substitutions in PrP or, in two cases, production of a stop codon resulting in expression of a truncated PrP; (b) insertions encoding additional integral copies of an octapeptide

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

Figure 26.8 Tonsil biopsy in vCJD

799 repeat present in a tandem array of ﬁve copies in the

HUMAN PRION DISEASES

clinical duration from onset to the development of normal protein. A suggested notation for these

akinetic mutism was around 5 years. There was no diseases is ‘inherited prion disease (PrP mutation)’,

periodic synchronous discharge on EEG but MRI

e.g. inherited prion disease (PrP 144 bp insertion) or scans showed atrophy of the motor cortex. On inherited prion disease (PrP P102L) (Collinge and

pathological examination there were plaques in the Prusiner, 1992). They are all autosomal dominantly

cerebral cortex, and neuronal loss but no spongiosis. inherited conditions. Kindreds with inherited prion

Neuroﬁbrillary tangles were variably present amongst disease have been described with phenotypes of

cases and no plaques were found in the cerebellum. classical CJD, GSS and also with a range of other neurodegenerative syndromes. Some families show

PrP A117V . This mutation was ﬁrst described in a remarkable phenotypic variability which can encom-

French family (Doh ura et al., 1989) and subsequently pass both CJD- and GSS-like cases, as well as other

in a US family of German origin (Hsiao et al., 1991b). cases which do not conform to either CJD or GSS

The clinical features are presenile dementia associated phenotypes (Collinge et al., 1992). Such atypical prion

with pyramidal signs, parkinsonism, pseudobulbar diseases may lack the classical histological features of a

features and cerebellar signs. Neuropathologically, spongiform encephalopathy entirely, although PrP

PrP immunoreactive plaques are usually present. This immunohistochemistry is usually positive (Collinge et

mutation has also been identiﬁed in a large family in al. , 1990). Progressive dementia, cerebellar ataxia,

the UK.

pyramidal signs, chorea, myoclonus, extrapyramidal features, pseudobulbar signs, seizures and amyo-

PrP Y145STOP . This mutation was detected in a trophic features are seen in variable combinations.

Japanese patient who had a clinical diagnosis of Relatively little clinical or pathological information

Alzheimer’s disease. She developed memory distur- is available on some of these mutations; indeed, some

bance and a slowly progressive dementia at age 38. The have to date been described in only a single family, or

duration of illness was 21 years. Histological examina- indeed individual, and evidence for pathogenicity is

tion revealed typical Alzheimer pathology without unclear. Brief details on some of the more frequently

spongiform change (Kitamoto et al., 1993). Many recognised subtypes is given below.

amyloid plaques were seen in the cortex, along with diﬀuse neuropil threads of paired helical ﬁlaments. However, the plaques were immunoreactive with PrP antisera. A4 immunocytochemistry was negative. The

Missense Mutations clinicopathological ﬁndings in this case emphasise the importance of PRNP analysis in the diﬀerential

PrP P102L . This mutation was ﬁrst reported in 1989

diagnosis of dementias.

in a UK and US family and has now been demonstrated in many other kindreds worldwide. Progressive

PrP D178N . ataxia is the dominant clinical feature, with dementia This mutation was originally described in two Finnish families with a CJD-like phenotype,

and pyramidal features. However, marked variability although without typical EEG appearances (Goldfarb at both clinical and neuropathological level is apparent

et al. , 1991c), and has since been demonstrated in in some families, and has recently been extensively

additional CJD families in Hungary, The Netherlands, documented in the original Austrian GSS family

Canada, Finland, France and the UK. The Finnish (Hainfellner et al., 1995). A family with marked

pedigree included 15 aﬀected members in four genera- amyotrophic features has also been reported

tions. The mean age of onset was 47 and mean (Kretzschmar et al., 1992). Cases with severe dementia

duration was 27.5 months. Brain biopsy and autopsy in the absence of prominent ataxia are also recognised.

specimens showed spongiform change without amyloid Histological examination reveals PrP immunoreactive

plaques.

plaques in the majority of cases. Transmissibility to This mutation was also reported in two unrelated experimental animals has been demonstrated.

families with fatal familial insomnia (FFI) (Lugaresi et al. , 1986; Medori et al., 1992a). The ﬁrst cases

PrP P105L . The Pro–Leu change at codon 105 has described had a rapidly progressive disease charac- been found in four patients from three Japanese

untreatable insomnia, families (Kitamoto et al., 1993) and in a single UK

dysautonomia and motor signs, and neuropathologi- family (unpublished). The patients presented with a

cally by selective atrophy of the anteriorventral and history of spastic paraparesis and dementia. The

mediodorsal thalamic nuclei. There is marked thalamic

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

astrocytosis. Mild spongiform change is seen in some diﬀerent alleles. This disease has not been transmitted cases and protease-resistant PrP can be demonstrated,

to laboratory animals.

albeit weakly, by immunoblotting. Proteinase-K treatment of extracted PrP Sc from FFI cases has shown a

PrP T183A . Reported in a single Brazilian family diﬀerent sized PrP band on Western blots than PrP Sc

with a frontotemporal dementia of mean onset 45 from CJD cases (Monari et al., 1994), suggesting that

years and duration 4 years (Nitrini et al., 1997). FFI may be caused by a distinct prion strain type. In a

Parkinsonian features were also present in some recent study, Goldfarb et al., 1992, reported that in all

patients. Neuropathological examination revealed se- the codon 178 families they studied with a CJD-like

vere spongiform change and neuronal loss in deep disease, the codon 178 mutation was encoded on a

cortical layers and putamen while there was relatively valine 129 allele while all FFI kindreds encode the

little gliosis. PrP immunoreactivity was demonstrated same codon 178 mutation on a methionine 129 allele.

in putamen and cerebellum. No transmission studies They suggested that the genotype at codon 129

have been reported to date.

determines phenotype. However, they have not demonstrated that the families they describe are

A variant form of GSS was described in unrelated and that therefore their comparison may

PrP F198S .

a large Indiana kindred which has been traced back to only be based on two extended families. Insomnia is

1792. Unlike other GSS patients with presenile onset not uncommon in CJD patients and FFI and CJD may

of neurological disability, the Indiana kindred had represent extremes of a spectrum of related disease

widespread Alzheimer-like neuroﬁbrillary tangles, phenotypes. Recently an inherited case with the E200K

composed of paired helical ﬁlaments, in the cortex mutation, which is normally associated with a CJD-

and subcortical nuclei, in addition to amyloid plaques. like phenotype, has been reported with an FFI

The amyloid plaques were composed of PrP and not phenotype (Chapman et al., 1996). An Australian

b A4. Aﬀected individuals in this kindred have a codon family has also been reported with the FFI genotype

198 T–C transition, resulting in a phenylalanine to but in which aﬀected family members have a range of

serine conversion (Dlouhy et al., 1992). There is an phenotypes encompassing typical CJD, FFI and an

apparent codon 129 eﬀect with this mutation, in that autosomal dominant cerebellar ataxia-like illness

individuals who were heterozygous at codon 129 had a (McLean et al., 1997). It is of interest that the CJD-

later age of onset than homozygotes. Transmission of like codon 178 cases have frequently transmitted to

this disease to laboratory animals has not yet been experimental animals, while the FFI type did not

reported.

transmit to laboratory primates (Brown et al., 1994). Recently, transmission of an FFI case to mice has been

PrP E200K . This mutation was ﬁrst described in reported, although this case was unusual in that a

families with CJD. Aﬀected individuals develop a single octapeptide repeat deletion was present on the

rapidly progressive dementia with myoclonus and same allele (Tateishi et al., 1995). This individual came

pyramidal, cerebellar or extrapyramidal signs and a from an extensive kindred in which other family

duration of illness usually less than 12 months. The members, with the same PRNP genotype, had a

average age of onset for the disease is 55. Histologi- CJD-like phenotype (Bosque et al., 1992). However,

cally, these patients are typical of CJD; plaques are two cases of FFI, one a British case and the second an

absent but PrP Sc can be demonstrated by immunoblot- Italian case, both with the usual FFI genotype of

ting. In marked contrast to other variants of inherited D178N, 129M, transmitted to transgenic mice expres-

prion disease, the EEG usually shows the characteristic sing human prion protein (Collinge et al., 1995a).

pseudoperiodic sharp wave activity seen in sporadic CJD. Interestingly, this mutation accounts for the three reported ethnogeographic clusters of CJD where

PrP V180I . This mutation was identiﬁed in two the local incidence of CJD is around 100-fold higher Japanese patients with subacute dementia and myo-

than elsewhere (amongst Libyan Jews and in regions of clonus (Kitamoto et al., 1993). The period from onset

Slovakia and Chile) (Hsiao et al., 1991a; Brown et al., to akinetic mutism was 6–10 months. No family

1992; Goldfarb et al., 1990). Now that cases can be history was noted. EEG did not show pseudoperiodic

diagnosed by PrP gene analysis, atypical forms of this sharp wave activity. Neuropathological examination

condition are being detected with phenotypes other demonstrated spongiform change, neuronal loss and

than that of classical CJD. Of interest also are reports astrocytosis. Interestingly, one of the patients with PrP

that peripheral neuropathy can occur in this disease Ile 180 also had PrP Arg 232 (see later). These were on

(Neufeld et al., 1992). Elderly unaﬀected carriers of the

801 mutation have been reported. Chapman et al. (1994)

HUMAN PRION DISEASES

Insertional Mutations

have made a detailed analysis on 52 mutation-carrying patients with deﬁnite or probable CJD and 34

A single unaﬀected mutation carriers. They conclude that the

PrP 24 bp insertion (one extra repeat) .

octapeptide repeat insertion has been reported in a cumulative penetrance reaches 50% at the age of 60

single French individual who presented at age 73 with and 80% by the age of 80. However, there was a group

dizziness. He later developed visual agnosia, cerebellar of patients, aged 69–82, with possible CJD containing

ataxia and intellectual impairment and diﬀuse periodic ﬁve proven and two obligate carriers of the mutation,

activity was noted on EEG. Myoclonus and cortical

i.e. the patients were demented but did not fulﬁl the blindness developed and he progressed to akinetic clinical criteria for probable CJD. If the analysis was

mutism. Disease duration was 4 months. The patient’s carried out assuming that these possible cases were

father had died at age 70 from an undiagnosed actually CJD, then the penetrance reaches 100% by the

neurological disorder. No neuropathological informa- age of 80. Individuals homozygous for the mutation

tion is available.

have been identiﬁed and are phenotypically indistinguishable from heterozygotes, indicating that this condition is a fully dominant disorder (Hsiao et al.,

PrP 48 bp insertion (two extra repeats) . This 1991a). Patients with this condition have now been

mutation has been reported in a single family from reported in several other countries outside the well-

the USA (Goldfarb et al., 1993). The proband had a recognised clusters, including the UK. At least one of

CJD-like phenotype, both clinically and pathologi- the UK cases does not appear to be related to the

cally, with a typical EEG and an age at onset of 58. ethnogeographic clusters mentioned above, suggesting

However, the proband’s mother had onset of cognitive

a separate UK focus for this type of inherited prion decline at age 75 with a slow progression to a severe disease (Collinge et al., 1993). Goldfarb et al. (1991b)

dementia over 13 years. The maternal grandfather had have found this mutation amongst 46 out of 55 CJD-

a similar late onset (at age 80) and slowly progressive aﬀected families studied at the National Institutes of

cognitive decline over 15 years. Health. The codon 129 genotype does not appear to

aﬀect age at onset of this disorder. Transmission to PrP 96 bp insertion (four extra repeats) .

A 96 base experimental animals has been demonstrated.

pair insertional mutation, encoding four octapeptide elements, was ﬁrst reported in an individual who died aged 63 of hepatic cirrhosis (Goldfarb et al., 1991a).

PrP Q217R . Reported to date only in a single There was no history of neurological illness and it is Swedish family, the presentation is with dementia

unclear whether this ﬁnding indicates incomplete followed by gait ataxia, dysphagia and confusion

penetrance of this mutation. This is the only recorded (Hsiao et al., 1992). As with the inherited prion disease

case of a PRNP insertional mutation other than in an with codon 198 serine mutation (Indiana kindred),

aﬀected individual with a prion disease or an at-risk there are prominent neuroﬁbrillary tangles. Transmis-

individual from an aﬀected kindred. Two separate four sibility to experimental animals has not yet been

octapeptide repeat insertional mutations have been demonstrated in this condition.

reported in aﬀected individuals, each diﬀering in the DNA sequence from the original four-repeat insertion, although all three of the mutations encode the same

PrP M232R . This mutation was ﬁrst found on the PrP. Laplanche et al. (1995) reported a 96 bp insertion opposite allele to a codon 180 mutation in a Japanese

in an 82 year-old French woman who developed patient with prion disease (Kitamoto et al., 1993). It

progressive depression and behavioural changes. She was further demonstrated in two additional Japanese

progressed over 3 months to akinetic mutism with patients with dementia. Both of the latter cases

pyramidal signs and myoclonus. EEG showed pseu- appeared to present as sporadic cases with no family

doperiodic complexes. Duration of illness was 4 history of neurological disease. Both patients had

months. There was no known family history of progressive dementia, myoclonus and periodic syn-

neurological illness. Another 96 bp insertional muta- chronous discharges in the EEG. The mean duration

tion was seen in a patient with classical clinical and of illness was 3 months. Neuropathology showed

pathological features of CJD, with the exception of the spongiform change, neuronal loss and astrocytosis.

unusual ﬁnding of pronounced PrP immunoreactivity PrP immunostaining revealed diﬀuse grey matter

in the molecular layer of the cerebellum (Campbell et staining, but no plaques.

al. , 1996).

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

PrP 120 bp insertion (five extra repeats) . An nine 129 PrP allele, there are two possible codon 129 additional ﬁve octapeptide repeat mutation was

genotypes for aﬀected individuals, methionine 129 reported in a US family with an illness characterised

homozygotes or methionine 129/valine 129 heterozy- by progressive dementia, abnormal behaviour, cere-

gotes. Heterozygotes have an age at onset which is bellar signs, tremor, rigidity, hyper-reﬂexia and myo-

about a decade later than homozygotes (Poulter et al., clonus. The age at onset was 31–45, with a clinical

1992). Limited transmission studies to marmosets were duration of 5–15 years (Goldfarb et al., 1991a). EEG

unsuccessful. Transmission to transgenic mice expres- showed diﬀuse slowing only. Histological features were

sing human prion protein has been achieved (Collinge of spongiosis, neuronal loss and gliosis. Transmission

et al. , in preparation). Further families with 144 bp has been demonstrated.

insertions, of diﬀerent nucleotide sequence, have now been reported in the UK (Nicholl et al., 1995) and Japan (Oda et al., 1995).

PrP 144 bp insertion (six extra repeats) . This was the ﬁrst PrP mutation to be reported and was found in

PrP 168 bp insertion (seven extra repeats) . This

a small UK family with familial CJD (Owen et al., mutation has been reported in a US family. The 1989). The diagnosis in the family had been based on

clinical features described include mood change, an individual who died in the 1940s with a rapidly

abnormal behaviour, confusion, aphasia, cerebellar progressive illness characteristic of CJD (Meyer et al.,

signs, involuntary movements, rigidity, dementia and 1954). The reported duration of illness was 6 months.

myoclonus. The age at onset was 23–35 years and the Pathologically there was gross status spongiosis and

clinical duration 10–413 years. EEG showed diﬀuse astrocytosis aﬀecting the entire cerebral cortex, and

slowing in two cases; a third showed slow wave burst this case is used to illustrate classic CJD histology in

suppression. Neuropathological examination showed Greenﬁeld’s Neuropathology. However, other family

spongiform change, neuronal loss and gliosis to members had a much longer-duration GSS-like illness.

varying degrees (Goldfarb et al., 1991a). Experimental Histological features were also extremely variable. This

transmission has been demonstrated. observation led to screening of various cases of neurodegenerative disease and to the identiﬁcation of

PrP 192 bp insertion (eight extra repeats) . This

a case classiﬁed on clinical grounds as familial mutation has been reported in a French family, with Alzheimer’s disease (Collinge et al., 1989). More

clinical features which include abnormal behaviour, extensive screening work identiﬁed further families

cerebellar signs, mutism, pyramidal signs, myoclonus, with the same mutation which were then demonstrated

tremor, intellectual slowing and seizures. The disease by genealogical studies to form part of an extremely

duration ranged from 3 months to 13 years. The large kindred (Collinge et al., 1992; Poulter et al.,

EEG ﬁndings include diﬀuse slowing, slow wave 1992). Clinical information has been collected on

burst suppression and periodic triphasic complexes. around 50 aﬀected individuals over seven generations.

Neuropathological examination revealed spongiform Aﬀected individuals develop, in the third to fourth

change, neuronal loss, gliosis and multicentric decade, onset of a progressive dementia associated

plaques in the cerebellum (Goldfarb et al., 1991a; with a varying combination of cerebellar ataxia and

Guiroy et al., 1993). Experimental transmission has dysarthria, pyramidal signs, myoclonus and occasion-

been reported.

ally extrapyramidal signs, chorea and seizures. The dementia is often preceded by depression and aggres-

PrP 216 bp insertion (nine extra repeats) . The sive behaviour. A number of cases have a long-

ﬁnding of a nine-octapeptide insertional mutation was standing personality disorder, characterised by aggres-

ﬁrst reported in a single case from the UK (Owen et al., sion, irritability, antisocial and criminal activity and

1992). The clinical onset was around 54 years, with hypersexuality, which may be present from early

falls, axial rigidity, myoclonic jerks and progressive childhood, long before overt neurodegenerative disease

dementia (Tagliavini et al., 1993a). Although there was develops. The histological features vary from those of

no clear family history of a similar illness, the mother classical spongiform encephalopathy (with or without

had died at age 53 with a cerebrovascular event. The PrP amyloid plaques) to cases lacking any speciﬁc

maternal grandmother died at age 79 with senile features of these conditions (Collinge et al., 1990). Age

dementia. EEG was of low amplitude but did not show at onset in this condition can be predicted according to

pseudoperiodic sharp wave activity. Neuropathologi- genotype at polymorphic codon 129. Since this

cal examination showed no spongiform encephalopa- pathogenic insertional mutation occurs on a methio-

thy but marked deposition of plaques, which in the thy but marked deposition of plaques, which in the

MOLECULAR DIAGNOSIS OF PRION DISEASE While sporadic CJD can often, following the exclusion

of other causes, be diagnosed with a high degree of conﬁdence on the basis of clinical criteria, atypical forms, which present much greater diagnostic diﬃ- culty, are not uncommon (Collinge, 1998). A widening of the recognised phenotypic range of vCJD from that based on the earliest patients can be anticipated and indeed is already emerging. Other phenotypic presentations of BSE prion infection in humans, particularly involving other PRNP genotypes, are to

be anticipated. Furthermore, current clinically-based diagnostic criteria for vCJD, used for surveillance, require the evolution of disease over at least 6 months and the development of several signs indicative of extensive cerebral damage (http://www.doh.gov.uk/ cjd/cjd_stat.htm). However, early diagnosis, before extensive irreversible brain damage has occurred, is crucial in such patients as they may be suffering from an alternative treatable disorder. Brain biopsy may well be considered, particularly in younger patients, to exclude such conditions as cerebral vasculitis. Early tonsil biopsy, if positive, obviates the need for further investigation. The need for early, specific diagnosis is now further emphasised by the arrival of potential therapies and clinical trials for CJD. The early clinical features of vCJD—depression, anxiety, behavioural change and sensory disturbances—are highly non- specific. Differentiation from much commoner psychiatric causes requires the arrival of overtly neurological features, such as ataxia, chorea and cognitive decline, although pre-existing use of neuro- leptics and other psychotropic drugs may initially delay their diagnostic recognition. While the diagnostic accuracy provided by a tonsil biopsy has to be balanced against the fact that it is an invasive procedure, early referral for investigation should allow much earlier diagnosis and access to clinical trials before extensive functional loss has occurred.

While neurologists have until recent years had to rely largely on clinical features to differentiate neurodegenerative disorders, the major advances in molecular genetics and in understanding molecular pathogenesis increasingly enable diagnosis using criteria higher in the diagnostic hierarchy of pathology. Around 15% of recognised prion disease is an inherited Mendelian disorder associated with one of the more than 30 recognised coding mutations in PRNP (Collinge, 2001). For a single-gene inherited disorder of high penetrance, such as inherited prion disease, the diagnostic supremacy of direct demonstration of causative mutation by DNA analysis is clear. Indeed, the availability of such definitive diagnostic markers has long allowed diagnosis of inherited prion disease in patients not only atypical on clinical grounds, but in whom classical neuropathological features are absent (Collinge et al., 1989, 1990). Kindreds are documented in which some individuals have the classical syndromes of ‘CJD’ and ‘Gerst- mann–Straüssler–Sheinker disease’, while others do not fit these rubrics at all (Collinge et al., 1992). Neuropathology in such patients is no longer the ‘Gold Standard’: rather, the recognised clinicopathological manifestation of a particular inherited condition simply widens. The acquired prion diseases, such as vCJD, although not contagious in humans, are infectious diseases. In infectious disease, while clinical and histopathological features may again be key, confirmation of diagnosis, not least in life-threatening conditions, is by identification of the infectious pathogen itself or a specific immune response to it. Isolation and strain typing of the pathogen is at the apex of the diagnostic hierarchy. Strain typing in particular may allow the source of an outbreak to be identified and the best available prognostic and therapeutic advice to be provided. While it is essential to balance the potential risks and discomfort involved in an invasive diagnostic test against the improved diagnostic accuracy, it will only be by progressing steadily to greater use of molecular analysis of neurological disease that we will be able to deliver the diagnostic and ultimately therapeutic advances to patients with neurodegenerative diseases that are so desperately needed.

PRESYMPTOMATIC AND ANTENATAL TESTING

Since a direct gene test has become available, it has been possible to provide an unequivocal diagnosis in

HUMAN PRION DISEASES

803

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

patients with inherited forms of the disease. This has from patients with classical CJD are infrequent and also led to the possibility of performing presympto-

have been questioned (Brown, 1995). Infectivity of matic testing of unaﬀected but at-risk family members,

blood from patients in the clinical phase of vCJD is as well as antenatal testing (Collinge et al., 1991a).

unknown. UK policy is now to leukodeplete all whole Because of the eﬀect of PRNP codon 129 genotype on

blood, a practice already in use (for other health the age of onset of disease associated with some

reasons) in some countries, and to source plasma for mutations, it is possible to determine within a family

plasma products from outside the UK. whether a carrier of a mutation will have an early or

A further possible route of transmission of vCJD is late onset of disease. Most of the mutations appear to

via contaminated surgical instruments. Prions resist

be fully penetrant; however, experience with some is conventional sterilisation methods and neurosurgical extremely limited. In families with the E200K mutation

instruments are known to be able to act as vectors for and in D178N (fatal familial insomnia) there are

prion transmission: several cases of iatrogenic trans- examples of elderly unaﬀected gene carriers who

mission of sporadic CJD prions via neurosurgical appear to have escaped the disease.

instruments are documented (Bernoulli et al., 1977; Genetic counselling in prion disease resembles that

Blattler, 2002). Recent evidence suggests that classical of Huntington’s disease in many respects and those

CJD may also be transmitted by other surgical protocols established for Huntington’s disease can be

procedures (Collins et al., 1999). In the UK, all adapted for prion disease counselling. PrP gene

surgical instruments used on patients with suspected analysis may have very important consequences for

CJD are quarantined and not re-used unless an family members other than the individual tested, and it

alternative non-prion diagnosis is unequivocally con- is preferable to have discussed all the issues with the

ﬁrmed.

family before testing commences. Following the The pathogenesis of vCJD diﬀers sharply from that identiﬁcation of a mutation, the family should be

of sporadic and other forms of ‘classical’ CJD. In referred for genetic counselling. Testing of asympto-

particular, in vCJD there is extensive involvement of matic individuals should only follow adequate

the lymphoreticular system (LRS) (lymph nodes, tonsil counselling of individuals and will require their full

and spleen), with the highest levels of PrP Sc outside the informed consent. It is also important to counsel both

CNS being found in tonsil, where levels are typically those testing positive for mutations and those untested

5–10% of brain levels (Wadsworth et al., 2001). PrP Sc but at-risk that they should not be blood donors and

is also detectable, at lower levels, in thymus, rectum, should inform surgeons, including dentists, of their

adrenal and retina in vCJD (Wadsworth et al., 2001). risk status prior to any procedure, as precautions

This wider tissue distribution raises concerns about should be taken to minimise risk of iatrogenic

iatrogenic transmission of vCJD via surgical instru- transmission (see below).

ments previously used on patients with pre-clinical vCJD prion infection. The number of individuals incubating vCJD but currently asymptomatic is unknown, but may be substantial. Prions adhere

PREVENTION AND PUBLIC HEALTH avidly to stainless steel and transmit the disease readily MANAGEMENT

in experimental models (Flechsig et al., 2001). Tonsil- lar PrP Sc is readily detectable in all cases of vCJD

While prion diseases can be transmitted to experi- studied at autopsy and lymphoreticular involvement is mental animals by inoculation, it is important to

a very early feature of natural prion infection in sheep appreciate that they are not contagious in humans.

and in experimental scrapie models, where replication Documented case-to-case spread has only occurred by

in the LRS is detectable early in the incubation cannibalism (kuru) or following accidental inoculation

period and rises to a plateau, which considerably with prions. Such iatrogenic routes include the use of

precedes, and is maintained in, the clinical phase inadequately sterilised intracerebral electrodes, dura

(Fraser et al., 1992). This suggests that tonsillar mater and corneal grafting, and from the use of human

PrP Sc has probably been present for a considerable cadaveric pituitary-derived growth hormone or gona-

period, perhaps years, before clinical presentation of dotrophin.

vCJD in humans and therefore tonsil biopsy should Considerable concern has been expressed that blood

enable diagnosis at the earliest stages of clinical and blood products from asymptomatic donors

suspicion. In addition, this pathogenesis forms the incubating vCJD may pose a risk for the iatrogenic

basis of prevalence screening of the general popula- transmission of vCJD. Reports of infectivity of blood

tion for infection. Several anonymous screens of

805 tonsil and appendix tissues, removed during routine

HUMAN PRION DISEASES

PROGNOSIS AND TREATMENT surgery, are under way. Tonsil appears a more

sensitive reporter of vCJD prion infection than All forms of prion diseases that are currently appendix (Joiner et al., 2002), and it is a cause for

recognised are invariably fatal following a relentlessly concern that a positive appendix has already been

progressive course. No currently available treatment reported in these ongoing studies (Hilton et al.,

alters the clinical course of the disease and all that 2002). National-scale studies are now being orga-

can be oﬀered at present is general supportive care nised by the UK Department of Health. It is

for the patient and family with hospitalisation in the unknown as to whether there is signiﬁcant prionae-

later stages. The duration of illness in sporadic mia in vCJD. Blood is infectious in some rodent

patients is very short, with a mean duration of 3–4 scrapie models and, more recently in BSE-infected

months. However, in some of the inherited cases the sheep (which have been proposed as a model for

duration can be 20 years or more (Collinge et al., vCJD as the tissue distribution of infectivity is

similar), the disease has been transmitted by transfu- Various compounds, some known to bind PrP Sc , sion of whole blood (Hunter et al., 2002).

including Congo red (Ingrosso et al., 1995), polyene Certain occupational groups are at risk of exposure

antibiotics (Pocchiari et al., 1987), anthracycline to human prions, e.g. neurosurgeons and other

(Tagliavini et al., 1997), dextran sulphate, pentosan operating theatre staﬀ, pathologists and morticians,

polysulphate and other polyanions (Ehlers and Dirin- histology technicians, as well as an increasing number

ger, 1984; Farquhar and Dickinson, 1986; Kimberlin of laboratory workers. Because of the prolonged

and Walker, 1986) and b-sheet breaker peptides (Soto incubation periods to prions following administration

et al. , 2000) have been shown to have limited eﬀects in to sites other than the central nervous system (CNS),

animal models of prion disease. Unfortunately, most which is associated with clinically silent prion replica-

show a signiﬁcant eﬀect only if administered long tion in the lymphoreticular tissue (Aguzzi, 1997),

before clinical onset (in some cases with the inoculum) treatments inhibiting prion replication in lymphoid

and/or are impractical treatments due to toxicity or organs may represent a viable strategy for rational

bioavailability. Quinacrine, an agent formerly used secondary prophylaxis after accidental exposure. A

widely as an antimalarial agent, has been shown to preliminary suggested regimen is a short course of

block PrP Sc accumulation in scrapie-infected neuro- immunosuppression with oral corticosteroids in indi-

blastoma cells at concentrations that might be viduals with signiﬁcant accidental exposure to human

achievable clinically (Korth et al., 2001). Clinical trials prions. Urgent surgical excision of the inoculum might

are under way or planned in several countries to also be considered in exceptional circumstances. There

evaluate any eﬀect of this drug on disease progression. is hope that progress in the understanding of the

The prion diseases are now amongst the best peripheral pathogenesis will identify the precise cell

understood of the degenerative brain diseases and the types and molecules involved in colonisation of the

development of rational treatments is appearing organism by prions. The ultimate goal will be to target

realistic. Recently, two anti-PrP monoclonal antibo- the rate-limiting steps in prion spread with much more

dies have been shown to protect mice indeﬁnitely from focused pharmacological approaches, which may

developing prion disease when infected by the intra- eventually prove useful in preventing disease even

peritoneal route (White et al., 2003). It is not yet after iatrogenic and alimentary exposure (Collinge and

known whether this approach can interfere with CNS Hawke, 1998).

disease, which would in any case require infusion of Subclinical prion infections have been described in

antibody into the CSF, but these data provide a proof experimental animals, where high prion levels (com-

of principal for antibody-based prion therapeutics and parable to those in end-stage clinically aﬀected

the possibility of secondary prevention by passive animals) are present in animals living a normal

immunisation.

lifespan. While the diﬀerentiation between pre- and The precise molecular events which bring about the sub-clinical infections is perhaps semantic in diseases

conversion of PrP C to PrP Sc and the molecular nature where the incubation period (at least when crossing

of the neurotoxic species remain ill-deﬁned, a fact species barriers) can approach the natural lifespan of

which at ﬁrst sight would preclude screening for the species concerned. Nevertheless, the possibility that

compounds which inhibit the process. However, any asymptomatic carrier states of natural human and

ligand which selectively stabilises the PrP C state will animal prion infection occurs must be considered

prevent its rearrangement and might reasonably be (Asante et al., 2002; Hill et al., 2000).

expected to block prion replication (and presumably

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

production of any putative toxic intermediate forms of Aguzzi A (1997) Neuro-immune connection in spread of PrP on the pathway to PrP Sc formation). Such an

prions in the body. Lancet, 349, 742–743. approach has recently been applied to block p53

Alper T, Cramp WA, Haig DA and Clarke MC (1967) Does conformational rearrangements which are involved in the agent of scrapie replicate without nucleic acid?, Nature,

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small size of the scrapie agent. Biochem Biophys Res disease to provide eﬀective intervention before exten-

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Prion diseases appear to be diseases of protein messengers meet. Proc Natl Acad Sci USA, 90, 10909– 10913.

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mission dynamics and epidemiology of BSE in British progress with tackling key public health issues posed

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by vCJD, be of wider signiﬁcance in pathobiology. Arnold JE, Tipler C, Laszlo L, et al. (1995) The abnormal Common themes are emerging in neurodegenerative

isoform of the prion protein accumulates in late-endosome- like organelles in scrapie-infected mouse brain. J Pathol,

diseases, many of which are also associated with

aggregates of misfolded protein. Also, the apparent Asante EA, Linehan JM, Desbruslais M et al. (2002) BSE ability of a single polypeptide chain to encode

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Bell, JE, Gentleman, SM, Ironside, JW et al. (1997) Prion serve as the essential model by which the mechanism of

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Creutzfeldt–Jakob disease. Lancet, 345, 1609–1610.

27 GBV-C and TTV

Shigeo Hino

Department of Virology, Tottori University, Yonago, Japan

INTRODUCTION The discovery of the hepatitis A–E viruses has led to

the identification of most blood-borne hepatitis viruses. In an attempt to identify any remaining such viruses, researchers are still striving to find novel blood-borne hepatitis viruses. Toward the end of the last decade, newly established molecular biological techniques were used to find two candidate viruses, known as GBV-C and TTV, although the real impact of these viruses on liver disease is still unclear. Due to the lack of a suitable cell system in which to culture these viruses, their biology remains obscure. While GBV-C is closely related to the hepatitis C virus, a member of the Flavivirus group, TTV, is the first human virus to be found with a single-stranded circular DNA genome similar to that of chicken anaemia virus. The prevalence of GBV-C is 1–4% in the general population, while that of TTV is over 90%. Animals have been found to harbour similar viruses that were specific to each species. Although posttransfusion hepatitis continues to occur in countries that screen blood donors for hepatitis B and C, the incidence of post-transfusion hepatitis is not sufficiently high to correlate with the high prevalence of these viruses. This review attempts to summarise the current status of the related research.

The story of the GB virus (GBV) started in 1967 (Deinhardt et al., 1967). Elevation of serum transaminase levels was observed in all of four tamarins (Saguinus spp.) that had been inoculated with day 3 serum obtained from a surgeon (GB) who was suﬀering from acute hepatitis. The GB agent (GBV) that passed sequentially through the tamarins main-

tained its pathogenicity. For a long time after it was first observed, however, it was not easy to distinguish GBV from the enteric hepatitis A virus. In 1995 representational difference analysis was used to identify two distinct RNA viral sequences in the GB agent (Simons et al., 1995a, 1995b). Both are partially related to Hepatitis C virus (HCV). Nevertheless, the original GB serum was found to contain another related virus, namely, GBV-C (Simons et al., 1995a). An independent group discovered Hepatitis G virus (HGV) in the same cluster as GBV-C (Linnen et al., 1996). In contrast, 1997 saw differential display technology being used to identify directly TT virus (TTV) in the serum of a hepatitis patient without any marker for known hepatitis viruses (Nishizawa et al., 1997). Approximately half of the studies of these viruses have attempted to identify the pathogenicity specific to each. However, the real impact on liver disease of either of these two viruses remains uncertain. Since the prevalence of each virus in humans is significant, it is important to continue to study the true biology of these viruses.

HISTORY OF GBV-C

In 1967, Deinhardt et al. (1967) reported that four tamarins (Saguinus spp.) had been inoculated with an acute-phase serum sample obtained from a surgeon (whose initials were G.B.) suﬀering from acute hepatitis with jaundice. The serum transaminase levels were elevated in all four tamarins, starting at 14–53 days after the inoculation, with hyperbilirubinaemia observed in one. The agent (GBV) continued to be

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

hepatogenic in the tamarins after several passages. The necrosis in liver biopsies (Schaluder et al., 1995). Peak tamarins proved susceptible to the agent, but common

viraemia levels exceeded 10 9 copies/ml, after which marmosets (Callithrix jacchus) and baboons (Papio

viral clearance was achieved within 14–16 weeks. A spp.) were not (Parks et al., 1969a). In the early phases

strong protective immune response after the initial of the study, it was not easy to distinguish between

infection was suggested by the appearance of brief GBV and Hepatitis A virus even though Parks et al.

viraemia following a challenge by the same virus (1969a) suggested in 1969 that GBV is small, heat-

(Beames et al., 2000). While some tamarins are infected labile and ether-sensitive. They also suggested that the

persistently, the induction of chronic liver disease in virus is of tamarin origin, because some of the control

tamarins has not been reported. GBV-A does not animals exhibited similar symptoms, and the GBV-

cause a signiﬁcant elevation of alanine aminotransfer- associated disease was not transmitted easily between

ase (ALT), even in tamarins (Schaluder et al., 1995). tamarins reared in the same cage (Parks et al., 1969b).

The questions raised by the original experiments However, their conclusions remain controversial

performed by Deinhardt et al. (1967) remain unan- (Deinhardt et al., 1975). At the time, the presence of

swered. If GBV-C does not replicate in tamarins, why indigenous pathogens in tamarins made the results

did all the tamarins inoculated with the GB serum ambiguous (Parks et al., 1969b). In 1989, Karayiannis

develop hepatitis? If GBV-C can replicate in tamarins, et al. (1989) reported that only the serum and liver

why did it disappear from the GB agent after several extracts of an infected tamarin were infectious,

passages?

whereas the faeces were not. In 1995, almost two decades after the ﬁrst observation of GBV, representational diﬀerence analysis was used to identify two related RNA viral sequences in the

GENOME OF GBV GB agent, which came to be designated as GBV-A and

GBV-B (Simons et al., 1995a, 1995b). The genome of GBVs have genomes consisting of around 9.3610 3 both viruses was over 9 kb long and had a limited

nucleotides, and a single large open reading frame that sequence similarity to the isolates of human Hepatitis

encodes a precursor polyprotein of around 2850 amino

C virus (HCV) (Simons et al., 1995a). The original acids. The genome is organised much like that of HCV, serum of GB did not contain either virus, but instead

with genes to encode the structural and non-structural contained another related virus, called GBV-C

proteins located at the 5’ and 3’ ends, respectively (Simons et al., 1995a). Another group independently

(Figure 27.1) (Simons et al., 1995a, 1995b; Linnen et al., discovered a viral sequence and designated it the

1996; Muerhoﬀ et al., 1995). The core protein of GBV- ‘hepatitis G virus’ (HGV), which was closely related, if

B and GBV-C seems to be truncated, while that of not identical, to GBV-C (Linnen et al., 1996).

GBV-A is almost missing (Linnen et al., 1996; Muerh- oﬀ et al., 1995; Simons et al., 1996). It would be interesting to know whether they produce infectious

GB AGENT IN ANIMALS virions, either with or without a truncated core protein. The 5’-termini of GBV-A and GBV-C contain

GBV-A was found in tamarins (Saguinus labiatus), 5’ non-translated sequences that are consistent with an mystax (Saguinus mystax), owl monkeys (Aotus trivirgatus ) and common marmosets (Saimiri spp.), while GBV-B was found in tamarins (Leary et al., 1996). GBVs from diﬀerent species are divergent from each other and cluster genetically according to their original animal species, suggesting that GBV-A and GBV-B originated in New World monkeys. GBV-C was also found in chimpanzees (Pan troglodytes), which also cluster within the host species and which are divergent from human GBV-C (Adams et al., 1998).

The natural course of a GBV-B infection in tamarins has been investigated extensively. Transient hepatitis in

Figure 27.1 Genomic structures of GBV-A, GBV-B and tamarins caused by GBV-B was conﬁrmed by elevated

HCV. Untranslated regions at the 3’- and 5’-termini are not liver enzyme levels and by inﬂammation and focal

shown shown

The phylogenetic analysis of human GBV-Cs revealed that they could be clustered into ﬁve groups: genotype 1 (West Africa); genotype 2 (USA/Europe), genotype 3 (Asia), genotype 4 (south-east Asia) and genotype 5 (South Africa) (Tucker et al., 2000). The hypervariability of the HCV E2 protein is one of the main factors in maintaining chronic HCV infection. However, GBV-C E2 seems to be much more stable than HCV. Orii et al. (2000) studied the variability of GBV-C RNA in six patients with acute hepatitis. The average amino acid substitution rate in the E2 region of GBV-C was less than 1/100 that of HCV. No amino acid substitution in the loop domain was observed in seven additional patients with persistent GBV-C viraemia over the course of 42 years. How does GBV-C maintain persistent viraemia in some individuals without any variation in the envelope protein?

GBV IN TISSUE CULTURE In tissue culture, GBV-C replicates in cells that

originate from CD4 + T cells (Ikeda et al., 1997), B cells (Shimizu et al., 1999) and hepatocytes (Seipp et al. , 1999). In every case, the replication of GBV-C was not sufficiently efficient to enable classical virological analysis. In contrast, GBV-B replicates in primary cultures of normal tamarin hepatocytes with the rapid amplification of the cell-associated viral RNA and the

secretion of around 10 7 copies/ml in the supernatant. In addition, the successful virus passage could be monitored by immunoﬂuorescence staining of the GBV-B non-structural NS3 protein (Beames et al., 2000). This system may prove useful for the in vitro screening of anti-HCV drugs. However, despite a reduction in the GBV-B replication in the tissue culture system, no signiﬁcant reduction in viraemia was observed in tamarins (Lanford et al., 2001).

REPLICATION SITE OF GBV-C The in vivo replication site of GBV-C is of interest,

because it may provide an insight into the possible pathogenicity of GBV-C. Fan et al. (1999) surveyed 17 patients who had undergone liver transplants and found that 70% had signiﬁcantly lower GBV-C RNA titres in the liver than in the serum, while the remainder had RNA only in the serum. Shimizu et al. (2001) could not detect GBV-C RNA in the liver, colon or gall bladder, but did ﬁnd it in the serum and

appendix. Tucker et al. (2000) investigated the negative-strand RNA using serum and 23 tissue samples taken from four individuals who had died in accidents. The spleen and bone marrow were invariably positive. Individual instances of a positive kidney and liver were also found. No negative strands were detected elsewhere. Radkowski et al. (2000) detected negative-strand RNA in ﬁve bone marrow samples. Although most of these studies suggest that GBV-C replicates mainly in the extrahepatic tissues, the samples were probably obtained from chronically infected people. Seipp et al. (1999) reported on the presence of negative-strand RNA in 4/6 explanted liver specimens, as well as on the hepatocyte-restricted infection caused by in situ hybridisation. Given that the hepatitis induced by GBV-B in tamarins is acute and transient, signiﬁcant intrahepatic replication might occur only in the acute phase of the infection.

GBV-C IN THE HEALTHY POPULATION

Most studies of GBV-C in the healthy population have been based on voluntary blood donors, using RT-PCR for the 5’-untranslated region of GBV-C to detect the GBV-C viraemia and antibody assay against the E2 protein. Most individuals exposed to GBV-C were either RNA-positive or antibody-positive (GBV-C marker-positive). The prevalence of GBV-C viraemia has been found to be 1–4% in most countries (Table 27.1): the high rate of GBV-C viraemia is usually associated with a high rate of HCV-positive donors in the respective regions. Among those individuals with the GBV-C marker, 13–27% were GBV-C viraemic. Of the GBV-C viraemic population, 50–100% remained viraemic throughout the 1–3 year observation period. Persis- tence of the high viraemic rate was also observed in children (Fischler et al., 1997; Zanetti et al., 1998). This might suggest that most of the viraemic population is infected during infancy. Some genetic background, such as HLA, could have an inﬂuence on the infection, the persistence of the viraemia, or seroconversion. This alone, however, cannot explain the persistence of this virus in the human community, because the mother-to- child transmission rate is far less than 100% (discussed later).

Most reports refute the association of GBV-C infection with hepatic diseases, but Bjorkman et al. (2000) observed mild portal inﬂammatory lesions in 6/

11 donors with persistent and isolated GBV-C viraemia, as well as steatosis in 10/13. The spouses of the viraemic population showed a high prevalence of

GBV-C AND TTV

815

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

Table 27.1 Prevalence of GDV-C RNA viraemia among GBV-C marker-positive individuals and persistency of viraemia in healthy population

Country

% Viraemia of

Canada 1 13 Giulivi et al. (2000) USA

1 13 Handa et al. (2000) USA

Gutierrez et al. (1997) West Indies

4 21 Cesaire et al. (1999) Iceland

4 22 Love et al. (1999) Norway

2 19 Nordbo et al. (2000) Sweden

3 61 Bjorkman et al. (1998) The Netherlands

1 27 Jongerius et al. (1999) France

3 26 Mercier et al. (1999) France

3 18 Cantaloube et al. (1999) Poland

3 12 Brojer et al. (1999) Taiwan

2 50 Wang et al. (1998) Taiwan

3 27 Yu et al. (2001) Japan

4 21 Akiyoshi et al. (1999) South Africa

11 Casteling et al. (1998) Australia

3 16 83 Hyland et al. (1998) Australia

4 88 Moaven et al. (1996) 1 Percentage of viraemic population among those with GBV-C markers (RNA or anti-E2 antibody).

viraemia: 7/32 spouses (22%) (Akiyoshi et al., 1999) GBV-C IN PATIENTS and 4/12 partners (33%) (Nordbo et al., 2000).

However, 5/7 spouses in the former series exhibited Among acute non-A–E hepatitis patients, most studies parenteral risk factors, such as blood transfusions,

found that the prevalence of GBV-C viraemia, at less acupuncture or major surgery. Even in the apparently

than 6%, is not signiﬁcantly higher than that of the healthy population, people apparently at risk of

general population (Table 27.2). However, Frider et al. parenteral infection exhibited a much higher preva-

(1998) found 19 GBV-C viraemic cases (31%) among lence, usually in excess of 20%. Thus, GBV-C does not

62 acute non-A–E hepatitis patients. Most studies seem to be eﬃciently transmitted via sexual inter-

could not ﬁnd evidence of signiﬁcant ALT elevation, course.

either. In a study of renal transplant recipients, De

Table 27.2 Prevalence of GBV-C and its viraemia in patients Category

No. tested

% Viraemia of

Acute hepatitis non-A–E 62 31 Frider et al. (1998) Acute hepatitis non-A–E

53 4 Chu et al. (1999) Acute hepatitis non-A–E

34 6 40 Jongerius et al. (1999) HCV hepatitis

Acute hepatitis non-A–E 98 3 Romano et al. (2000)

11 19 Hassoba et al. (1997) CLD

HCV CLD 1 123

9 26 Bjorkman et al. (2001) Healthy

22 Bjorkman et al. (2001) Chronic haemodialysis

76 16 43 Chu et al. (2001) Kidney transplantation

24 59 De Filippi et al. (2001) 1 CLD: chronic liver disease.

817 Filippi et al. (2001) reported that 14% of the GBV-C

GBV-C AND TTV

the rate of mother-to-child infection. Hino et al. (1998) RNA-positive and HCV-negative patients exhibited

reported a high transmission rate of 7/11 children born persistently elevated ALT, although 8% of the control

to mothers co-infected with HCV and GBV-C and patients who were free of GBV-C and HCV also had

signiﬁcantly higher GBV-C RNA titres in mothers the same proﬁle.

with infected children. The nucleotide sequence of the Blood transfusions are one of the major transmis-

NS3 region was identical in each mother–child pair. sion pathways for GBV-C. Among those patients

Ohto et al. (2000) reported that 26/34 (77%) babies transfused with GBV-C RNA-positive blood, 8/21

born to viraemic mothers and almost all of those born (transmission rate, 38%) became viraemic after trans-

to mothers with a titre of 410 6 copies/ml (23/24) were fusion (Wang et al., 1998). However, they reported a

infected. They claimed that elective Caesarean section rate of persistent viraemia of 19% (4/21), lower than

reduced the rate of transmission, but that doing so the persistency rate expected in the healthy population

solely to avoid GBV-C transmission would not be as quoted above. Adult infection may lead to a lower

warranted because of minimal evidence, if any, for probability of persistent viraemia. The prevalence of

signiﬁcant pathogenicity of this virus. the viraemic population in patients with chronic liver

In contrast, Fischler et al. (1997) reported that only disease, chronic haemodialysis, and those who have

1/8 infants born to (HGV-GBV-C) RNA-positive had kidney transplants was apparently higher than

mothers was persistently infected (42 months), with that in the healthy population. The ratio of viraemic

no signs of liver disease. Menendez et al. (1999) individuals to the marker-positive patient population

reported a transmission rate of 7/18 children born to appeared higher, especially in haemodialysis and

GBV-C RNA-positive Tanzanian mothers in contrast transplant patients. Factors inﬂuencing these ﬁgures

to 4/42 children born to non-infected women included lifestyle and associated risk factors and/or

(p=0.01): the rate of mother-to-infant transmission nosocomial infections. This may be explained by the

was molecularly assessed in only 3/7 children born to fact that those patients with chronic medical interven-

viraemic mothers. Considering the possibility of tions are more vulnerable to recent and repeated

transmission through saliva, GBV-C RNA was infections. Co-infection of GBV-C might moderate the

detected at titres of 10 7 2 –10 7 4 of that in the course of HCV infection, since the HCV RNA levels

corresponding serum (Seemayer et al., 1998). were signiﬁcantly lower in the 15 HCV/GBV-C co- infected patients than those in the 48 patients with HCV infection alone (2.2 vs. 10.8 copies/ml; p=0.02;

HISTORY OF TTV De Filippi et al., 2001). The role of GBV-C in HIV

infection may prove more interesting. GBV-C was not The TT virus (TTV) was found in 1997, again by the found to aggravate the course of patients with HIV

use of diﬀerential display technology (Nishizawa, infections. Moreover, carriage of GBV-C RNA was

1997). The starting serum was obtained from a associated with the slower progression of HIV. All

hepatitis patient with the initials T.T. who did not parameters (survival, CDC stage B/C, HIV RNA load,

possess any markers for known hepatitis viruses. In CD4 T cell count) showed signiﬁcant diﬀerences in

spite of intensive studies to identify TTV-speciﬁc terms of the cumulative progression rate between those

pathogenicity, the real impact of TTV on liver disease individuals who were positive and those who were

still remains uncertain (Moriyama et al., 2001). The negative for GBV-C RNA (Tillmann et al., 2001).

vast majority of virological eﬀorts have been focused on the molecular epidemiology of the diverse spectra of the TTV genome (Biagini et al., 2001; Okamoto et al.,

MOTHER-TO-CHILD TRANSMISSION OF GBV-C 2001). The considerable diversity of TTVs and the lack of suitable conventional virological systems to study

In a multicentre study conducted by Zanetti et al. TTV limit our knowledge of TTV. (1998) 34/175 (19.4%) anti-HCV positive mothers were found to be positive for GBV-C RNA. All 21 (61.8%) babies to whom GBV-C was transmitted remained

CLASSIFICATION OF TTV persistently viraemic for 3–19 months after birth, except for one who seroconverted at 18 months.

The genome of TTV is the ﬁrst human virus to be Seven (35%) babies developed marginally elevated

found with a single-stranded circular DNA genome levels of ALT, excluding one dually-infected baby.

(Mushahwar et al., 1999; Miyata et al., 1999). Viruses Elective Caesarean section did not signiﬁcantly reduce

possessing single-stranded circular DNA genomes

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

Table 27.3 Viruses with single-stranded circular DNA genome Family

Genus

Representative viruses

Strandedness Number of circles

Microviridae

Sense 2 Geminiviridae

X174 (bacteria)

Ambisense 1–2 Circoviridae

Maize streak virus (plant)

Circovirus

Porcine circovirus (PCV)

Ambisense 2

Psittacine beak and feather disease virus (PBFDV) Pigeon circovirus (PICV) Goose circovirus (GoCV) Canary circovirus (CaCV) Banana bunchy top virus (BBTV) Coconut foliar decay virus (CFDV) Subterranean clover stunt virus (SCSV)

Gyrovirus

Antisense 2 Aneliovirus

Chicken anaemia virus (CAV)

TT virus (human, non-human primate, and other animals)

Antisense 3

have been known for some time, e.g. the bacterial by the antigenomic strand (Figure 27.2) (Niagro et al., viruses Microviridae, the plant viruses Geminiviridae,

1998). An earlier member of Circovirus, viz. chicken and the animal and plant viruses Circoviridae (Table

anaemia virus (CAV), is now classiﬁed into the genus 27.3). Members of Geminiviridae have more than one

Gyrovirus , since its Rep protein is coded by the circular DNA genome. Within the Circoviridae, the

antigenomic strand and the genomic strand has no genus Circovirus contains animal and plant viruses,

signiﬁcant ORFs (Pringle, 1999). CAV does not have such as the porcine circovirus (PCV), psittacine beak

the nine-nucleotide stem–loop structure that is com- and feather disease virus (PBFDV) and the banana

mon in Circovirus. In this sense, the genome of TTV bunchy top virus (BBTV). The Circovirus has a

has a feature that is common to CAV. Although the common nine-nucleotide stem–loop structure at its

TTV and CAV genomes have a common 36 nt stretch replication origin. The genome of Circovirus is

with around 80% identity, other regions of their ambisense, as the largest Rep protein is coded by the

genomes have no signiﬁcant similarity with each other. genomic strand while the other two proteins are coded

TTV diﬀers from CAV in its genome size, 3.9 kb vs. 2.3 kb, and by its extraordinary diversity. A working group set up to establish a nomenclature for circoviruses is working currently on a proposal to name a new genus of TTV and its related viruses Anellovirus (ring). The spelled-out name for TTV will be ‘Torque- TenoVirus’, while the TTV-like mini virus (TLMV) (Takahashi et al., 2000a; Takahashi et al., 2000b) will

be ‘TorqueTenoMiniVirus’ (TTMV). The acronyms were devised to incorporate torque (necklace) and tenuis/teno (thin), while trying to retain the already widespread term, TTV. It is emphasised that the acronym TTV is based on the initials of the original patient and it is not an abbreviation for transfusion- transmitted virus.

GENOMIC STRUCTURE OF TTV

Figure 27.2 Strands of TTV and TTMV in comparison with the chicken anaemia virus (CAV, genus Gyrovirus) and

Okamoto et al. (1998) reported a sequence of linear porcine circovirus type 2 (PCV2, genus Circovirus). The

3739 nt as the complete genome of TTV (TA278). sizes of the circles correspond to the genome size of each

However, it was not likely to constitute a complete virus. Clockwise arrows: translations on the antigenomic

genome because of the lack of a terminal repeat. A strand; counterclockwise arrows: those on the genomic strand

113 nt GC-rich stretch was added to complete its

GBV-C AND TTV

Figure 27.3 Partial genome of TTV (27852, nt 3740–3852). The GC-rich 113 nt region is shown in white with black characters. The similarity to the chicken anaemia virus (CAV, M55918) is indicated by asterisks. Multiple transcription modiﬁer motifs include

SP-1 (solid-line box), NF-kB (dotted-line box), ATF/CREB (solid underline) and AP-2 (dotted underline)

circular 3852 nt genome (Figure 27.3) (Mushahwar analysis of TTMV. A single host can be infected with et al. , 1999; Miyata et al., 1999). Within this GC-

both virus groups, but there are some hosts with only rich stretch, the 36 nt region (nt 3816–3851 of

one virus, indicating that the replications of these TA278) has a signiﬁcant similarity to nt 2237–2272

viruses are independent.

of CAV, and no signiﬁcant similarity was observed TTVs are composed of a variety of viruses, and are elsewhere.

classiﬁed into at least 16 subgroups with over 30% This region and its immediate vicinity constituted a

nucleotide diversities (Okamoto et al., 2000). TTMVs stem–loop structure, suggesting the origin of DNA

also share their divergent characters with TTV (Biagini replication (Mushahwar et al., 1999; Okamoto et al.,

et al. , 2001). Why and how these viruses can survive 1999). One-third of the genome containing this region

with such a wide spectrum of diversity is still unclear. is non-translating, and has a high degree of similarity

A single host can be infected with diﬀerent clusters of within the extremely divergent TTVs. Moreover,

TTVs (Okamoto et al., 1999; Takayama et al., 1999). multiple transcription modiﬁer motifs, such as ATF/

Do TTV and TTMV continue to mutate within a host CREB, AP-2, SP-1 and NF-kB binding sites, can be

to escape from the immunosurveillance system of the found in this region (Miyata et al., 1999). However, the

host, as is the case of HCV and Lentivirinae? details of the biological signiﬁcance of these motifs

TTVs are prevalent in non-human primates, while remained unclear, as no functional survey had been

the human TTV can cross-infect chimpanzees (Abe et attempted. Detailed studies of these structures may

al. , 2000). Furthermore, TTV sequences have been reveal the control mechanism of TTV replication and

detected in 19% of chickens, 20% of pigs, 25% of cows lead to the development of a suitable target cell system.

and 30% of sheep (Leary et al., 1999). The taxonomic

THE TTV GROUP The TTV group consists of two distinct clusters; the

3.9 kb original TTV and the 2.9 kb miniature TTV (TTMV) (Figure 27.4) (Takahashi et al., 2000a, 2000b). Although the genomic sizes of these two viruses are very diﬀerent, they share several common features. The non-coding regions surrounding the GC- rich stretch and occupying approximately one-third of the genome are similar to each other, as is the structure of the putative replication origin. The structures of the coding regions in these two virus groups are also similar to each other. The largest, ORF1, which accounts for approximately two-thirds of the viral

ORF structures of TTV genome, is common to all these viruses. Two other

Figure 27.4 Frame

and

(AB008394+AB017911) and TTMV (AB38625). Short double-spliced mRNAs have been found in TTV, and

vertical lines represent ATGs, while long vertical lines are also expected to be revealed by the DNA sequence

correspond to stop codons

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

divergence of these animal viruses relative to human et al. , 2001). Kikuchi et al. (2001) suggested the TTVs and their pathogenicity has yet to be clariﬁed.

possible association of TTV with aplastic anaemia, which might be interesting in conjunction with haematological disorders caused by CAV and Parvo-

TARGET CELLS OF TTV virus B19. Again, the detailed genotyping of TTVs might help us to understand the pathogenicity of

TTV was ﬁrst reported as a candidate agent for non-

TTVs.

A–E hepatitis (Nishizawa et al., 1997). Ukita et al. found that the concentration of TTV is 10–100 times greater in bile than in peripheral blood, suggesting the replication of TTV in the liver (Ukita et al., 1999).

PROTEINS OF TTV Okamoto et al. (2000) found the presence of the

replicative form of TTV in liver and bone marrow. The genome conﬁgurations of TTV and CAV are However, because of the wide spectrum of TTVs, the

similar to each other in spite of their diﬀerent genome real target of each TTV remains unclear. The high

sizes (Figure 27.5). The largest ORF constitutes two- prevalence and viral load of TTV in saliva suggest that

thirds of the entire genome and resides on the TTV might also replicate in the oropharyngeal tissues

antigenomic strand associated with a TATA box (nt and/or the salivary glands (Deng et al., 2000).

85–90 of TA278; Accession No. AB008394) and a Although TTV was found originally in serum, the

polyA signal (nt 3073–3079). This protein probably detection of TTV in stool suggested that it is

serves as a replicase and the major structural protein of transmitted by a faecal–oral route (Okamoto et al.,

TTV, because its N-terminus region contains a highly 1998). This pathway is more consistent with its

basic stretch consistent with other circoviral capsid ubiquity. TTV can be transmitted by mother-to-child

proteins and several conserved Rep protein motifs infection. The presence of TTV in cord blood, as

(FTL and YXXK) (Niagro et al., 1998; Takahashi reported in the literature, was 0–1% of N22 PCR (Goto et al., 2000; Kazi et al., 2000). Within 6 months of birth, the prevalence of TTV in children born to TTV-positive mothers was signiﬁcantly higher than that of children born to TTV-negative mothers. However, once the child was 1 year old, the prevalence in children born to TTV-negative mothers caught up to that of children born to TTV-positive mothers and to that of adults, even without breast-feeding (Kazi et al., 2000). This implies that the milk-borne transmission of TTV is insigniﬁcant. The low TTV titre in CSF suggested that the central nervous system is less likely to be a target of TTV replication (Maggi et al., 2001).

Although 20–60% of patients with idiopathic fulminant hepatic failure (Charlton et al., 1998; Simmonds et al. 1998) were reported to be TTV- positive, the signiﬁcance of TTV in the development of fulminant hepatic failure remains questionable because of the inconsistency of the patient selection and methods used for investigation and the high prevalence of TTV in the general population. At present, the pathogenicity of TTV and TTMV remains in question. Most reports suggest that there is no signiﬁcant association with liver diseases such as chronic hepatitis or hepatocellular carcinoma (Cossart, 2000). However, in addition to the original report of

Figure 27.5 Comparative arrangement of ORFs expected TTV, there have been several reports suggesting the

from the TTV genome (AB008394 + AB017911) and CAV potential aggravation of underlying hepatic diseases

(AF372658). SP-1 site (arrow), TATA box (closed triangle), by TTV viraemia (Cleavinger et al., 2000; Sampietro

polyA signal (open triangle) and GC-rich region (closed box)

821 et al. , 1998). The post-translational modiﬁcation

GBV-C AND TTV

the genome (Figure 27.6). The other two species of should be investigated further.

mRNA, 1.2 and 1.0 kb, had a second splicing at nt There were additional putative ORFs on the

712–2373 and 712–2566, respectively. The second exon antigenomic strand without legitimate initiation

was common to these mRNAs on ORF2 of frame 2. codons. To explain these incomplete ORFs, the

The third exons of the 1.2 and 1.0 kb mRNAs are presence of several spliced mRNAs was expected

located on frames 2 and 3, respectively. Our pre- with the common usage of the TATA box and the

liminary data suggested that these two mRNAs use polyA signal. Kamahora et al. constructed a plasmid

A 353 TG as the initiation codon (as suggested by T. containing the promoter region through the polyA

Kamahora). Given this assumption, we calculated an signal continuously (nt 2762–3852, 1–3770) (Kamahora

expected protein size of 286 and 290 amino acids. The et al. , 2000). Three species of mRNAs with sizes of 3.0,

real nature of these three proteins has yet to be

1.2 and 1.0 kb were expressed in the transfected COS1

elucidated.

cells. All 15 clones obtained from the cDNA library of Asabe et al. reported phosphorylation of the VP2 the polyA RNAs had the 5’-terminus at nt 123–135

(ORFs 2–4) product in the COS1 cells using an adjacent to the TATA box and the 3’-terminus at 6–9

expression plasmid, and claimed a similarity to nt downstream of the polyA signal. All three species of

NS5A of HCV (Asabe et al., 2001). Yokoyama et al. the mRNA clones exhibited a common splicing at nt

(2002) reported on transgenic TTV mice, which 186–276. The 3.0 kb mRNA has an initiation codon at

expressed mRNA with a splicing corresponding to

A 589 TG downstream of the splicing and codes for the the 1.0 kb mRNA. However, the protein in the Rep protein with 770 amino acids, as expected from

transgenic mice was corded by ORF1 on frame 1 to ORF4 on frame 2, instead of ORF2 on frame 2 to ORF5 on frame 3 in those cells that are transfected with the full-size genome. Theoretically, this type of reading frame may take place in natural infection. However, because they intentionally deleted the region including the authentic initiation codon of the natural

1.0 kb mRNA (A 353 TG), their construct was forced to use A 589 TG as the initiation codon.

PROBLEMS WITH THE DETECTION OF TTV In spite of the initial introduction of TTV as a putative

hepatitis virus, there is little data for specifying the pathogenicity of TTV. TTV may be non-pathogenic, or only a limited genotype of TTV may be pathogenic. By applying hemi-nested PCR to the N22 region (nt 1939–216) (Okamoto et al., 1998) we were able to detect genotype 1–6 TTVs, while that applied to the 5’- non-coding region (NTR; nt 26–184) (Takahashi et al., 1998) can detect most genotypes. By introducing the

Figure 27.6 Schematic of the TTV genome (AB008394+ NTR PCR, the TTV-positive population expanded AB017911) and its mRNAs. Three reading frames of the

genome are shown in the upper panel. The open triangle from 10–30% to 90% (Okamoto et al., 1999; Chan et al., indicates the position of the cap site while the closed triangle

2001). Some reports have claimed a higher sensitivity indicates that of the polyA signal. The short and long vertical

to NTR PCR, but were actually detecting a wider lines indicate ATGs and stop codons, respectively. Predicted

spectrum of TTVs. If only a certain genotype of TTV is ORFs are indicated by numbers. The shaded area represents

pathogenic, a PCR that can detect only the responsible the ﬁrst splicing common to all mRNAs of three diﬀerent

genotype should be used. The use of a broader assay sizes. The lower panel indicates the frames that are used and

conﬁgurations of 3.0 kb, 1.2 kb, and 1.0 kb mRNAs. The solid system will dilute the possible pathogenicity, even by

lines indicate exons, the dotted lines represent introns, and the N22 PCR detecting genotypes 1–6. Furthermore, the boxes indicate coding regions. Because of the alternative

sensitivities of PCR have not been carefully controlled splicing for the 1.0 kb mRNA, the 5’-terminal nt 2567 is

in most studies. It may take years to determine the real labelled with an asterisk

pathogenicity of TTV, especially if it is noted that

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

more than 15 years elapsed between the discovery of Charlton M, Adjei P, Poterucha J et al. (1998) TT-virus the porcine circovirus (PCV) (Tischer et al., 1982) and

infection in North American blood donors, patients with the discovery of pathogenic PCV2 (Allan et al., 1998).

fulminant hepatic failure, and cryptogenic cirrhosis. Hepatology , 28, 839–842.

Chu C, Hwang S, Luo J et al. (2001) Clinical, virological, immunological, and pathological signiﬁcance of GB virus

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28 Emerging Virus Infections

Brian W. J. Mahy

National Center for Infectious Diseases, Atlanta, GA, USA

INTRODUCTION diseases that threaten public health throughout the world and are not limited in their potential geo-

Although infectious disease mortality declined sharply graphic range. As pointed out in the ﬁrst report on throughout the twentieth century, apart from a

emerging infections, in the context of infectious dramatic increase following the 1918 pandemic of

diseases, there is nowhere in the world from which inﬂuenza virus, in the last twenty years a small but

we are remote and no one from whom we are noticeable increase in mortality from infectious

disconnected (Lederberg et al., 1992). diseases can be observed (Armstrong et al. 1999;

Mahy, 2000; Figure 28.1). The start of this increase roughly coincides with the appearance in the popula-

FACTORS CONTRIBUTING tion of human immunodeﬁciency virus (HIV)

TO EMERGENCE infection and associated cases of acquired immune

deﬁciency syndrome (AIDS), but there is good Considering infectious diseases as a whole, the evidence suggesting that many factors contributed to

committee of the IOM recognised 13 factors contrib- this situation.

utory to emergence, and these are listed in Table 28.1. In 1991, the Institute of Medicine (IOM) of the

Some of these are natural events that can be responded US National Academy of Sciences convened a multi-

to, but not prevented from occurring, such as disciplinary committee to consider emerging infec-

microbial adaptation and change, and climate and tious diseases and the factors responsible for them,

weather. Others are societal and political in origin and and to make recommendations for future actions to

could be eliminated, such as the breakdown of public

be taken in response to their threat to public health health measures, and poverty and social inequality. (Lederberg et al., 1992). In 2001, the IOM convened

Finally, ‘intent to harm’ refers to acts of bioterrorism,

a new committee on Microbial Threats to Health in such as the deliberate release of smallpox (variola) the Twenty-ﬁrst Century (Smolinski et al., 2003),

virus (Mahy, 2003).

with a similar mandate to review the situation 10 This chapter will consider the factors responsible for years later. It is noteworthy that during the period

the emergence of new virus infections under four broad covered by the two sets of recommendations more

headings: (a) virus evolution; (b) human demo- than 60 new virus diseases have been recognised in

graphics, susceptibility to infection and behaviour; (c) the human population (Figure 28.2). These range

improved technology for the detection of virus infec- from hepatitis C virus (HCV), ﬁrst recognised in

tion; and (d) increased contact with vectors of virus 1988, to the new human coronavirus, ﬁrst recognised

infection. By considering examples of virus emergence in 2003, which is responsible for severe acute

under each category, the factors responsible for the respiratory syndrome (SARS). As with most emer-

remarkable global increase in human virus infections ging virus infections, these are examples of virus

over the last 15 years (Figure 28.3) will become clear.

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

Figure 28.1 Infectious disease mortality in the USA, 1900–1996. Reproduced from Armstrong et al. (1999)

Virus Evolution mechanisms do not exist for RNA, and so RNA viruses are much more mutable.

There are a number of diﬀerent mechanisms by which viruses may evolve. In general, RNA viruses are much more likely to evolve by genome changes than DNA

Mutation

viruses, and this in part reﬂects the proof-reading functions present in the host cell that preserve the

The most obvious mechanisms for evolution involve integrity of host cellular DNA. Such proof-reading

point mutations in the genome RNA nucleotide

Figure 28.2 Emergence of viruses aﬀecting humans

827 Table 28.1 Factors in emergence

EMERGING VIRUS INFECTIONS

likely that rubella virus was originally generated in this way, and we see many examples of recombination in

Microbial adaptation and change Human vulnerability

action, especially with the picornaviruses, such as Climate and weather

poliovirus. For example, an outbreak of poliomyelitis Changing ecosystems

in Hispaniola in 2001 was caused by a recombinant Economic development and land use

virus between the live Sabin poliovirus vaccine strain Human demographics and behaviour

and an enterovirus, which restored the pathogenicity Technology and industry

of the vaccine virus (Kew et al., 2002). International travel and commerce

Breakdown of public health measures As a general rule, recombination events seem to be

Poverty and social inequality much more common with viruses having a positive- War and famine

strand RNA virus genome, and such events are rarely Lack of political will

reported for negative-strand viruses, although they Intent to harm

certainly can occur.

Reproduced by permission from Smolinski et al. (2003) sequence, either random (spontaneous) or in response

Human Demographics, Susceptibility to Infection, to a selective pressure, such as inhibition by an

and Behaviour antibody or an antiviral drug. For example, inﬂuenza viruses undergo frequent mutations in response to

Changes within the host population have contributed humoral antibodies against inﬂuenza which exist in the

greatly to the emergence of virus diseases during the general population: this phenomenon produces ‘anti-

past 20 years.

genic drift’. Another example is HIV, which, in the presence of a drug which prevents DNA replication, such as

Increases in World Population and Global zidovudine (azidothymidine), rapidly generates mutant

Travel

viruses that are resistant to the drug. The rapid increase in the world population has combined with the greatly enhanced opportunity for global travel to increase the opportunity for persons to

Reassortment come into contact with virus diseases formerly considered to be exotic to the developed world (such

A second mechanism for genetic change is reassort- as Lassa fever, which causes 5000 deaths/year in West ment, which is a common feature of those viruses that Africa), e.g. in 1989 a patient died from Lassa fever in have a segmented genome, such as inﬂuenza viruses

a hospital in Chicago (Holmes et al., 1990). The patient and rotaviruses. In cases where two viruses of distinct was an American citizen who had visited Nigeria to genetic lineage infect the same cell, it is possible for attend his mother’s funeral, and became ill 2 days exchange of genome segments to occur, giving rise to a before his return to Chicago. Although 102 people had new reassortant virus that may have altered properties contact with him in the Chicago hospital, fortunately of transmission or pathogenesis. This is of particular none of these contacts became infected. importance clinically with the influenza viruses, and it Global travel also provides the opportunity for very has been clearly demonstrated that both the 1957 rapid movement of a newly emerged virus, as we ‘Asian’ influenza pandemic and the 1968 ‘Hong Kong’ witnessed when severe acute respiratory syndrome influenza pandemic resulted from viruses that were (SARS) was first recognised in Guangdong Province, apparently generated by reassortment between human China, on 16 November 2002, where it had caused 300 and avian influenza virus strains (Webster and cases of unknown aetiology and five deaths. This was Kawaoka, 1994). reported to the World Health Organization (WHO) on

11 February 2003, and then on 26 February a WHO oﬃcial working in Hanoi, Vietnam, Dr Carlo Urbani, Recombination

reported an unusual case of severe acute respiratory disease to WHO. Other cases were soon identiﬁed in

The third most common mechanism of virus evolution Hanoi and in Hong Kong, and many of these were is recombination between the genomes of two diﬀerent

health care workers. On 12 March WHO issued a viruses. Although not absolutely proven, it seems most

global alert about these cases, and on 14 March cases global alert about these cases, and on 14 March cases

be linked to a hotel in Hong Kong (Hotel M), where the index case of the disease, a 65 year-old medical doctor from Guangdong Province in China, had arrived on 21 February. He had noted his onset of symptoms on 15 February, and he was hospitalised on

22 February and died the following day. Although he only stayed in the hotel one night to attend a family wedding, this single individual infected 12 other guests in the hotel, and from them several hundred persons in widely disbursed geographic areas became infected within a few weeks, resulting eventually in more than 300 deaths between February and June 2003. By the end of the epidemic in August 2003, WHO reported more than 8000 probable cases of SARS worldwide, of whom ten percent, some 800 persons, died (World Health Organization, 2003). Most of those who died were elderly patients (Centers for Disease Control and Prevention, 2003b).

Increased Numbers of Elderly Persons This brings us to another demographic change which is

aﬀecting the emergence of infectious diseases. The elderly population is growing faster than any other segment of society in most highly developed countries, but elderly people may have impairment of normal defence mechanisms against infection, resulting in greater susceptibility to infection.

Immunosuppression Other factors that aﬀect susceptibility to infection

include immune suppression, due to genetically inherited traits, malnutrition, use of immunosuppressive drugs following organ transplantation or as part of cancer treatment, or infection with human immunodeficiency virus (HIV). Worldwide, many millions of persons are now infected with HIV, and this has created a large segment of the global population in which emerging virus infections may spread and become amplified. In addition, a person infected with HIV may also suffer from reactivation of latent virus infections that are normally held in check by the immune system. These latent infections may include several members of the herpesvirus group, such as cytomegalovirus, or the human polyomavirus JC, which may result in progressive multifocal leukoencephalopathy.

Human Behaviour Human behaviour is a major factor in the emergence

of virus diseases. HIV, for example, would not spread within the human population if obvious precautions were taken by all individuals to prevent transmission. In addition, disease outbreaks have been recognised as resulting from unusual human behaviour. The emergence of monkeypox in the USA in 2003 is an example of a disease that only emerged because of the demand by US citizens for importation of exotic animal pets. In this case, the importer brought in giant rats from Ghana, and these rats were housed together with other animals, especially prairie dogs, burrowing rodents of the genus Cynomys, which are a popular pet animal in the mid-west of America. One or more of the giant rats was infected with monkeypox virus, which spread to the prairie dogs, and from them to people (Centers for Disease Control and Prevention, 2003a).

A more serious example of virus emergence due to human behaviour occurred in the early 1980s, when a decision was made to alter the production process of bovine meat and bone meal, a dietary supplement for young calves, by omitting a lipid extraction phase. As a result, a new prion disease, bovine spongiform encephalopathy (BSE), became established in the UK cattle population and eventually led to human cases of the disease, called new variant Creutzfeldt–Jakob disease (nvCJD) (Collinge et al., 1996). The exact mechanism by which humans acquire this disease remains uncertain, but it is believed to be the result of eating meat from an infected bovine animal. Certainly the virus found in the brains of humans who have died of nvCJD is identical to the virus causing BSE (Almond and Pattison, 1997).

Improved Technology for Virus Detection

In recent years a variety of molecular techniques, especially the polymerase chain reaction (PCR), have been used to identify viruses causing a variety of diseases that were formerly of unknown aetiology. Examples of these are listed in Table 28.2.

Screening cDNA Expression Libraries One of the most important discoveries that used

molecular technology was the identity of the virus causing hepatitis non-A non-B, now known as Hepatitis C virus . It had long been recognised that an important cause of serum-transmitted hepatitis was

828

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

due to neither hepatitis A nor hepatitis B viruses. In 1989, Bradley at the Centers for Disease Control and Prevention (CDC) collaborated with workers at Chiron Corporation. They used the blood of a chimpanzee that had been experimentally infected with a preparation known to transmit non-A non-B hepatitis. RNA extracted from the chimpanzee’s blood was reverse-transcribed using random primers to make cDNAs, which were then cloned into a bacteriophage expression vector. The resultant bacterial colonies were then screened using sera from patients with non-A non-B hepatitis. Thousands of clones were screened in this way, until one was found that reacted with antibodies in the sera of infected patients. DNA from the positive clone was then used to screen other clones by DNA hybridisation, and eventually the full-length sequence of Hepatitis C virus was elucidated (Choo et al ., 1989). This enabled the development of diagnostic tests that are crucial to screening the blood supply, and to preventing transmission of hepatitis C by blood transfusion. A very similar molecular approach was used later to identify Hepatitis E virus, responsible for occasional large outbreaks of water-borne viral hepatitis (Purdy and Krawczynski, 1994).

Consensus PCR Direct application of the PCR technique proved very

valuable when an outbreak of cases of acute respiratory disease syndrome occurred in the south-western USA, initially on a Navajo Indian reservation. Initial serological studies suggested that the serum of patients who had experienced the disease contained antibodies that cross-reacted with Hantaan virus, a bunyavirus that was a known pathogen transmitted by rodents and causing haemorrhagic fever with renal syndrome (HFRS) in Asian countries such as Korea, but Hantaan virus was not suspected of causing fatal pulmonary disease in the USA.

Because of the serological cross-reaction, the known nucleotide sequence of Hantaan virus was used to make

consensus primers for PCR amplification, and by this means a new group of hantaviruses, causing severe pulmonary distress syndrome, was identified (Nichol et al ., 1993). Once the consensus primers had amplified the RNA genome of the new hantaviruses, more specific primers could be designed, based upon sequence analysis of the new hantaviruses. The prototype virus of what has proved to be a very large group of viruses present in rodents of the subfamily Sigmodontinae was named Sin Nombre virus.

Representational Difference Analysis In addition to PCR, sequence representational diﬀer-

ence analysis (RDA) was used to identify Human herpesvirus 8 (HHV8), a rhadinovirus found in association with Kaposi’s sarcoma (KS), a rare skin tumour found in AIDS patients. In this technique, DNA extracted from KS tissue was compared with DNA extracted from normal tissue, and DNA sequences of a new herpesvirus were only found in the Kaposi’s sarcoma tissue (Chang et al., 1994; Moore et al ., 1996). Similar virus sequences were subsequently detected in tissue from patients with multicentric Castleman’s disease and the rare primary eﬀusion lymphoma, and HHV8 is believed to be causally associated with these tumours.

The RDA approach has also been successfully used in the identiﬁcation of viruses which are apparently common human infections, but for which no clear association with disease has been found (Mushahwar, 2000). Three viruses, known as GBV-A, GBV-B and GBV-C , were found as a result of studies on the serum from a surgeon (George Barker) with acute non-A non-B hepatitis. When injected into marmosets (tamarins) of Saguinus spp., his serum caused hepatitis which could be serially passed in marmosets: the causative virus was immunologically and structurally distinct from the known Hepatitis A, B, C and E viruses. Two viruses (GBV-A and GBV-B) were isolated from serum of the infected marmoset using

EMERGING VIRUS INFECTIONS

829 Table 28.2 Examples of molecular approaches to pathogen discovery

Virus

Type of approach

Reference Hepatitis C virus

Expression library

Choo et al. (1989) Sin Nombre virus

Consensus PCR

Nichol et al. (1993) Human herpesvirus 8 and KS

RDA Chang et al. (1994) GB hepatitis viruses

RDA Simons, et al. (1995) TT virus

RDA Nishizawa et al. (1997) Human metapneumovirus

RAP-PCR van den Hoogen et al. (2001)

the RDA technique, and found to be new members of the family Flaviviridae, and associated with GB agent hepatitis.

A third newly discovered ﬂavivirus, GBV-C, was identiﬁed in human sera by reverse transcription and consensus PCR, using primers based upon the GBV-A, GBV-B and Hepatitis C virus helicase gene, and appears to be identical to a virus that was independently isolated and called Hepatitis G virus (Simons et al ., 1995; Linnen et al., 1996). For this reason, the virus is usually called GB virus C/Hepatitis G virus. This latter virus is widely distributed in the human population, but has not been shown to cause hepatitis. It seems able to infect both liver and spleen cells without causing obvious disease symptoms.

The RDA approach was also used to identify a completely diﬀerent class of viruses in the human population. Once again, the initial discovery of the virus occurred during attempts to isolate a new agent causing transfusion-related hepatitis that was not due to hepatitis viruses A, B, C, D, E, or G. Using RDA, DNA clones were isolated from a patient (initials TT) before and after transfusion. The diﬀerential DNA analysis revealed a small non-enveloped virus with a DNA genome, and it was named TT virus (TTV) after the initials of the patient (Nishizawa et al., 1997). Although no association of TTV has been found with any human or non-human primate disease, the virus appears to be ubiquitous in the healthy human population, with multiple variants (Mushahwar et al., 1999; Okamoto et al., 2000; Khudyakov et al., 2000). The name Circinoviridae has been proposed to describe this large family of viruses that have a circular, single-stranded, negative- sense DNA genome approximately 3850 nucleotides in length. Analysis of a large collection of TT virus genomes showed nearly 50% of them to be recombinant (Worobey, 2000).

Conclusions Regarding Molecular Techniques It seems likely that as molecular techniques continue to

be applied in this manner, many more viruses that have no obvious association with pathogenesis will be found in the human population. In addition, it is likely that, using molecular approaches, a number of diseases presently of unknown aetiology will prove to be associated with causative viruses that have yet to be discovered.

Despite the availability of these sophisticated molecular approaches for the identiﬁcation of emerging viruses, the most recently identiﬁed emerging

virus was found to be a human coronavirus causing severe acute respiratory syndrome (SARS) by the classical technique of electron microscopy of the virus growing in cell culture (Ksiazek et al., 2003). Only once the virus was discovered were molecular techniques used to establish that it was a newly identiﬁed coronavirus not hitherto suspected in the human population.

Increased Contact with Vectors of Virus Infection As the global human population continues to expand,

there is increased opportunity for human contact with natural virus vectors, such as arthropods, birds or rodents, and in recent years we have seen the emergence of a number of new virus diseases, as well as a resurgence of other diseases that were formerly well controlled. Some instances involve the invasion by humans of a new ecological niche which brings them into closer contact with the vector. As an example, in South America, deforestation of large land areas may deprive rodents carrying arenaviruses or hantaviruses of their usual habitat, resulting in infestation of houses and closer contact with the human population. Although there has been no large pandemic of influenza since 1968, it is highly likely that reassortment of an avian influenza virus with a human one, as happened in the last two pandemics, will occur again. There is a vast reservoir of influenza viruses in the avian population (Webster and Kawaoka, 1994) and our ability to control such a pandemic, if it occurs, has not significantly improved in the last 30 years.

Arthropod-borne Viruses Another important factor that has contributed to the

emergence of virus diseases is the vector population density. This is particularly true of mosquito-borne viruses. Once well-controlled, the mosquito population, including Aedes aegypti and A. albopictus, has expanded greatly since the cessation of the use of DDT, resulting in increasing cases of dengue fever worldwide (Figure 28.4) and in particular a resurgence of dengue haemorrhagic fever, a severe form of dengue involving infection with two or more serotypes of dengue virus.

Translocation of a vector-borne virus into a new geographical region can result in the emergence of a dramatic disease outbreak. In 1999 there was an outbreak of West Nile viral meningoencephalitis, ﬁrst detected in New York City. This virus had only been

830

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

detected previously in Africa and the Middle East, and once the virus found in New York was sequenced, it was found to be identical in sequence to a virus isolated from diseased birds in Israel. How the virus was imported into the USA is unknown—an infected person, mosquito or bird are all possibilities. However, once established in a virgin population with the appropriate vector, the virus soon spread both south and west from New York, and has now moved south into the Caribbean and Mexico and north into Canada. By 2002, West Nile virus had spread to 39 states and DC, and caused 2741 human cases of meningoencephalitis and 263 deaths. More than 124 000 dead birds were reported, as well as more than 12 000 cases in horses. The virus was recovered from 37 species of mosquito, and is now an endemic disease requiring considerable public health eﬀorts for its control.

Rodent-borne Hantaviruses In 1993 an epidemic of acute respiratory disease

occurred in south-western USA, and was identiﬁed as hantavirus pulmonary syndrome (HPS), a new clinical disease of high mortality (40%), spread by a virus in the deermouse (Peromyscus maniculatus) population in the area. It soon emerged that climatic conditions in the summer of 1993 had favoured an explosion in the population of deermice, and this was the major factor which had contributed to a large number of HPS cases from which the epidemic became recognised. Once recognised, other hantaviruses were identiﬁed in both North and South America, many causing severe and often fatal HPS in the human population (Figure 28.5).

Although most rodents worldwide appear capable of housing and transmitting hantaviruses, the only cases of severe HPS have been associated with rodents of the subfamily Sigmodontinae, which is conﬁned geographically to the Western hemisphere, North and South America. In other parts of the world hantaviruses cause milder disease, with few fatalities.

Bats as Virus Vectors Another important group of animals that act as

vectors for virus diseases are bats, which comprise a quarter of living mammalian species. In the USA, insectivorous bats are the most important vector species for human rabies, often transmitting the disease silently, by a bite of which the human is unaware.

In recent years fruit-eating bats have been recognised as important disease vectors. There are some 170 species of fruit-eating bats (Megachiroptera) in the tropical regions of Asia.

In 1994 a horse trainer and a stablehand who worked on a farm in Hendra, a suburb of Brisbane, Australia, became ill while nursing a sick pregnant mare that had recently been brought onto the property. The disease spread to other horses on the property, and 14/21 infected horses died of pulmonary disease with haemorrhagic manifestations. Of the two infected humans, the trainer died, but the stablehand survived the infection after a lengthy illness. One year later, another horse farmer died 600 miles away in Mackay, from encephalitis caused by a similar virus. The virus, named Hendra virus, was found to be a new paramyxovirus with a large genome, about 19 kb in length, morphologically similar to other paramyxo-

viruses but with a very long nucleocapsid. Subsequently, the only time this virus was recognised as causing disease again was in 1997, when it caused the death of a horse in Cairns, in the far north of Australia. However, in the meantime work carried out in the Australian Animal Health Laboratory in Gee- long had identiﬁed the virus in fruit bats, and a survey revealed that a high proportion of fruit bats had antibodies against the virus.

Beginning in 1997, an outbreak of a new respiratory disease in pigs was noticed by farmers in Malaysia. In late 1998 and 1999 many farmers and other persons having close contact with pigs developed severe symptoms of encephalitis. In all there were 265 human cases, of whom 105 persons died. The disease did not appear to be transmissible between humans, and was stopped in May 1999 by the slaughter of more than a million pigs, causing great economic loss to the Malaysian pig industry. In March 1999 a virus was isolated in cell culture from the brain of a patient in Sg Nipah village, Bukit Pelandok, Malaysia. The virus caused syncytia during growth in Vero cells, and gave a positive test by immunoﬂuorescence assay carried out at CDC using an antiserum made against Hendra virus. By thin-section electron microscopy of infected Vero cells, a paramyxovirus with a long nucleocapsid was found. Complete sequencing of the virus genome showed that it was a large negative-stranded RNA molecule, 18 246 nucleotides in length, with about 80% homology to the genome of Hendra virus. The new paramyxovirus, called Nipah virus (Chua et al., 2000), was subsequently found in large fruit-eating bats, and it was from these that the virus is presumed to have spread ﬁrst to pigs, and then to humans in close contact with infected pigs. Curiously, the mortality

EMERGING VIRUS INFECTIONS

831

rate in pigs was less than 5%, much less than was seen amongst the human cases. Pigs develop rapid, laboured breathing and an explosive non-productive cough as the main symptoms, with occasional neurological changes, such as lethargy or aggressive behaviour. Humans develop a febrile encephalitis which rapidly progresses to multisystem involvement with vasculitis and syncytial giant cell formation at various sites. Spread to the brain is by the vascular route and leads to discrete small foci of necrosis and neuronal degeneration.

Non-human Primates as Virus Vectors It is now recognised that both HIV-1 and HIV-2 have

their origin in chimpanzees and sooty mangabey monkeys, respectively (Holmes, 2001), which can be infected with these viruses but do not themselves routinely develop AIDS. The transmission of these zoonotic infections has been ascribed to the practice of killing and eating the primates (as bushmeat), and it is clear that other viruses can emerge in the same way. Viruses causing severe haemorrhagic fever, such as Ebola virus and Marburg virus, have certainly passed from non-human primates to man by this means on several occasions, but it is generally believed that another natural reservoir exists for these viruses since, like man, non-human primates are highly susceptible to both these ﬁloviruses. Because they cause extremely high mortality in man (up to 90% of infected persons),

a great deal of concern is generated when an outbreak occurs. The original outbreak of Marburg virus was caused by direct transmission of the virus from infected monkeys by persons handling fresh monkey tissues, but in general ﬁlovirus outbreaks are largely ampliﬁed by person–person transmission, usually in a hospital setting, as occurred in the last major outbreak caused by Ebola virus in Kikwit, Democratic Republic of the Congo, in 1995.

Conclusions Regarding Contact with Vectors It is clear from the experience gained over the past 20

years that animal, bird or arthropod reservoirs are extremely important sources for the emergence of new diseases. This has led to an assumption that most newly discovered virus diseases have their origin in another species. The outbreak of SARS coronavirus may be a case in point, and suspicion has been placed on various exotic animal species that are eaten by the Chinese and may have started the epidemic in

Guangdong. This remains to be proven, but it is widely believed that the next pandemic of human inﬂuenza will occur as a result of reassortment between an avian and a human inﬂuenza virus.

Only by continued global surveillance and prompt reporting to WHO can we hope to control the outcome of such an event.

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29 Hospital-acquired Infections

INTRODUCTION chickenpox, etc. Preventing the nosocomial spread of viral respiratory and gastrointestinal pathogens, how-

The previous chapters have covered the important ever, with very short incubation periods of only hours clinico-pathological aspects of all the major groups of

to a few days, relies heavily on the staﬀ admitting the human viruses, including their epidemiology and

patient recognising the potential for transmission and control at a population level, yet little has been said

adhering strictly to basic infection control measures, concerning the prevention of nosocomial virus infec-

such as hand-washing. The admission to single rooms tion. This chapter aims to provide the practical

of patients experiencing viral prodromes of, for knowledge and approaches required to enable micro-

example, parvovirus or measles during epidemic biologists and virologists to control the spread of virus

seasons or local upsurges, and cohort nursing are infection in hospitals. Indeed, the entire subject of

very eﬀective but require thought from admitting nosocomial virus infection has not had the prominence

teams. To facilitate this approach, clear, concise it deserves and has been a largely neglected area.

guidance from the virology, infection control and This is surprising, as hospitals are a particularly

occupational health departments on how to manage easy target for several virus groups and may act as

individual virus infections and staﬀ exposures to them centres of virus ampliﬁcation for the further spread

is required.

of virus in the wider community. This has been Control of virus infections in hospitals should also exempliﬁed recently in the case of SARS and

be an active process. Although rapid diagnosis and previously with Ebola virus outbreaks being centred

excellent communication with staﬀ in the clinical areas in hospitals in Africa. There is also an ever-

are essential if nosocomial infection is to be prevented, increasing burden of extremely vulnerable patient

anticipating recurring problems, e.g. the annual RSV groups, including pre-term neonates and those with

winter epidemic, by informing staﬀ through contin- immunodeﬁciency secondary to HIV or solid organ

uous education programmes and pro-actively if local and bone marrow transplants. Global travel has also

outbreaks are occurring, is of additional beneﬁt. meant that increasingly the staﬀ who work in health

In this last chapter we cover the burden of services come from all over the world. This brings with

nosocomial virus infection of the most important it other unique problems, such as increased suscept-

viruses. Section A discusses blood-borne viruses, whilst ibility rates to chickenpox if staﬀ are from equatorial

section B illustrates the problems, and some of the climes.

solutions to controlling the spread of viruses by all The prevention of nosocomial virus infections

other routes and means of contact. Practical measures requires a team eﬀort. Very often virology is able

that should be taken to prevent an outbreak or limit its only to prevent secondary cases where the incubation

spread and guidance on the management of staﬀ period is relatively long, e.g. 10–14 days for measles,

susceptible to particular agents are also given.

Table 29.1 Incubation periods, communicability and transmission modes for common virus infections 836 Virus

Mode of transmission

Incubation period Duration of shedding

Risk of transmission

Prevention

Adverse outcomes

in hospitals 1

Rubella Respiratory droplet

15–20 days

7 7 to +2 days

Low

Screening

Congenital Rubella Syndrome

Highest before or at

Vaccination

rash onset

Reporting

Measles Respiratory droplet

7–18 days

7 2 to +5 days

Death in I/C pts

PRINCIPLES Mumps

Reporting

Respiratory droplet

14–19 days

7 6 to +4 days

Low (may increase in

Screening

Meningitis

certain cohorts)

Chickenpox Respiratory droplet

Congenital chickenpox Physical contact

10–21 days

7 2 to +7 days

Moderate

Screening

Death in I/C pts 2 AND Airborne

(normally 14)

(or until scabbed)

Vaccination

Reporting

Parvovirus Respiratory droplet

13–18 days

7 6 to 73 days

Low/moderate

Reporting

Miscarriage PRACTICE

before symptoms

Aplastic crises Chronic anaemia

Enteroviruses Faecal–oral

Severe neonatal disease Fomites

5–7 days

1–8 weeks

Low/moderate

Reporting

OF Airborne

CLINICAL Inﬂuenza

Respiratory droplet

Death (I/C, elderly) Airborne

2–3 days

7 1 to 7 days

High

Reporting

Ward closure Parainﬂuenza

Vaccination

Respiratory droplet

1–7 days

7–21 days

Very high

Reporting

Ward closure Death in I/C

VIROLOGY RSV

Death in I/C HSV

Respiratory droplet

Death in neonates Physical contact

Saliva

Few days

1–8 weeks

High if breakdown in hand

Reporting

(mean=6)

washing/personal hygiene

Fomites CMV

Physical contact

None with virus at mucosal surfaces

Hand washing

Rotavirus Faecal–oral

Prolonged stay in hospital ? Respiratory

Hand washing

SRSV Faecal–oral

Ward and hospital closure Airborne

1–3 days

Up to 2 weeks

Very high

Reporting

(allow back to work 72 h

Isolation

after symptoms resolved)

2 Refers to overall risk of transmission, modiﬁed by level of immunity in the population. I/C pts, Immunocompromised patients.

29A Infections Acquired by the Blood-borne

Route

Anthea Tilzey

Guy’s, King’s and St Thomas’ School of Medicine, London, UK

INTRODUCTION It has long been recognised that HCWs are at increased risk of BBV infection. A study among The blood-borne viruses (BBVs), which are associated

orthopaedic surgeons in the USA showed that the with persistent replication and a persistent viraemia,

prevalence of markers of both HBV and HCV are well-recognised causes of nosocomial infection.

infection increased with age (Shapiro et al., 1996). The most commonly reported nosocomial BBVs are

Both in this study and in a number of others, a Hepatitis B virus (HBV), Hepatitis C virus (HCV) and

signiﬁcantly higher prevalence of HCV antibodies in Human immunodeﬁciency virus (HIV). Transmission

HCWs compared to control volunteer blood donors may occur from patient to health care worker (HCW)

has been demonstrated. For example, a survey of New or from HCW to patient; the greater risk is from the

York dentists (Klein et al., 1991) showed an anti-HCV former but the greater publicity tends to surround the

prevalence of 1.75% in 456 dentists compared to latter. Patient-to-patient transmission can also occur,

0.14% in 723 blood donor controls. The prevalence usually only where there is a deﬁciency in infection

was particularly high (9.3%) in 43 oral surgeons. control procedures.

With regard to HIV, as of June 1999, 319 HCWs Nosocomial infection may occur when blood, other

worldwide were reported to have acquired HIV body ﬂuids (see Table 29A.1) (Department of Health,

infection through occupational exposure (CDSC, 1998), unﬁxed tissues or organs are transferred from an

1999), 102 following a documented seroconversion infected source to an uninfected recipient.

after a speciﬁc occupational exposure, and 217 where In the health care setting, transmission usually

seroconversion was not demonstrated but no other risk follows percutaneous exposure. The relative risk of

factors were identiﬁed.

transmission following a single percutaneous exposure There have been a number of documented outbreaks is normally quoted as approximately 1/3 for HBV,

of HCW-to-patient transmission of HBV, HCV and with an ‘e’ antigen-positive source, 1/30 for HCV and

HIV. There have been over 45 reports of transmission 1/300 for HIV. The risk of transmission for all three

of HBV from HCW to patient, resulting in more than will depend to a large extent on the amount of virus

400 infected patients (Gunson et al., 2003). Initially, all inoculated, and this in turn will depend on the viral

were associated with ‘e’ Ag-positive HCWs; most were load in the source and the volume and type of body

associated with dental or surgical procedures, particu- ﬂuid transferred. Mucosal exposure carries a lower

larly cardiac surgery and obstetrics and gynaecology. risk, e.g. the risk of acquiring HIV after a single

Look-back exercises demonstrated transmission rates

varying from 1.5% (Hadler et al., 1981) to as high as (Department of Health, 1998).

mucocutaneous exposure to blood is 5 1/2000

24% (Welch et al., 1989). More recently there have

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

Table 29A.1 Body ﬂuids other than blood that may and contaminated immunoglobulin preparations, most transmit nosocomial infection

resulting from inadequate infection control procedures High-risk body fluids

or inadequate disinfections (Henderson, 2003). How do we prevent or at least minimise the risk of CSF

Low-risk body fluids

BBV transmission in the health care setting? Preven- Peritoneal ﬂuid

Saliva

tion in this context may be divided into prevention of Pleural ﬂuid

Faeces

exposure and prevention of infection, which will apply Pericardial ﬂuid

Urine

to patient-to-HCW, patient-to-patient and HCW-to- Synovial ﬂuid

Vomit

Amniotic ﬂuid patient transmission. Recommendations currently in Semen

place and/or under discussion to protect patients from Vaginal secretions

infected HCWs are discussed below. Breast milk Saliva in association with dentistry Low-risk body ﬂuid containing

visible blood

PREVENTION OF EXPOSURE Preventing or minimising BBV exposure is achieved

through establishing and practising high standards of been a number of documented transmissions of HBV infection control, i.e. general measures to minimise from ‘e’ Ag-negative surgeons (Heptonstall et al., percutaneous or mucocutaneous exposure to blood, 1997); all had detectable HBV DNA by nested PCR tissues and other body ﬂuids. and pre-core mutants of HBV which failed to produce

HBeAg. All employing authorities should produce written There are also a number of reports of transmission of

protocols based on risk assessments of the work by HCV from HCW to patients; to date, six published

diﬀerent health care staﬀ, and national guidelines such reports, resulting in the infection of 14 patients (Gunson

as those produced by the UK Health Departments or et al. , 2003). Additional cases, as yet unpublished are

the Health and Safety Executive. The aim of such under investigation. Transmission rates are lower than

protocols is to prevent, or at least control, exposure to for HBV (0.13–0.18%) (Henderson, 2003). However,

hazardous substances. HCWs should be trained in safe one should perhaps mention the atypical outbreak of

and practicable ways of performing routine tasks and HCV in Spain, in which a Spanish anaesthetist, who

these should be kept under regular review. was a morphine addict, infected 171 of his patients by

In the context of BBVs, ‘universal precautions’ sharing needles and opiates with them (Henderson,

should be applied, i.e. all blood, tissues and body ﬂuids 2003).

should be regarded as potentially infectious. The With regard to HIV, apart from the ﬁrst recorded

following general measures to reduce the risk of occupational exposure are recommended by the UK

outbreak, involving a Florida dentist with AIDS who Health Departments (Department of Health, 1998). appeared to transmit infection to six of his patients

(0.5% of 10 evaluated) (Centers for Disease Control, . Wash hands before and after contact with each 1991; Ciesielski et al., 1992), hundreds of look-back

patient, and before putting on and after using exercises, carried out on many thousands of patients

rubber gloves.

following the identiﬁcation of HIV-infected HCWs, . Change gloves between patients. have been very reassuring and have supported the view

. Cover existing wounds, skin lesions and all breaks that the risk of transmission is very small. Only two

in exposed skin with waterproof dressings. Wear nosocomial infections have been identiﬁed in two

gloves if hands are extensively infected. separate look-backs—one involving an orthopaedic

. Wear gloves where contact with blood can be surgeon in France and 1000 patients tested (Lot et al.,

anticipated.

1999) and one involving the Spanish anaesthetist . Avoid sharps usage where possible, and where mentioned above.

sharps usage is essential, exercise particular care in Finally, there have been many reports of patient-to-

handling and disposal.

patient transmission of BBVs, linked to a variety of . Avoid wearing open footwear in situations where medical and surgical procedures, including haemodia-

blood may be spilt, or where sharp instruments or lysis, orthopaedic, gynaecological and cardiothoracic

needles are handled.

surgery, endoscopy, colonoscopy, organ transplanta- . Clear up spillage of blood promptly and disinfect tion, anaesthetics, spring-loaded ﬁnger stick devices,

surfaces.

839 . Wear gloves when cleaning equipment prior to

HOSPITAL-ACQUIRED INFECTIONS

HCWs, including students, should be educated about sterilisation or disinfection, when handling chemi-

the possible risks from occupational exposure and be cal disinfectant and when cleaning up spillages.

made aware of the importance of seeking urgent advice . Follow safe procedures for the disposal of con-

following any possible exposure. Protocols on the taminated waste.

management of such exposures should be drawn up, made readily available and health care staﬀ should be

Safe handling and disposal of sharps involves the designated to whom HCWs may be referred immedi- adequate supply and positioning of sharps containers, ately for advice, counselling and management. HCWs the placing of all disposable sharps in such containers who sustain an occupational exposure should have immediately after use, not overfilling such containers been educated about the potential risks, the recom- and avoiding resheathing needles, unless done by the mended immediate first aid to the site of exposure, the single-handed technique with or without an appro- need for prompt reporting, and the sources available priate device. Specific guidance for reducing the risk of for urgent advice, management and treatment, both percutaneous and mucocutaneous exposure during within and outside normal working hours. surgical procedures is also given in this document.

Other general measures which should be employed The speciﬁc pre- and post-exposure measures by to prevent exposure to BBVs include adequate

which the risk of infection with BBVs in HCWs may be decontamination, i.e. sterilisation, disinfection and

reduced are discussed below for the three main BBVs. cleaning of equipment (Department of Health, 1996)

and cleaning and disinfecting of work surfaces. HBV

Pre-exposure PREVENTION OF INFECTION

All HCWs, including students and trainees, who have In addition to the general measures given above to

direct contact with blood, tissues or other body ﬂuids prevent or minimise exposure to BBVs in the health

should be immunised against HBV, and have their care setting, there are more speciﬁc pre- and post-

response to vaccine checked in order to ensure an exposure measures whereby the risk of infection with

adequate response. Non-responders should be given HBV, HCV and HIV may be reduced. In addition, all

appropriate advice regarding the management of

Table 29A.2 HBV prophylaxis for reported exposure incidents HBV status of

Non-signiﬁcant exposure person exposed HbsAg-positive

Signiﬁcant exposure

Continued risk No further risk source

Unknown source

HbsAg-negative

source

Initiate course of No HBV pre-exposure

4 1 Dose HB vaccine Accelerated course

Accelerated course Initiate course of

of HB vaccine*

HB vaccine prophylaxis HBIG61

of HB vaccine*

HB vaccine

Reassure 5 2 Doses HB vaccine

Finish course of No HBV pre-exposure (anti-

One dose of HB

Finish course of HB

HB vaccine prophylaxis HBs not known)

vaccine followed

vaccine

by second dose Reassure one month later Known responder

Consider booster No HBV to HBV vaccine

Consider booster

dose of HB prophylaxis (anti-HBs

dose of HB

vaccine Reassure 5 10 miU/ml) Known non-responder

HBIG61. Consider HBIG61. No HBIG. Consider No HBIG. No to HB vaccine (anti-

Consider booster prophylaxis HBs 510 miU/ml)

booster dose of

Consider

booster dose of HB

dose of HB Reassure 2–4 months post-

HB vaccine

booster dose of

vaccine

vaccine vaccination

HB vaccine

*An accelerated course of vaccine consists of doses spaced at 0, 1 and 2 months. A booster dose may be given at 12 months to those at continuing risk of exposure to HBV. Reproduced from PHLS Hepatitis Subcommittee (1992)

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

potential exposures, and should be investigated to with or without other anti-HIV drugs, may reduce the identify those who may pose a risk to their patients

risk of becoming infected with HIV following a during exposure-prone procedures (EPPs). Occupa-

percutaneous exposure (CDC, 1998). HIV PEP, there- tional health services are responsible for keeping

fore, usually as a combination of three antiretroviral accurate immunisation records for all employees and

drugs, is recommended following percutaneous expo- administering booster doses where appropriate.

sure or exposure of broken skin or mucous membranes to material known or strongly suspected to be infected with HIV. Details are given in the national guidelines

Post-exposure (Department of Health, 1997; Centers for Disease Control, 1998).

In the UK, guidance from the PHLS Hepatitis Sub- committee is summarised in Table 29A.2, which gives advice depending on the HBV status of the source and the HBV vaccination status of the recipient. Hepatitis

B immune globulin (HBIG) is available as post- PREVENTION OF HCW-TO-PATIENT exposure prophylaxis (PEP) for unvaccinated indivi-

TRANSMISSION duals or non-responders. Finally, I would like to discuss measures recommended to minimise the risk of transmission of BBVs from

HCV HCWs to patients. As mentioned previously, although the risks are small, such infections attract a dispropor-

Pre-exposure tionate amount of media publicity. Currently, in the UK, with regard to HBV, all

There is currently no vaccine available for HCV. HCWs who carry out EPPs must be screened for HBsAg and HBeAg. All found to be eAg-positive, or eAg-negative but with HBV DNA levels of 410 3

Post-exposure genome equivalents/ml, are precluded from EPPs

(Department of Health, 2003b). HCWs who feel that There is currently no eﬀective post-exposure prophy- they may be at risk of HIV or HCV infection are laxis for HCV, and any potential exposure should be encouraged to be tested. Any HCW who is HCV- or managed according to national guidelines. Close HIV-positive and found to have transmitted infection follow-up of exposed individuals is recommended with to patients must refrain from EPPs (Department of baseline and follow-up blood tests, e.g. HCV RNA Health, 2003a). Some countries have adopted similar PCR assays, HCV antibody tests or liver function tests policies; others are less Draconian. However, in view (LFTs). Recommended intervals vary according to the of the ever-increasing reports of BBV transmission proposed form of management. ‘Watchful waiting’ is from HCW to patient, many countries are reviewing likely to be replaced by pre-emptive therapy with

the situation.

immunomodulators as the evidence for their eﬃcacy in In the UK, the Department of Health recently reducing the risk of chronic infection accumulates

(Henderson, 2003). published a consultation document, Health Clearance for Serious Communicable Diseases: New Health Care Workers , in which it is recommended that all new

HIV HCWs who carry out EPPs will need to have standard health clearance for serious communicable diseases Pre-exposure and additional health clearance for BBVs. It seems likely that restrictions will be imposed on the working There is currently no vaccine available for HIV.

practices of HCWs infected with any of the three main BBVs, HBV, HCV or HIV.

Increasing concern about HCW-to-patient transmis- Post-exposure

sion of BBVs is also evident from the recent publication of a consensus statement from a European

Although the risk of infection with HIV following consensus group, the aim of which is to reduce the risk exposure is small, there is now evidence that post-

of transmission of HBV and HCV from infected exposure prophylaxis (PEP) with zidovudine (AZT),

HCWs to patients (Gunson et al., 2003).

841 REFERENCES

HOSPITAL-ACQUIRED INFECTIONS

Guidance for Consultation . UK Department of Health, London.

CDSC (1999) Occupational Transmission of HIV, Summary Department of Health (2003b) Hepatitis B-infected healthcare of Published Reports, PHLS AIDS & STD Centre at the

workers: http://www.doh.gov.uk/nhsexec/hepatitisB/html Communicable Disease Surveillance Centre and Collabora-

Gunson RN, Shouval D, Roggendorf M et al. and the tors, December 1999.

European Consensus Group (2003) Hepatitis B virus Centers for Disease Control (1987) Recommendations for

(HBV) and hepatitis C virus (HCV) infections in health prevention of HIV transmission in health care settings.

care workers (HCWs): guidelines for prevention of Morb Mortal Wkly Rep , 36, 1–8.

transmission of HBV and HCV from HCW to patients. J Centers for Disease Control (1991) Recommendations for

Clin Virol , 27, 213–230.

preventing transmission of human immunodeﬁciency virus Hadler SC, Sorley DL, Acree KH et al. (1981) An outbreak and hepatitis B virus to patients during exposure prone

of hepatitis B in a dental practice. Ann Intern Med, 95, invasive procedures. Morb Mortal Wkly Rep, 40 (RR-8),

1–9. Henderson DK (2003) Managing occupational risks for Centers for Disease Control (1998) Public Health Service

hepatitis C transmission in the health care setting. Clin Guidelines for the management of health-care worker

Microbiol Rev , 16, 546–568.

exposures to HIV and recommendations for postexposure Heptonstall J, on behalf of The Incident Investigation Teams prophylaxis. Morb Mortal Wkly Rep, 47 (RR-7), 1–28.

(1997) Transmission of hepatitis B to patients from four Ciesielski C, Marianos D, Ou CY et al. (1992) Transmission

infected surgeons without hepatitis B e antigen. N Engl J of human immunodeﬁency virus in a dental practice. Ann

Med , 336, 178–184.

Intern Med , 116, 798–805. Department of Health (1998) Guidance for Clinical Health

Klein RS, Freeman K, Taylor PE and Stevens CE (1991) Care Workers: Protection Against Infection with Blood-

Occupational risk for hepatitis C virus infection among borne Viruses. Recommendations of the Expert Advisory

New York City dentists. Lancet, 338, 1539–1542. Group on AIDS and the Advisory Group on Hepatitis . UK

Lot F, Segvier JC, Fegueux S et al. (1999) Probable Department of Health, London.

transmission of HIV from an orthopaedic surgeon to a Department of Health (1996) Sterilisation, Disinfection and

patient in France. Ann Intern Med, 130, 1–6. Cleaning of Medical Equipment: Guidance on Decontamina-

PHLS Hepatitis Subcommittee (1992) CDR Rev, 2, R97– tion . Microbiology Advisory Committee to the Department

R101.

of Health: Part 1, Principles. UK Department of Health, Shapiro CN, Tokars JI, Chamberland ME and the American London.

Academy of Orthopaedic Surgeons Serosurvey Study Department of Health (1997) Guidelines on Post-exposure

Committee, Atlanta, Georgia (1996) Use of the hepatitis Prophylaxis for Health Care Workers Occupationally

B vaccine and infection with hepatitis B and C among Exposed to HIV . UK Department of Health, London.

orthopaedic surgeons, J Bone Joint Surg, 78-A, 1791–1799. Department of Health (2003a) Health Clearance for Serious

Welch J, Webster M, Tilzey AJ et al. (1989) Hepatitis B Communicable Diseases: New Health Care Workers, Draft

infections after gynaecological surgery. Lancet, 1, 205–207.

29B Infections Acquired by Other Routes

Philip Rice

St George’s Hospital Medical School, London, UK

HERPES SIMPLEX VIRUS (HSV) identiﬁed, prophylaxis may be taken to cover these periods, e.g. the summer holidays.

The prevention of nosocomial HSV infections relies It is important to note, however, that although entirely on good standards of personal hygiene.

nosocomial transmission has been proven by Although virus may be shed in the genital tract and

restriction enzymatic digestion analysis of cultured in saliva without clinical lesions, adhering to hand-

isolates, often the identity of the presumed common washing with soap and water or alcohol-based gels is

source has remained undiscovered (Linnemann et al., suﬃcient to destroy infectious virus. The main reason

1978). Virus has also survived for more than 60 h on a for concern over nosocomial HSV infections is the

cot that had been used for a baby who died from potential for transmission to neonates, born either pre-

disseminated HSV infection. More than 60 h after this term or term, as neonatal HSV infection has extremely

baby died, the cot was re-used without being deconta- high rates of mortality and morbidity even with the

minated. A second neonate was then nursed in it. prompt use of antivirals. Approximately one-third of

Twelve days later this infant also died (Sakaoka et al., cases of neonatal HSV infection are thought to be

acquired from a non-maternal source. It is vital, therefore, that staﬀ employed on delivery suites, postnatal wards and neonatal units understand the

VARICELLA-ZOSTER VIRUS (VZV) importance of hand-washing and of the absolute requirement to absent themselves from work if they

Preventing patients and staﬀ from contracting develop an herpetic whitlow. The more commonly

chickenpox in hospitals forms a signiﬁcant part of faced scenario, however, is of the midwife or neonatal

the workload of a clinical virologist. Outbreaks cause nurse with a cold sore. The only truly safe way to

signiﬁcant disruption to wards, occasionally entire manage such occurrences is to remove the individual

hospitals, increase the workload of clinical virology from all clinical duties until the lesion has dried and

laboratories at very short notice, and necessitate crusted over. Although virus will still be shed in saliva,

considerable expense when susceptible staﬀ have to the likelihood of transmission is lower than with wet or

be excluded from work after exposure. Chickenpox vesicular skin lesions. One practical measure for staﬀ

can also occur at any age. We have personally seen who develop frequent recurrences could be to advise

primary infection in two 65 year-olds, a 78 year-old them to have readily available oral aciclovir, which can

and a man of 92 years!

be started immediately prodromal symptoms appear. When a patient with varicella-zoster virus (VZV) Such an approach, combined with strict hand-washing,

infection is identiﬁed, all staﬀ in contact with the case will help to further reduce the risk of transmission.

must know their immune status. A well-taken history Additionally, if trigger factors such as sunlight can be

of previous chickenpox correlates very well with

The transmission rate of chickenpox to susceptible staff from contact with VZV has been estimated to be between 4.7–29% after exposure (Myers et al., 1982; Wreghitt et al., 1996; Langley and Hanakowski, 2000). There are several factors that exert an effect on this rate, most of which are extremely difficult to quantify. However, there are general guidelines on the extent of exposure which can be helpful in deciding whether a real exposure has occurred. Examples of significant exposures have been defined as a face-to-face contact or being present in the same room for 1 h with an individual with chickenpox or disseminated/uncovered zoster (Salisbury and Begg, 1996). At the other end of the exposure spectrum, the likelihood of transmission taking place from cases of covered, e.g. thoraco- lumbar, zoster is remote. However, airborne transmission on open wards has been described, even, in our experience, to a susceptible neonatal unit registrar after

a patient with chickenpox had been placed in a single room and with whom he had had no direct contact, the implication here being that virus escaped into the corridor.

Although chickenpox is much more infectious than zoster, most of the practical problems involved with control of infection arise from patients who have zoster. This is simply because chickenpox is easily recognisable and such patients are admitted immediately into a side room. Zoster, however, often develops days or weeks after admission and has usually been present for several days before the diagnosis is clinically suspected. Thus, many more staﬀ and patients are exposed to VZV after contact with patients who have zoster than those with chickenpox. This was demonstrated clearly in a study from Cambridge, UK, where during a 5 year period, only 1/28 (3.6%) of

susceptible staﬀ developed chickenpox after an exposure to varicella, compared with 5/29 (17.2%) after exposure to zoster (Wreghitt et al., 1996).

It is wise to remember, however, that chickenpox can be mistaken for other vesicular exanthems due to enteroviruses or even allergy, contact dermatitis or eczema. If you are presented with the information that

a member of staff or a visitor/relative on the neonatal unit, for example, has developed chickenpox, the implications for the unit are considerable. Multiple doses of VZIG will almost certainly need to be administered, susceptible staff will need to be sent home, visiting may become restricted and it is possible that the unit will have to be closed to new admissions. Thus, although it is an extreme example of what might happen, before embarking on the above course of action it is essential that the diagnosis of chickenpox be confirmed by an experienced virologist or microbiol- ogist with access to laboratory facilities capable of providing rapid results, either by direct immunofluorescence or real-time PCR on vesicular lesion scrapings.

If a susceptible member of staff has been exposed, the simplest way to manage the exposure is to send that member of staff home, commencing on day 8–10 after contact until day 21 after the first exposure. This is because cases of chickenpox are infectious 2 days prior to rash onset. Another option used quite commonly is to allow staff to remain at work, provided that they feel well and are afebrile (Josephson et al., 1990; Weber et al., 1996). They should be instructed to take twice daily temperatures and to remain at home if they are febrile, as this could be the first sign of a prodromal varicella illness. This measure may be used more frequently in critical members of staff whose absence from a clinical area would be detrimental to patient care. Alternatively, the individual could perform duties that do not involve patient contact, although the usefulness of this approach for an experienced ward sister, for example, is questionable. Theoretically, however, and probably practically, the most cost-effective long-term way of managing such staff is through active vaccination against VZV, either before or after an exposure (Centers for Disease Control, 1999).

Live attenuated varicella vaccine has been in use for more than 20 years in Japan and the Far East and for almost a decade in the USA, but only recently have two varicella vaccine products been licensed in the UK for susceptible individuals 412 years of age.

Both products, one from Glaxo Smith Kline, the other from Aventis-Pasteur, are highly eﬀective at preventing chickenpox prior to exposure and produce

a seroconversion rate of 495% after a two-dose

844

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

schedule (Sharrar et al., 2000). Although pre-exposure vaccination has been shown to be cost-effective, this has been assessed only in mathematical models (Gray et al. , 1997) and there are several important practical considerations before advising a staff-wide vaccination policy. These include how to manage staff working in high-risk areas, as a small proportion of vaccinees will develop a rash both at the site of vaccination and outside of the injection area, and there is also a measurable rate of breakthrough infection after exposure to wild-type virus and thus the potential for ongoing transmission.

The rate of vesicular rash post-vaccine is ca. 8% (Arbeter et al., 1986). However, in the majority the rash is only maculopapular, and in the 30% where it is vesicular, the average number of lesions is only 14. The nature of the rash and the lack of evidence for pharyngeal excretion of virus probably explain why transmission of vaccine virus to another healthy susceptible individual occurs only extremely rarely. Nevertheless, to take account of this problem, staﬀ employed in high-risk areas should take particular care in the 2 weeks after both doses and report to occupational health immediately a possible varicella- like rash develops. Breakthrough infection, however, is potentially more troublesome, as this is a long-term problem and occurs at a rate of 5–8% after an exposure (Arbeter et al., 1986; Watson et al., 1993). The average timing for such breakthrough infections is

30 months post-vaccination (Watson et al., 1993). Despite breakthrough cases of infection being described after contact with VZV at work, where presumably the degree of exposure is less intense

compared with at home, transmission of wild-type virus from cases of breakthrough infection to susceptible contacts, even within the home, is only 12% (Watson et al., 1993). This should be compared with a household transmission rate of 80–90% for natural varicella. It should also be noted that breakthrough cases have far fewer lesions, on average 31 (1–100) compared with the mean number of lesions seen in ordinary varicella of 250–500. Such breakthrough infections may occur after a documented seroconversion to VZV vaccine, but in a study by Gershon et al. (1988) 9/12 (75%) of staﬀ experiencing breakthrough were shown to be seronegative after their initial vaccination. It is possible, therefore, that this group could be oﬀered a booster dose of vaccine post- exposure and re-tested in 7 days to assess the response. If antibody were then detected, they could almost certainly continue to work uninterrupted.

VZV vaccine as a post-exposure measure is also highly eﬀective, provided that it is given within 3 days of contact with infectious material. A study from Asano et al. (1977) examined 26 contacts in 21 families who were immunised within 3 days of the index case developing chickenpox. None of the vaccinees developed a rash. However, a control group of 19 unimmunised contacts in 15 families were all infected. In the same study, Asano (1977) also gave vaccine to

34 healthy siblings within 3 days of the index case rash; 2/34 (5.8%) got varicella. The protective eﬃcacy of post-exposure vaccination is therefore 490%. Whilst this approach may not necessarily allow the member of staﬀ to remain at work in very critical areas, such as bone marrow transplantation units, the exposure that

HOSPITAL ACQUIRED INFECTIONS

Table 29B.1 Arguments for and against the use of varicella vaccine for health care workers For

Against Eliminate natural varicella from staﬀ

Of great use especially in high-risk areas (paediatrics, HIV, transplant, neonatal units) Reduce potential for nosocomial transmission Much lower risk of transmission to pregnant staﬀ Very good safety record: in use for 20+ years Lower incidence of shingles in vaccinees Lower rate of vaccine-related events compared with natural infection Durable immunity with natural boosters Virtually no risk of transmission from one healthy person to another Less worry for ﬁnal exams in students (5% intake susceptible) Less risk of transmission for susceptible pregnant women (c. 25% of whom are in

ethnic minorities where the prevalence of adult immunity is much lower approximately 50%)

May be used post exposure (recent US and UK license) Potential case for complaint or worse, if transmission of wild-type virus occurred from

a susceptible staﬀ member to vulnerable patient (e.g. a pregnant woman)

Cost £80/person Administration costs Uncertain durability of immunity Vaccine-associated rash Latent virus reactivation as zoster Possible need to send oﬀ or restrict to

non-clinical work if breakthrough infection occurs

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

led to the use of VZV vaccine as a post-exposure sometimes oﬀered CMV screening, by other staﬀ or measure should be that individual’s last time of

occasionally occupational health departments, princi- needing to be sent home from work for 2 weeks.

pally as a means of providing reassurance. However, For the 3 years 2000–2002 at St George’s Hospital,

such screening tests are offered in ignorance of the data all cases of varicella in staff and students notified to the

concerning the risks of transmission and are erro- medical microbiology and occupational health depart-

neously believed to be capable of determining whether ments have been documented. A total of 25 cases have

a person is ‘immune’ to CMV infection. Moreover, the been notiﬁed from all staﬀ/student disciplines. Of these

potential implications and consequences of commen-

25 cases, 13/22 (59%) occurred in individuals born cing CMV antibody testing in pregnant members of outside of the UK, where the country of birth was

staﬀ are never considered by those requesting the tests. known. If pre-exposure vaccination had been adopted

Indeed, the ﬁrst time that a clinical virologist will be at St George’s, even amongst only the nursing and

involved is when the pregnant member of staﬀ contacts medical staﬀ and students, 85% of chickenpox cases

him/her to discuss the results of her own CMV would have been prevented (Devi and Rice, 2002).

serology and its signiﬁcance. This is where the One other problem with the issue of immunising

diﬃculties arise. Indeed, as discussed in the CMV staﬀ against chickenpox is that it is a live attenuated

chapter, the concept of CMV immunity, especially in vaccine and as such may reactivate as zoster later in

relation to pregnancy, is incorrect, since reactivation life. However, the vaccine probably oﬀers beneﬁt over

and reinfection may lead to congenital infection and wild-type infection, because the rate of zoster with

disease in newborns.

reactivated vaccine virus is about 50% lower than after Although the CMV serology may indicate past wild-type infection. The key problem with a wide-

infection or susceptibility, occasionally CMV IgM is spread staﬀ immunisation policy, even though it is

detected, which produces considerable anxiety. established that vaccinating susceptible staﬀ will

Further tests, such as IgG avidity, are then required, reduce considerably the risk to patients from nosoco-

as well as a hunt for earlier samples to try to determine, mial varicella, is that immunity to infection after

as accurately as possible, the timing of any CMV vaccination is not guaranteed as it is after natural

infection. Furthermore, when past infection is shown, chickenpox, and that it is not possible to identify which

the potential for reinfection or reactivation must then staﬀ may be at risk of breakthrough infection after an

be discussed. If the member of staﬀ is susceptible, the exposure.

discussion widens to include the potential beneﬁt and The decision has to be made as to how to manage

harm of follow-up testing and prenatal diagnosis and immunised staﬀ in these circumstances. The data

the possible outcomes for the baby if a primary indicate, that whilst transmission of breakthrough

infection is demonstrated.

wild-type varicella infection may occur, especially to It is obvious from this outline that screening of staﬀ immunosuppressed patients, this is less likely than

for CMV is a big undertaking and should only be done after natural varicella. If a staﬀ-wide vaccination

after a prolonged and informed discussion with the policy were to be instituted, this should be accom-

clinical virologist and an obstetrician. The member of panied by a concerted eﬀort to immunise

staﬀ should be advised that if she has been adhering to simultaneously the most vulnerable patient groups.

universal precautions and has washed her hands with Should such a policy be contemplated, the points set

soap and water or an alcohol-based solution after out in Table 29B.1 may assist in making the decision.

dealing with patients, serological testing for CMV should not be performed. This is because there are several well-conducted studies which all conclude that

CYTOMEGALOVIRUS (CMV) there is no evidence that CMV excreted by hospitalised patients pose a signiﬁcant additional risk for pregnant

CMV is one of the easiest viruses to control in hospital, staﬀ (Dworsky et al., 1983; Young et al., 1983; Balfour yet probably causes more anxiety, especially among

and Balfour, 1986; Demmler et al., 1987; Balcarek et pregnant staﬀ, than any other virus, apart from HIV.

al. , 1990).

The anxiety usually results from the identiﬁcation of a The annual incidence of CMV infection in hospital baby or infant with congenital infection, often on the

staﬀ is approximately 2% and is no diﬀerent between neonatal unit. As congenitally infected babies often

various occupational groups within and the general excrete a high titre of virus, there are understandably

population outside hospital. Balcarek et al. (1990) concerns at the potential risk that these babies pose to

showed, for example, that there was no difference in pregnant members of staff. At this stage such staff are

the rate of infection in staﬀ regardless of area of

847 work, job type (nurse, doctor, support services,

HOSPITAL ACQUIRED INFECTIONS

CMV serology can be fraught with diﬃculties if low or administrative staﬀ) or number of patient contact

even high level IgM is detected. hours of 10–40 h/week. Moreover, when the rates of

Environmentally acquired infection also does not CMV excretion by patients are known in diﬀerent

appear to be a problem. As CMV is an enveloped areas of the hospital, this does not appear to inﬂuence

virus, it does not survive well on inanimate surfaces or the rate of transmission to staﬀ. Demmler et al.

on hands. This was shown clearly in a study in a (1987) showed this even when rates of CMV excretion

paediatric hospital, where virus was not isolated from varied from 3% in newborns to 16% in a chronic

any equipment, toys or hard surfaces and, although it care unit for children, many with severe neurological

was isolated from the dry hands of staﬀ members, it and physical handicap, a proportion of whom would

was removed completely by hand-washing with soap have had congenital infection and be excreting high-

and water (Demmler et al., 1987). Simple hygienic titre virus in urine.

measures and the washing of surfaces with household Further proof that patients are not the source of

detergent is suﬃcient to remove infectious material infection has come from studies which have examined

from the environment.

virus strains detected in staff who are shown to seroconvert at work. In these cases either the strains of virus in staff and patient have been different or

MEASLES, MUMPS AND RUBELLA another source outside of hospital, often a sexual

partner, child at home, etc., has been shown to be shedding an identical virus (Onorato et al., 1985;

Measles Virus Demmler et al., 1987).

The risk of measles infection occurring in medical The single study that did show an increased

personnel is estimated to be 13-fold higher than in the incidence of infection among staﬀ, demonstrated this

general population (Davis et al., 1986; Atkinson et al., only for student nurses on their ﬁrst placement in

1991). Approximately 5–10% of all notiﬁed cases of paediatrics (Haneberg et al., 1980). Such staﬀ were

measles are reported to have been acquired in a reportedly observed to kiss drooling infants while

medical setting and over one-third of cases acquired in feeding them. Indeed, this paper highlights the fact, as

hospitals are in health care workers, mostly (485%) stated by others, that intimate and prolonged contact

previously unvaccinated (reviewed by ACIP/HICPAC, is required for CMV transmission to occur (Onorato et

al. , 1985). This would explain the increased rate of Measles virus infection is now a particular problem infection seen in pregnant mothers with a young child

partly because of the success of MMR vaccination, at home or in staﬀ employed in child day-care, where

because whilst this has dramatically reduced the the transmission rate may be 10–25%/year (Adler,

incidence of infection in the community, the clinical 1989).

skills required to diagnose measles have also waned. We would certainly not advise routinely screening

Moreover, as the population of immunosuppressed staﬀ after a possible exposure occupationally to CMV,

(organ/bone marrow transplantation, intensive care as considerable anxiety is experienced whilst waiting

and HIV) patients has continued to grow, atypical for the results and there can be diﬃculties in their

presentations are more likely to occur and there is a correct interpretation. Indeed, when this was

higher risk of the diagnosis being missed (Kidd et al., attempted in a hospital in the UK some years ago it

2003). Thus, as measles virus has a reproduction rate was a ‘disaster’ and led to ‘public relations, psycholo-

of 14:1, it can easily re-emerge in developed countries, gical and management problems’ (reviewed in Young

due largely to the importation of measles from et al. , 1983). The key to preventing infection relies

countries with less well-developed measles vaccination upon the education of staﬀ about how the virus is

programmes (Ramsay et al., 2003). This becomes even transmitted (sexually, kissing, lapses in hand-washing,

more a problem if the MMR uptake rate falls, as seen especially after changing nappies) and about the many

recently in the UK. Once imported, measles can then pitfalls of antibody testing. If a staﬀ member has

circulate without the need for further outside intro- requested to be tested after an exposure, ideally the

ductions (Thomas et al., 1998). virologist should see her to discuss all of the issues

There are, however, certain clinical criteria that can relating to the possible outcomes. The issues to be

assist in the diagnosis of measles. One acronym that we discussed would be the fact that CMV infection is

have found helpful is the acronym CCDC, already in ubiquitous, staﬀ are at no increased risk of infection,

use in the UK for Consultant in Communicable transmission is interrupted easily by hand-washing and

Disease Control (CCDC). When applied to measles it

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

becomes cough, coryza, diarrhoea, conjunctivitis, hospital employees. This is because, given recent falls accompanied by rash and fever. The diagnosis

in the uptake of MMR, we may be creating a cohort of becomes almost certain in the context of a children, at least in the UK, which will become large

community-wide outbreak or an epidemiological link enough to sustain not only local virus transmission but with other cases. Travel abroad or contact with

also be a source for susceptible infants who have yet to recently returned travellers also increases the like-

receive MMR. This is because as most mothers now lihood of the diagnosis, as does having had no or only

have vaccine-induced immunity, measles antibody will one dose of MMR vaccine. Other clues to the

have disappeared in infants by 9 months and infection

diagnosis are lymphopaenia (50.6610 9 /l) and slight

under 1 year of life presents a much greater risk of

developing Sub-Acute Sclerosing Panencephalitis ideally be admitted to a negative-pressure room or, if

thrombocytopaenia (5150610 9 /l). Patients should

(SSPE) as an older child.

this is not possible, to a single room, and be cared for To contain measles in a hospital, the only eﬀective only by staﬀ who are aware of their measles immunity

measures are to isolate the patient and to maintain status. A strict gloves, aprons and hand-washing policy

high uptake rates of primary measles immunisation. must be adhered to if further infection in staﬀ or

Even post-exposure vaccination may be of dubious patients is to be avoided. Transmission is by droplet

merit. When administered in an epidemic setting, the infection, although fortunately the degree of infectivity

eﬃcacy can be as low as 4% (King et al., 1991). Our reduces quickly after the rash appears and lasts for

experience has also been very similar when attempting only 3–5 days afterwards. The incubation period can

to prevent an outbreak in infants at a nursery. The

be as short as 7 days, but more typically is 10–13 days. same day the index case was notiﬁed, which was on the However, because it may be as long as 19 days,

day the rash appeared, four of the six contacts, all aged susceptible staﬀ should be excluded from work for 5–

4 1 but 52 years of age and previously unimmunised

21 days after contact. with MMR, were vaccinated; 10–14 days later all six Problems have arisen in the UK as at present there

children developed clinical measles, conﬁrmed by is no formal policy of establishing measles immunity

detection of wild-type virus in one of the vaccinees for all staﬀ (Mendelson et al., 2000). Guidelines

(Rice et al., 2004). Thus, it is essential to achieve the already in place in the USA, however, recommend

highest possible level of staﬀ immunity as post- that all health care workers provide evidence of

exposure prophylaxis, whilst advisable, should not be immunity to measles through either a documented

relied upon to prevent secondary cases. Failure to do physician-diagnosed measles, two doses of live

so will eventually result in ward closure and death in attenuated measles vaccine after their ﬁrst birthday

unimmunised immunocompromised patients (Kidd et or a positive measles antibody test result (Krause et

al. , 2003).

al. , 1994). A cut-oﬀ year of 1957 is suggested, as people born before this year are very likely to have had measles infection as a child. However, as the

Mumps Virus susceptibility rate in this age group is still 5–9%,

individuals in this group who cannot provide evidence Mumps outbreaks in closed communities, including for physician-diagnosed measles must undergo anti-

hospitals, have been described; especially when there body testing and, if susceptible, be given two doses of

are many opportunities for close contact (Wharton et MMR (Braunstein et al., 1990; Smith et al., 1990;

al. , 1990). One such outbreak occurred at trading Schwarcz et al., 1992). Even this approach may not

ﬂoors in Chicago in 1987, with over 100 cases (Kaplan always be successful, as shown in The Netherlands,

et al. , 1988). The reason for an increase in cases in where there was measles transmission to susceptible

young adults is again because of the success of MMR staﬀ even though the overall level of immunity was

vaccination. This has led to fewer exposures for a 98.5% (de Swart et al., 2000). Pre-exposure vaccina-

cohort of young adults who were ineligible for tion is known, however, to be highly eﬃcacious, as

immunisation, yet who have also escaped natural shown by two primary school outbreaks where the

infection. A similar problem is also being seen in the attack rate in unimmunised children was 26–46% yet

UK, where three-quarters of reported cases have been it was only 0.4% in children who had had MMR

in children and young adults aged 10–19 years (PHLS, (Richardson and Quigley, 1994).

2003). Controlling mumps virus in hospitals is still It is essential and, indeed, a responsibility of hospital

important because of the risk of meningitis and facilities to ensure that their staﬀ have made all

deafness. A high index of clinical suspicion, hand- practicable eﬀorts to eliminate measles from all

washing and admission to a single room with droplet washing and admission to a single room with droplet

Rubella Outbreaks of rubella in hospitals are now rare, due

largely to MMR vaccination, effective staff and student antibody screening and targeted immunisation (Greaves et al., 1982). The susceptibility rates in staff in the 1970s and 1980s varied from 5% to 18%, depending on age (Polk et al., 1980). With such high susceptibility rates, it was possible for rubella virus to circulate in hospitals and large outbreaks were described (Polk et al., 1980). However, as non-immune staff are now much less common, if transmission to a member of staff does occur it is just as likely that this merely reflects a larger community-wide outbreak. However, individuals born in developing countries where rubella vaccination programmes are non-exis- tent are at increased risk of infection in hospital if a health care worker develops rubella. Indeed, of particular concern are women from southern Asia and Africa, where susceptibility rates may be almost 20% in those of childbearing age (Miller et al., 1990; Devi et al., 2002). Furthermore, infants with congenital rubella syndrome are a potential source of infection for susceptible staff and other infants, due to the excretion of high-titre virus in urine for prolonged periods, approximately 2 years (Aitken and Jeffries, 2001). Indeed, vigilance to prevent nosocomial infection must be maintained, since it was recently demonstrated that a baby with congenital rubella syndrome (CRS) infected another baby in a neighbouring cot (Sheridan et al., 2002). The only way that transmission could have taken place is by non- adherence to hand-washing by staff after dealing with urine and other bodily fluids from the baby with CRS. The best way to prevent nosocomial rubella is to fully investigate staff members with rash illness, remembering that in the first 3 days after the rash has appeared they may be IgG- and IgM-negative. Once identified, colleagues in close daily contact should be tested for rubella IgG, with the focus on pregnant staff 518 weeks’ gestation. Although post-exposure rubella

vaccination is ineﬀective in preventing infection, any non-pregnant susceptible staﬀ should then be immunised and tested 2–3 months later for a response.

PARVOVIRUS B19

Infection with human parvovirus B19 occupies a unique place among nosocomial virus infections. This is because once a single case of symptomatic infection has been identified in a member of staff, secondary cases will have already been infectious for several days and have probably infected further tertiary cases who, in turn, will be infectious in the next few days. Thus, unlike all other virus infections, once identified, the index case poses no further risk of infection to patients or staff; the problems lie with the contacts (staff and patients), whose infectivity or immunity status is as yet unknown. It is this group which presents a significant risk in the ongoing spread of infection. There are two reasons for this. First, the typical symptoms of rash and arthralgia manifest only when virus antigen– antibody complexes are formed, so that the index case presents when no longer infectious. Second, volunteer studies have shown that the period from exposure to such symptoms is normally 13–18 days (maximum 21 days), yet the period from exposure to infectiousness is only 7 days. In addition, as an estimated 30–40% of adults are susceptible and 20– 30% of adult cases are symptomless, it would appear likely that virus transmission would pose significant problems within hospitals.

Nosocomial transmission may also be especially hazardous, as there are particularly vulnerable patient groups at risk of adverse outcomes: pregnant women, the immune-compromised and those with reduced red blood cell survival, who may develop a life-threatening aplastic crisis. However, the available information suggests that whilst hospital outbreaks do occur, they are uncommon. Moreover, before ascribing cases detected within a hospital to be nosocomial, they need to be examined carefully in the context of a wider community epidemic that may be occurring concur- rently. Indeed, a hospital pseudo-outbreak was described in a maternity unit after several cases among staff and patients during the preceding weeks had been notified (Dowell et al., 1995). However, when cases of acute infection on other wards, different hospitals and even healthy control subjects were sought, it was demonstrated that the percentage of individuals with recent infection, both in and out of hospital, was not significantly different, being 23–30%

HOSPITAL ACQUIRED INFECTIONS

849

in all locations or wards. The most likely explanation put forward was the presence of the largest community-wide parvovirus outbreak for 18 years (Dowell et al. , 1995).

When hospital outbreaks have been described they appear initially to be explosive, since they are noticed only after many cases in staff and patients have occurred, often over a period of several days. This is demonstrated by the attack rate of 27–50% in susceptible individuals, both staff and patients (Bell et al. , 1989; Pillay et al., 1992; Seng et al., 1994; Miyamoto et al., 2000; Lui et al., 2001). What is less clear is whether or not any infection control measures can prevent further virus transmission. This is important, since the measures proposed are potentially very costly in terms of staff absences or redeployment and possible ward closure. Indeed, to limit the spread of infection some authors have proposed ward closure, transfer of only immune staff to any affected ward, and restriction of the nursing staff to working only on the affected ward (Pillay et al., 1992). However, by the time that an outbreak has been notified it is highly likely that the majority of virus transmissions that are going to occur have already taken place. This was clearly demonstrated in an outbreak at a UK London teaching hospital (Seng et al., 1994). Over a 1 month period on a single ward, a total of 18 cases of acute parvovirus B19 infection were detected in 15 staff, of whom 12 were symptomatic; three patients were also affected. However, of the 12 symptomatic staff and the three affected patients, all who had known dates of symptom onset, 10 either were or had recently been symptomatic prior to outbreak notification. Moreover, of the five further cases that were going to occur, four developed symptoms within 8 days of notification of the index case. Thus, only one case was possibly preventable by the institution of infection control measures (Seng et al., 1994). Another outbreak on a paediatric ward demonstrated that 50% of cases might have been prevented had notification to the infection control team been made earlier (Pillay et al., 1992). These hospital outbreaks mirror the experience from community outbreaks, e.g. in schools. The high transmissibility of parvovirus B19 was shown in an outbreak investigated in a UK primary school for children aged 3–11 years during the epidemic year of 1994 (Rice and Cohen, 1996). This showed that 75% of the total number of cases had already occurred by the time the first few cases had been notified and, of the remaining 25%, most would have been infected and have also been incubating the infection by this time. Any efforts aimed at limiting the spread of infection would thus have had little impact.

The high infectivity of parvovirus in an outbreak setting is also illustrated by demonstrating that after this single outbreak in the school, the prevalence of parvovirus IgG rose from 15% to 60–70%, i.e. to an adult level of immunity (Rice and Cohen, 1996). It is our experience that when single cases of infection are identified among staff, further transmission to other staff occurs probably only as a result of social contact. This was also raised as one of the reasons for the extensive ward outbreak referred to earlier (Seng et al., 1994). Moreover, given the reasonably high attack rate of 50% in close, social and household contacts and the long incubation period before symptoms appear, it may be possible to detect virus in susceptible contacts by PCR or even by using standard electron microscopy. This is an additional measure which may be undertaken to limit the spread of infection, by identifying those individuals who are in the infectious phase before they have mounted an antibody response.

Nosocomial transmission is a particular problem from patients with aplastic crises, since they present earlier in the course of their illness when they are still infectious. This was clearly demonstrated in a paediatric ward outbreak, where 12 health care workers were infected, most probably as a result of a failure to recognise the patient’s infection early enough (Bell et al. , 1989). This outbreak was, however, probably made more likely since in the two outbreaks, only 12% and 31% of staﬀ had pre-existing immunity before the outbreak. Transmission has also been described from immunocompromised patients with chronic infection, due to an inability to clear the viraemia, and who presumably still shed oropharyngeal virus (Lui et al., 2001).

As parvovirus can cause signiﬁcant harm to vulnerable patient groups and pregnant staﬀ, the following appear to be reasonable precautions to prevent virus spread:

. Patients with reduced red cell survival, especially children, should be admitted to a single room if they experience a sudden drop in haemoglobin consistent with a possible aplastic crisis. Acute infection should be assumed, especially if the reticulocyte count is normal or low, indicating a shutting down of erythropoeisis. They should not

be nursed by pregnant staﬀ and hand-washing should be re-emphasised (Bell et al., 1989; Pillay et al. , 1992, Crowcroft et al., 1999).

. Any staﬀ with rash, and especially those with arthralgia, should be investigated for parvovirus and rubella and their household and close social contacts oﬀered testing for recent or past infection.

850

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

However, not all laboratories offer testing and those that do may only perform them in weekly batches, and it will often be necessary to make a judgement on whether or not staff should be re- deployed or furloughed before test results are available. Those who are found to be susceptible, however, can then be tested for the presence of viraemia. Decisions may then be made about furloughing of such staff.

. Pregnant staff who are at less then 20 weeks of gestation are at an increased risk of miscarriage or fetal hydrops. They should be advised before testing of the low probability of any adverse outcome, due to the low transmissibility, moderate levels of adult immunity and low risk (ca. 12%) of miscarriage. However, advising whether an individual staff member may wish to remain at work may also be influenced by other factors, including whether or not the pregnancy was achieved using IVF.

RESPIRATORY VIRUSES Respiratory viruses are well established as agents

capable of nosocomial transmission. They are particularly well suited, for many reasons. They are highly infectious, with reproduction rates of 5–15:1, can spread by both droplet and aerosol routes and have short incubation periods. Explosive outbreaks are thus possible. Furthermore, during the epidemic season high community attack rates of 10% for influenza and 40% for RSV in the groups at highest risk of infection make possible multiple introductions into hospitals. They are capable of survival on surfaces and unwashed hands for hours and among immunocompromised patients infectious virus may be shed for weeks. Finally, vaccination is available only for influenza and effective antiviral agents are available for only two agents (RSV and influenza). They represent a significant financial burden on hospitals by prolonging hospital stays, increasing mortality in vulnerable patient groups, and increasing staff absences, with the need for hiring of temporary replacements at additional cost. Ward closure may sometimes be necessary to terminate an outbreak. There are also no tests for checking the immune status of staff. Indeed, for influenza and RSV, reinfection is common.

To prevent outbreaks, close cooperation between the microbiology department and the infection control team is essential. A key element of this is rapid communication of positive results on all patients in a

hospital, especially at the start of the winter season. To facilitate this, rapid diagnostic assays should be employed. A variety of methods are available, but regardless of sensitivity and specificity their proper use is the key to success in controlling infection. Near- patient testing, if used properly, has sensitivity approaching that of conventional immunofluorescent tests, but it still requires knowledgeable staff capable of collecting an adequate specimen. We undertook a small study to assess the quality of nasopharyngeal aspirates taken from an accident and emergency department. Almost one-third of samples contained insufficient cells capable of achieving a result with good negative predictive value. An alternative approach to the testing of all symptomatic infants during the RSV season, for example, would be to test those whose symptoms and presentation are atypical (Salgado et al. , 2002).

Newer PCR protocols involving real-time assays are capable of providing results as fast as immunofluorescent tests, with enhanced sensitivity. A recent paper demonstrated that real-time PCR using the light cycler could generate a result in less than 2 h after specimen receipt, and it was significantly more sensitive in older children and adults (Whiley et al., 2002). This study showed that of 77 samples positive by light cycler RT- PCR, seven were negative by immunofluorescence testing. Of these seven samples, however, six were from adults or children aged 412 years. This is compatible with lower viral loads in these individuals, most probably as a result of reinfection. Thus, PCR may offer advantages in certain groups, e.g. less cooperative children or adults who may be harder to sample. However, immunofluorescence assays are still the simplest way to test for multiple pathogens simultaneously.

Respiratory Syncytial Virus

The most effective methods for preventing nosocomial infection with RSV are through regular hand-washing by staff, parents and visitors, the wearing of gloves and gowns and either the cohorting of infected patients or admission directly into single cubicles. This limits spread mostly by introducing an ethos among all involved in patient care, that preventing nosocomial infection and hand-washing are important. Indeed, the effectiveness of such measures has been shown by several studies. Isaacs et al. (1991) studied hospital- acquired infection among children with congenital cardiac and lung disease. They showed a nosocomial

HOSPITAL ACQUIRED INFECTIONS

851

infection rate of 35% prior to the introduction of enhanced infection control measures of hand-washing and patient cohorting. Not only were they able to demonstrate a 66% reduction in the rate of nosocomial RSV, but among patients admitted for 414 days the rate was even higher. During the year prior to the intervention of 11 patients staying longer than 14 days in hospital, eight (73%) developed nosocomial RSV compared with two of 77 (2.6%) during the period of intervention.

The prevention of RSV infection is paramount on bone marrow transplant units, where the mortality from RSV lower respiratory tract infection may be 30–100% (Harrington et al., 1992). Moreover, since infected patients shed virus for longer, they act as potential reservoirs for the maintenance and cascading of infection in the ward.

Whether or not cohorting of infected patients is done on the basis of speciﬁc virus type or on respiratory symptoms alone is debatable. It was noteworthy that in the study by Madge et al. (1992), in which all children were initially cohorted on the basis of respiratory symptoms alone, they showed that no symptomatic child initially placed in an RSV infected cohort, but who was subsequently shown to

be RSV-negative, became infected as a result of this contact. The underlying reason for this may in part be due to the protective eﬀect of viral interference of a pathogen other than RSV in the upper or lower respiratory tracts. For more details on nosocomial RSV infection, please see Chapter 7.

Influenza Inﬂuenza is a particular problem in hospitals because

of the explosive nature of outbreaks and the especially vulnerable patient groups that are heavily exposed during the epidemic season. During the winter, up to 10% of the general population are infected with influenza; in hospitals during outbreaks the attack rate may be as high as 50% in patients on affected wards and up to 20% in the general hospital population. All types of wards have been affected and, whilst the mortality can be low, in geriatric wards it can be as high as 16% (Gowda, 1979) and in transplant centres mortality due directly to influenza is 30–60% (Weinstock et al., 2000). These figures are particularly worrying, since it has been estimated that up to 70% of influenza infections in transplanted patients are nosocomial in origin. The staff are also at high risk of infection, with transmission rates of 11–

60% in those caring for patients with influenza (reviewed in Salgado et al., 2002). In hospitals with a particular interest in influenza and a well-developed strategy for preventing nosocomial infection, however, the attack rate in staff is significantly reduced to only 2% (reviewed in Salgado et al., 2002).

There is good evidence that most cases of influenza are originally infected by respiratory droplets of small sizes which are inhaled. The most frequently quoted paper demonstrating the airborne route concerned a 72% attack rate in passengers in the cabin of an aeroplane with an inoperative ventilation system (Moser et al., 1979). All four individuals who decided to leave the plane escaped infection. However, those that chose to remain were free to move around the passenger compartment, indicating that infection by large droplets may also have occurred. However, small particle aerosols seem to be the most important mode of infection, since volunteer studies have shown that the dose required to establish infection is 10–100-fold lower for such aerosols compared with large droplet nasal administration of virus. Furthermore, intrana- sally administered influenza only uncommonly leads to influenza under experimental conditions and, whilst intranasal zanamivir prevents infection introduced by the same route, in ambulatory patients who, by implication, are more likely to come into contact with aerosolised virus, zanamivir requires delivery by inhalation to prevent infection.

Inﬂuenza virus is stable for 24–48 h after aerosolisa- tion and for at least 5 min on unwashed hands. Virus shedding begins 1 day prior to symptom onset and continues for 4–7 days in immunocompetent patients, although extended periods of virus excretion of several weeks are seen in the immune-compromised.

Preventing the spread of influenza in hospitals is difficult because of the multiple routes of entry of virus in staff, patients and visitors. However, low nosocomial rates are achievable through a concerted action by all staff and the infection control and occupational health departments. Staff must understand that they should not report for work if they are ill and that they will be sent home if they develop an influenza-like illness. The ethos should be one of protecting patients as well as working as a team.

Prior to the influenza season it is good practice for occupational health departments to actively encourage staff, especially those employed in high-risk areas (ITU, HIV, transplant, paediatrics, pathology) to be immunised against influenza. The way to achieve good uptake rates is, first, to educate staff about the benefits of vaccination and the risk that influenza poses to them, their families and their patients. This can be

852

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

achieved, for example, by posters and messages via hospital e-mail or in pay slips. The final stage is to offer the vaccine in as convenient a form as possible, e.g. by immunising staff in their workplace. By adopting such an approach, uptake rates can increase from 10% to 70% (reviewed in Salgado et al., 2002).

The overall efficacy of vaccine in patients and healthy staff is 70–90%. Vaccination of staff is probably the most effective measure in reducing mortality in hospital from influenza, as shown in a study from Scotland (Carman, 2000), especially as some studies in elderly institutionalised patients have demonstrated much lower rates of vaccine efficacy of 30–40% for preventing infection.

Patients who are admitted with suspected inﬂuenza should be placed in a single room and placed on respiratory precautions, i.e. plastic apron, gloves and hand-washing. A mask should be worn because of the risk of small particle aerosols. Specimens for rapid diagnosis should be sent as soon as possible, so that cohorting may be possible if the demand for single rooms becomes too great. There is no absolute need to place such patients in negative pressure, as the experience over a 15 year period from a hospital in Virginia, USA, has been that admitting patients with inﬂuenza to private rooms at slight positive pressure has not resulted in temporally related secondary cases (reviewed in Salgado et al., 2002).

If there appears to be an increase in the number of cases of proven inﬂuenza or inﬂuenza-like illnesses on

a particular ward, consideration should be given to immunising staff as rapidly as possible. Whilst this takes effect, antiviral prophylaxis should also be considered. Amantadine, the first of the antiviral agents active against influenza, was shown many years ago to be an effective prophylactic regimen. However, it does suffer from two major problems; rapid development of drug resistance and significant CNS toxicity. Even during a short treatment course of only 2–5 days, up to one-third of influenza virus isolates will have developed resistance. These are as transmissible as wild-type virus and have been known to cause fatalities. CNS toxicity is also problematic and, when compared with rimantadine, 18% of patients suffered from CNS side-effects compared with just 2% taking rimantadine. The newer neurami- nidase inhibitor agents, zanamivir and oseltamivir, given by inhalation or orally respectively, have an improved side-effect profile and so are better tolerated. Furthermore, when used in the home as post-exposure prophylaxis, they are extremely effective in preventing influenza illness, with efficacies of 74% and 90%, respectively.

Parainfluenza Virus

Like other respiratory viruses, parainfluenza (PIV) virus may cause outbreaks in nurseries, neonatal units and bone marrow transplant units. Infection in high- risk patients can have considerable mortality of approximately 40–50% in bone marrow transplant (BMT) patients (Zambon et al., 1998). When outbreaks occur it is always in the context of a wider community epidemic, which for parainfluenza virus usually runs from May to September each year. Thus, it is possible that what may appear to be an outbreak is merely the result of multiple introductions of virus into the ward by patients, staff and visitors. However, the available data from two large outbreaks on a paediatric ward and a bone marrow transplant unit showed the introduction of one PIV strain and its spread and propagation from person to person (Karron et al., 1993; Zambon et al., 1998). How virus may be transmitted is not easy to determine with absolute confidence, but transmission by unwashed hands, contaminated fomites and large droplets are all possible. In a report from the UK, one of two outbreaks on a BMT unit started 9 weeks after the index case had been identified and who was still shedding infectious virus (Zambon et al., 1998). This emphasises the absolute necessity for extreme vigilance when adhering to hand-washing and for prolonged periods when patients are immune-compromised. Indeed, virus excretion for 4 months has been demonstrated in this patient group (Zambon et al. , 1998). What was also clear from this study, and another in Canada, is that hospital parainfluenza virus outbreaks can, unlike those due to influenza where the median duration is only 7 days, be protracted and ultimately only terminated by closure of the unit to new admissions (Moisiuk et al., 1998). When the first case of a parainfluenza virus is discovered on a ward, especially those with high- risk patients, full adherence to all necessary infection control measures (gowns, gloves, patient cohorting and hand-washing), including reiterating the standing instructions to staff not to come to work if unwell, must be rigorously enforced.

Severe Acute Respiratory Syndrome (SARS)

The spread of SARS, now known to be caused by a previously unknown coronavirus, has highlighted the extreme dangers that are posed by emerging viruses, particularly respiratory pathogens, to virgin-soil

HOSPITAL ACQUIRED INFECTIONS

853

populations. It is also unfortunate that hospitals were unwittingly involved in amplifying the spread of the epidemic (Tomlinson and Cockram, 2003). This was seen very clearly in the Prince of Wales Hospital in Hong Kong, China. Within just 3 weeks of admission of the index case, 156 further cases had been diagnosed, all capable of being traced back to this single case. The major reasons given for the spread of infection in this hospital were failure to apply appropriate isolation precautions to cases not yet identified as SARS and breaches of such precautions. Staff were also appearing for work with a mild fever, so potentially exposing other patients and staff, and of especial importance in the rapid dissemination of infection in this hospital was the use of a nebulised bronchodilator in the index case, thereby hugely increasing the droplet contamination of the patient’s environment.

A case-control study in five Hong Kong hospitals demonstrated that adhering to such precautions as use of a mask, gloves, gowns and hand-washing resulted in no secondary cases in 69 staff who followed these prevention-of-infection measures. However, all of the 13 staff who became infected after caring for SARS cases reported not adhering to at least one of these measures (Seto et al., 2003). When each of the four infection control measures were examined, however, only the wearing of N95 masks was shown to be essential for protection against infection. This agrees with the principal mode of spread of SARS being by respiratory droplets. Unlike most of the other respiratory viruses, however, wearing an N95 mask capable of trapping 95% of all particles adds a further level of protection to staff. The wearing of simple paper masks did not afford any additional protection.

The World Health Organization (WHO) has given recommendations to control the spread of SARS. When a suspected case enters the hospital, droplet precautions must be strictly enforced, the patient must

be isolated to a negative-pressure room and, after transfer to an appropriate facility capable of handling Category 4 pathogens, rapid diagnosis with the least invasive specimen should be attempted. To prevent the spread of SARS in hospitals, patients who may represent possible cases should be given a face mask to wear, preferably one that ﬁlters exhaled air. The staﬀ involved in the initial contact must wear a mask,

e.g. N95 with 95% ﬁlter eﬃciency, and goggles. They must wash hands before and after contact and wear gloves. Standard disinfectant solutions, such as household bleach and alcohol gel preparations, should be readily available in the immediate vicinity of the

patient. The WHO has advised that there must be strict adherence when caring for SARS patients. All precautions against airborne, droplet and contact transmission must be employed. Further infection control measures include restricting visitors and super- vising those in the use of protective equipment. It has also been suggested that the wearing of footwear that can be easily decontaminated should be considered. The removal of linen should be done by staﬀ wearing full protective equipment (goggles, N95 or N100 masks, gloves, disposable gowns/aprons). The linen must be placed in biohazard bags and destroyed by incineration. Room cleaning should then take place using a broad-spectrum disinfectant, e.g. household bleach.

SMALL ROUND STRUCTURED VIRUSES (SRSV S OR NOROVIRUSES)

Of all the viruses capable of causing nosocomial infection, noroviruses probably present the greatest challenge to clinical virologists and infection control teams. Although initially termed ‘winter vomiting disease’ after a school outbreak in Norwalk, Ohio in 1966, these agents are now known to circulate all year round. To an individual they are of little consequence, as the illness is short-lived, self-limiting and only in exceptional cases does it lead to any complications. In hospitals, however, when outbreaks occur, almost without exception, ward closure and occasionally the entire closure of the hospital results (Stevenson et al., 1994). This is because there is often considerable spread to contacts (staff and patients), resulting in an attack rate of more than 50% (Caul, 1994). Indeed, it is our experience that by the time an outbreak is notified to the virology department, so many staff are absent from work because of illness that this alone results in ward closure. There are many factors, both host and viral, that contribute to this.

It is known from volunteer studies and outbreak investigations that the incubation period is short, often 12–48 h. Indeed, volunteers exposed to infectious virus orally have been found to be shedding virus in faeces only 15 h later (reviewed in Chadwick et al., 2000). The illness commences abruptly, with projectile vomiting in over half of cases and profuse watery diarrhoea. However, because the occasional case of vomiting and diarrhoea is not unusual among patients, many of whom are debilitated, elderly or on antibiotics, this may be initially overlooked. The environment can then become contaminated quite quickly and, as the

854

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

infectious dose is low (10–100 particles) and the viruses resist low-dose chlorine disinfection and a temperature of 608C, any breakdown in hand-washing leads very quickly to secondary cases occurring via the faecal– oral route (Caul, 1994). Fomite transmission is also possible because of the environmental stability of virus and it has even been suggested, although not actually conﬁrmed, that infection may occur via airborne transmission (Caul, 1994).

Evidence from PCR detection of viral RNA indicates that virus excretion persists for longer than previously thought. Although virus shedding in stool is maximal at 24–72 h after exposure, virus can be detected for almost 2 weeks in both symptomatic and asymptomatic persons (reviewed in Chadwick et al., 2000). The epidemiological signiﬁcance of this new ﬁnding is unclear. Finally, because of the strain diversity among the noroviruses, there is incomplete cross-protection and

no long-term

immunity.

Repeated infections throughout life are therefore common.

The main focus of the infection control eﬀort should

be that, after outbreak notification on a ward, attempts should not necessarily be made to try to stop the virus from spreading to other patients or staff already on that ward, because the transmission rate is so high. The primary consideration should be to stop it affecting other wards and parts of the hospital. Indeed, an outbreak described in Salford, UK, in 1994 is a typical case (Chadwick and McCann, 1994). By the time the problem was highlighted to microbiology, over 80% of the eventual total number of cases on the first ward to

be affected were either symptomatic or incubating the infection. Establishing an early aetiological diagnosis with laboratory techniques is often difficult with noroviruses, as the sensitivity of electron microscopy is as low as 20%. Moreover, unless stool samples are collected during the acute phase of the illness, when they are liquid or semi-solid, the detection rate declines further. Specimens from six affected persons should be tested in one batch to maximise the efficiency of electron microscopy. However, there are new EIA tests for antigen becoming available, with much better rates of virus detection, ca. 60–70%, and RT-PCR is even more sensitive. Fortunately, to counteract this key difficulty, a clinical diagnosis of a norovirus outbreak is usually reliable if the following criteria are adhered to: stool samples are negative for bacterial pathogens, the percentage of cases with associated vomiting is

4 50%, the duration of illness is short (1–3 days), the incubation period, if available, is only 24–48 h with both patients and staﬀ are aﬀected.

All aﬀected patients should be nursed either in single rooms or cohorted together. Strict hand-washing must

be observed and gloves and gowns must be worn. The risk of infection has been shown to increase in a linear fashion with the number of patient contacts or exposure to nearby vomiting. Although there is no evidence that the wearing of masks reduces the risk of infection, they could be considered for individuals who clear up and dispose of vomit and faeces. All dirty linen should be disposed of with minimal agitation (to prevent further environmental contamination), horizontal surfaces and floors should be cleaned with 1000 ppm of available chlorine, approximately equivalent to a 10% solution of household bleach. There is no need to clean the walls unless visibly contaminated. The rate of PCR detection of environmental norovirus RNA during an outbreak was found to be 30% but this was confined to the immediate vicinity of symptomatic patients, so supporting the need for patient cohorting (Green et al., 1998). This environmental cleaning should continue once the outbreak has subsided. As the number of staff affected is often high, personnel shortages may lead naturally to ward closure. If they do not, however, the ward must be closed to new admissions for a period of 72 h after the last case. Furthermore, the staff on the ward at the onset of the outbreak must not work on other wards, and neither should patients be transferred to other areas unless it is medically necessary to do so (Caul, 1994). Even then, consultation should take place with the infection control team. Staff must be made aware of the critical importance of not coming to work unless well. Visitors, too, must be made aware of the necessity for hand-washing before and after entering the ward and non-essential staff should be excluded.

ROTAVIRUSES

Rotaviruses are spread in an identical manner to noroviruses, although infection is largely restricted to young children (55 years old) and the elderly. In the context of an outbreak, however, it is also possible for staﬀ to not only carry virus from one patient to another on unwashed hands but also to become symptomatic cases (Cubitt and Holzel, 1980). As a group, the rotaviruses have been shown to account for approximately half of all cases of nosocomially- acquired diarrhoea in children (Ford-Jones et al., 1990). Outbreaks on neonatal units and elderly care wards are well documented, with high attack rates of 40–60%, with the elderly at particular risk of complications because of underlying disease.

HOSPITAL ACQUIRED INFECTIONS

855

PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

Diagnosis is much easier than with noroviruses, been a vertically infected baby. The most common because of the much higher titres of virus in stool

serotype noted has been Echovirus 11 (Modlin, 1986), and the wider availability of latex agglutination and

but other types, such as 4, 7, 9, 6 and 30, are as enzyme immunoassays.

pathogenic and as capable of nosocomial transmission The principal reasons for the propagation of such

(Bailly et al., 2000). As it is not always possible to outbreaks are lapses in basic hygienic and infection

provide enough single rooms to isolate neonates with control procedures, as shown in a very protracted

suspected virus infections, rapid diagnosis of entero- outbreak lasting 5 months on a neonatal unit in The

virus infections by PCR has been proposed to assist in Netherlands (Widdowson et al., 2002). Although the

preventing transmission (Chambon et al., 1999; Bailly detection of a novel strain of rotavirus probably

et al. , 2000). Moreover, the phylogenetic analysis of contributed to the scale of the outbreak, suspicion

VP1 sequences of Echovirus 30 has also enabled also centred on the fact that the neonates were being

epidemiological connections to be established over fed nasogastrically by staﬀ who were not wearing

long periods where nosocomial transmission may not gloves. Only after the wearing of gloves was enforced

necessarily have been originally suspected (Bailly et al., and the unit was closed for 7 days was the outbreak

2000). Universal precautions (rigorous hand-washing, terminated. Of additional concern was the ﬁnding that

gloves) should always be employed and as soon as a environmental contamination persisted even after

case of infection is identiﬁed on a neonatal unit, cleaning (Widdowson et al., 2002).

virological surveillance should commence to ascertain Another such outbreak demonstrated that rotavirus

the degree of spread and assist in cohorting. could be detected in stool within 5 days of infants being newly admitted to the neonatal unit (Grehn et al. , 1990). These outbreaks all emphasise the necessity for timely and close liaison between high-risk wards

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Index

Page numbers in italics indicate ﬁgures and tables.

abacavir, HIV 745, 747

respiratory infection 347, 348–50 abortion

gene therapy 357

ribavirin 257, 357 Lassa fever 604, 605

genome detection 355–6

serology 355 parvovirus B19 713

genotype 7h 349

serotypes 344 Rift Valley fever 573

genotypes 344

sexual transmission 352 aciclovir

haemorrhagic cystitis 351

structure 345 CMV 113

hexons 345

swimming pool outbreaks 347, 350 herpes zoster 77, 78

histopathology 355

taxonomy 344 HHV-6 and HHV-7 161

history 343–4

transmission mode 347 HSV 44, 45, 46, 47, 48, 49, 50

HIV 352

treatment 357 IM 136

humoral response 354

tumour necrosis factor inhibition ophthalmic zoster 78

hybridisation 355

354 prophylactic 79

immune evasion 354

vaccine vectors 357 varicella 75, 77

immune response 353–4

vaccines 356–7 VZV resistance 78–9

immunocompromised 351–2

virus-associated gene transcription acute respiratory disease, adenoviruses

immunoﬂuorescence 355

347 in military recruits 344, 349–50,

immunohistochemistry 355

adult T cell leukaemia-lymphoma 357–8

immunotherapy 357

(ATLL) 759, 768, 769, 770–1, 773 adefovir, hepatitis B 227, 228, 230–1

inactivation 345

adult T cell leukaemia virus (ATLV) adenoviruses (AdVs) 343–60

interferon inhibition 354

isolation 355

Aedes mosquito antigen detection 355

AIDS 352

keratoconjunctivitis 347, 350–1

Barmah Forest virus 517 bone marrow transplant 351–2

latent infection 347, 353

Chikungunya virus 518 capsid proteins 345

lytic infection 347, 353

dengue 541 cellular response 354

measles pneumonia 349

Eastern equine encephalitis virus cidofovir 257, 357

meningoencephalitis 352

521 clinical manifestations 347–8

MHC class I downregulation 354

emerging infections 830 clinical syndromes 347–8

military recruits 344, 349–50, 357–8

Rift Valley fever virus 570 coxsackie-adenovirus receptor

myocarditis 352

Ross River virus 515 (CAR) protein 346

obesity 352

Wesselsbron virus 539–40 cytopathic eﬀect 355

oncogenic transformation 347

yellow fever 536 diagnosis 354–6

oncogenicity 354

African green monkeys 614, 621 disseminated disease 355

pathogenesis 353

AG7088, rhinoviruses 373 DNA 345

PCR 355–6

Aichivirus , gastrointestinal 262 replication 346–7

pentons 345

persistent infection 347, 353

AIDS

EIA 355

adenoviruses 352 electron microscopy 355

pertussis-like syndrome 349

anogenital squamous carcinoma encephalitis 352

pharyngoconjunctival fever 347, 350

738–9 enteric 249, 256–7, 347, 351

prevention 356–7

Burkitt’s lymphoma 143 epidemiology 347

prototype strain 344–5

case deﬁnition 732 eye infection 347, 350–1

replication 346–7

cervical cancer 739 gastroenteritis 249, 257, 347, 351

resistance to physicochemical agents

children 739–40

AIDS (cont.) clinical disease 732–43 CMV 100, 116, 118 CMV retinitis 118, 735 EBV 143–4 global burden 725 HHV-6 and HHV-7 158–9 historical events 724 Kaposi’s sarcoma 177, 737, 738 malignancies 736–9 management 732–43 multicentre Castleman’s disease 181 non-Hodgkin’s lymphoma 737–8 primary eﬀusion lymphoma 180–1 progressive multifocal leukoence-

phalopathy (PML) 693, 694, 735 VZV retinitis (PORN) 71 Aino virus 568 Akabane virus 568 alanine aminotransferase (ALT) 201 alcohol intake, hepatitis C 240 Alenquer virus 569 allergic response, rhinoviruses 367 Alphaherpesvirinae 24 alphaviruses 509–29

antibody response 513 antigenic properties 511–12 biochemical properties 510–11 biophysical properties 510–11 capsid proteins 510 case deﬁnition 513 diagnosis 512–13 E1 and E2 509, 510–11 E3 511 encephalitis 520–6 fevers 514–20 genome 509, 510 genotypic properties 511–12 IgM and IgG antibodies 513 isolation 513 laboratory diagnosis 513 lipid bilayer 509 management 513 morphology 509–10 non-structural proteins 509, 510 polyarthritis 514–20 prevention 513 replication 511 RT-PCR 513 single-stranded RNA 510, 511 spectrum of diseases 512

amantadine, inﬂuenza 289, 290, 853 amdoxovir, HIV 747–8 amitriptyline, herpes zoster 78 amniocentesis, CMV 101, 109 amprenavir, HIV 745, 749 Andes virus 582 angiogenesis, KSHV 185–6

anogenital squamous carcinoma,

AIDS 738–9 Anopheles mosquito, o’nyong-nyong

virus 519 antibody

avidity tests 5 response evolution 4

antigen detection 2–3 antigenaemia 101–2, 105 anti-HBc 217–18 antiviral therapy

drugs resistance 16–18 monitoring 14–15

Apeu virus 565 aplastic anaemia, TTV 820 aplastic crisis 706, 711–12 apnoea, respiratory syncytial virus 330 Apodemus ﬁeld mice 579, 580 arboviruses, deﬁned 509 Arenaviridae 590 arenaviruses 589–609

ambisense coding 594, 595 antibodies 598, 599 antigenic relationships 596–7 cell-mediated immunity 599 cellular immunity 598 chemical composition 593–4 diagnosis 596 electron microscopy 591 ELISA 596 emerging infections 606–7 genome 593 geographical distribution 590 glycoprotein precursor polypeptide

593 GP1 and GP2 593 hosts 590 immune response 597–8 immunoﬂuorescence 596 interferon 598 intracytoplasmic inclusion bodies

592 L protein 593, 594 monoclonal antibodies 596–7 N protein 593, 594, 595 natural history 590–1 natural killer cells 598 nomenclature 590–1 nucleic acid 593–4 pathology 599–606 PCR 596 persistent infection 599 phylogenetic analysis 594 plaque reduction neutralisation 597 proteins 593 replication 594–6 T cell response 598 ultrastructure 591–2

Z protein 593 Argentinian haemorrhagic fever 601–2 arthropathy, parvovirus B19 711 arthropod-borne viruses, emerging

infections 830–1 ascorbic acid, rhinoviruses 373 aseptic meningitis, enteroviruses 477 aspartic aminotransferase (AST) 201 asthma

coronaviruses 388–9 rhinoviruses 369 RSV bronchiolitis 331–2

astroviruses 249, 260–2 classiﬁcation 261 clinical course 261 detection 250 diagnosis 261 epidemiology 262 genome 260–1 multiple serotypes 262 pathogenesis 261 prevention 262 replication 261 structure 260 treatment 262

atazanavir, HIV 750 atopy

respiratory syncytial virus 332 rhinoviruses 368

Aura virus 512, 523 aural zoster 70 Australian bat lyssavirus 632, 637, 648,

655

B cell proliferation, KSHV 185–6 B lymphoproliferative disease 131

Babanki virus 514 Bangui virus 585 Barmah Forest virus 511, 512, 516–17 Batai virus 564 bats

emerging infections 831–2 ﬁloviruses 619 rabies 637, 641–2, 647–8, 657

Bcl-2, KSHV 187 BDprobeTEC # 11 Bear Canyon virus 607 behaviour, emerging infections 828 Bell’s-type palsy 70 Betaherpesvirinae 24 Bhanja virus 585 bilirubin 201 bioterrorism 3, 825 birds

Eastern equine encephalitis virus 521 Sindbis virus 514 Birnaviridae 263

860

INDEX

bismuth subsalicylate, diarrhoea 250 BK virus 675

associated diseases 692–3 asymptomatic activation 691 bone marrow transplant 692 gene expression control 679 genome 677 genomic analysis 686 haemagglutination inhibition 698 haematogenous spread 688 humoral immune response 698 IgM 698 JCV co-infection 690–1 nephropathy (BKN) 692, 700 oncogenicity 695–6 primary infection 686–7 renal transplant 692 treatment of associated disease 700

blood-borne viruses exposure prevention 838–9 healthcare workers 16, 837–9, 840 hospital-acquired 837–41 infection prevention 839 parvovirus B19 709 patient-to-patient transmission 838 prions 787 sharps handling and disposal 838 universal precautions 838

blood donor screening, HTLV-I/II 774 blood products, serology 6 blood transfusion

CJD transmission 795 CMV 92–3 GBV-C 817 serology 5–6

body cavity-based lymphoma (BCBL) 180–1 body ﬂuids, nosocomial infection 838 Bolivian haemorrhagic fever 601,

602–3 Bolomys obscuris 607 bone marrow transplantation

adenoviruses 351–2 BKV infection 692 HHV-6 and HHV-7 155 see also transplantation patients

Bornholm disease 479 bovine PIV3 (BPIV3) 299, 304, 306,

315 bovine spongiform encephalopathy (BSE) 779, 788, 798 human behaviour 828 species barrier 786 vCJD link 796

branched chain DNA assay 11–12 Brazilian haemorrhagic fever 606 break bone fever 542 break heart fever 542

breast-feeding 726

HIV 726, 739 HTLV-I 768

breast milk

CMV 90, 92 HTLV-I 768 mumps virus 461

brivudin, herpes zoster 78 bronchiolitis

parainﬂuenza viruses 311 respiratory syncytial virus 329,

331–2 bronchitis

inﬂuenza 282 respiratory syncytial virus 329 rhinoviruses 369

buﬀalopox 492, 498–9 Bunyamwera virus 563 Bunyaviridae 555–88

ambisense RNA 556 biological characteristics 557–60 cell culture 557 classiﬁcation 557, 558–60 clinical syndromes 561 cross-reactivity 562–3 detection 562–3 extrinsic incubation period 561 G1 and G2 proteins 556 genome 556 hibernation 561 identiﬁcation 562–3 IgM antibody 563 L protein 556 laboratory diagnosis 561–3 N protein 556, 557 RNA 556–7 RT-PCR 562 serology 563 structure 556–7 transmission 560–1

Burkitt’s lymphoma 123, 136–8

AIDS 143 chromosomal abnormalities 137–8 clinical features 138 co-factors in pathogenesis 138 cyclophosphamide 138 derived cell lines 137 diagnosis 138 EBV association 123, 131, 137 geographical distribution 137 HIV 143 malaria association 123, 136, 138 pathogenesis 137–8 prevention 138 relapses 138 seroepidemiology 136–7 treatment 138

Bwamba virus 565

Cache Valley virus 564 caliciviruses 249, 257–60

capsid 258 classiﬁcation 258 clinical course 259 epidemiology 260 genome 258 immune response 259 laboratory diagnosis 258–9 open reading frames 258 pathogenesis 258 prevention 260 replication 258 RT-PCR 258–9 structure 258 vaccine 260 viral shedding 260

California encephalitis virus 565–6 Calomys callosus 603 Calovo virus 564 cancer

HPV 668–70 HTLV-I 768–9 VZV vaccine 81

candidiasis, HIV 735 Candiru virus 569 canine distemper virus 398 capravirine, HIV 748 Caraparu virus 565 Catu virus 567 CCR5 inhibitors, HIV 751 cDNA expression library screening

828–9 cell culture 1, 2 Central European encephalitis virus

(CEE) 535, 550–1 Cercopithecus aethiops 614 cervical cancer

HIV/AIDS 739 HPV 670

cervix, HPV infection 668–70 Chagres virus 569 chicken anaemia virus (CAV) 818, 820 chickenpox, derivation 53 see also

varicella Chikungunya virus 512, 515, 517–19 Chittoor virus 564 chronic fatigue syndrome

enteroviruses 481–2 IM diﬀerential diagnosis 135

cidofovir

adenoviruses 257, 357 BKV infection 700 CMV 112, 113 HHV-6 and HHV-7 161 HSV 47 human polyomaviruses 700 KSHV 190

INDEX

861

INDEX

cidofovir (cont.)

MRI 796 poxviruses 505

transcription 383–5

neuropathology 797 Circinoviridae 830

transcription-associated sequence

PrP Sc 798 Circovirus 818

(TAS) 383, 385

tonsil biopsy 798, 804–5 cirrhosis, hepatitis B 220–1

upper respiratory tract disease

PrP Sc 784, 793 clevudine, hepatitis B 231

sporadic 788, 789, 790, 791–3 Coe virus 480

vaccines 393–4

Crimean-Congo haemorrhagic fever cold sores 35

virion assembly 383

virus 575–8 common cold, rhinoviruses 361, 368

virion composition 380, 382

clinical pathology 577 complement ﬁxation test 4

corticosteroids

control 578 condylomata acuminata 668

IM 136

distribution 575 congenital malformations

respiratory syncytial virus 335

ﬁbrin 577 inﬂuenza 284

cot death syndrome, inﬂuenza 285

incubation period 577 parvovirus B19 713

Councilman bodies 538

isolation 578 congenital varicella syndrome 68–9

cowpox 492, 499–500

liver lesions 577 conjunctivitis, enteroviruses 480

Coxsackie A virus 474, 475

onset of disease 577 contagious pustular dermatitis 501

aseptic meningitis 477

rash 577 cornea, HSV 36, 49

Bornholm disease 479

ribavirin 578 corneal transplant, HSV 49

cellular receptor sites 471, 472

treatment 578 coronaviruses

hand, foot and mouth disease 480

cromolyns 373 accessory genes 383

parainﬂuenza viruses 305, 308–10, antigenic structure 385–6

aminopeptidase N (APN) 386

murine pathogenicity 475

311, 315 asthma 388–9

rashes 480

respiratory syncytial virus 329 CEACAM 1 387

respiratory infections 480

cryotherapy, genital warts 672 cell attachment 387

Coxsackie-adenovirus receptor 346,

Cryptococcus neoformans , HIV 735 cell attachment proteins 387

Culex mosquito cell receptors 386–7

Coxsackie B virus 474, 475

Barmah Forest virus 517 central nervous system 391

antigenic heterogeneity 475

Eastern equine encephalitis virus clinical features 387–91

aseptic meningitis 477

521 diagnosis 391–3

Bornholm disease 479

Getah virus 526 electron microscopy 380, 392

cellular receptor sites 471

Japanese encephalitis virus 545 enteric tract 390–1

chronic fatigue syndrome 482

Ross River virus 515 epidemiology 389–90

conjunctivitis 480

St. Louis encephalitis virus 547 fusion processes 387

isolation 483

Venezuelan equine encephalitis virus gastrointestinal 263

murine pathogenicity 475, 479

524 genome 380

myocarditis 477, 478

West Nile virus 548 glycans 387

neonatal infection 479

Culiseta mosquito group-speciﬁc genes 383

pancreatitis 481

Eastern equine encephalitis virus groups 380

Creutzfeldt-Jakob disease (CJD) 791–3

521 growth in vitro 386

amyotrophic variants 792

Highlands J virus 523 HCoV-229E 379, 383, 385, 386

ataxic 792

cyanovirin-N 625 HCoV-OC43 379, 382, 383, 385,

atypical 792

cyclophosphamide, Burkitt’s 386, 392

blood transfusion 795

lymphoma 138 host range 390

EEG 791–2

Cynomolgus monkeys 618, 621 IBV vaccine 394

history 779

cystitis, haemorrhagic 351 infection initiation 386–7

iatrogenic 788–9, 790, 794–5

cytology 4 isolation 392

new variant (vCJD) 789, 795–800

cytomegalic inclusion disease 98, 108 lower respiratory tract disease 388–9

BSE link 796

cytomegalovirus (CMV) 85–122 microarray analysis 393

clinical presentation 796

aciclovir 113 multiple sclerosis 391

contaminated surgical instruments

AIDS 100, 116, 118 otitis media 387–8

amniocentesis 101, 109 prophylaxis 393–4

diagnosis 790

antigenaemia assay 3, 101–2, 105 replication 383–5

EEG 796

antiviral drug strategies 113–19 RT-PCR 393

ﬂorid plaques 797

blood transfusion 92–3 SARS see SARS coronavirus

human behaviour 828

breast milk 90, 92 serology 392–3

iatrogenic transmission 804

canarypox-gB 111 therapy 394

lymphoreticular system 804

molecular diagnosis 784, 803

cell cultures 103, 105

863 cell-mediated immunity 97

INDEX

control 544 cidofovir 112, 113

misleading impressions 95

diagnosis 543–4 clinical features 98–101

monitoring 15

epidemiology 541–2 CMV syndrome 100

morphology 85

history 540 complement 90

neonatal screening 107

host range 541 congenital 91, 95–6, 97, 98–9, 106–8,

non-infectious enveloped particle 85

surveillance 544 110

nucleic acid 85–6

vaccine 544 cytopathic eﬀect 103

owl’s eye inclusions 85, 102

vector control 544 dense body 85

pathogenesis 93–8

virus isolation 543–4 detection of early antigen ﬂuores-

PCR 104–5

virus properties 540–1 cent foci (DEAFF) 104, 105

perinatal infection 90, 92, 97, 99, 108

dengue fever 540 diagnosis 101–6

porcine 159

clinical features 542 DNA detection 104–5

postnatal infection 92, 99–100,

complement ﬁxation 543 drug resistant 118

diagnosis 543–4 early proteins 89

pre-emptive therapy 115–16

haemagglutination inhibition 543 EIA 104

pregnancy 91, 92, 95–6, 101, 108–9

IgG assay 543 electron microscopy 103–4

prevention 110–12

IgM antibodies 543 epidemiology 90–1

prophylaxis 113–15

MAC ELISA 543 examination site selection 101–2

proteins 88–9

dengue haemorrhagic fever/dengue Fc receptors 90

prototype conﬁguration 85

shock syndrome (DHF/DSS) 540 foscarnet 112, 113, 116, 118

qualitative detection 13

clinical features 542 ganciclovir 107–8, 112–13, 115, 116,

reactivation 91

complement ﬁxation 543 118

rectal intercourse 92

deﬁnition 542 gastrointestinal 263

recurrent 91

diagnosis 543–4 genital secretions 92

replication 94

haemagglutination inhibition 543 genome 85–6

retinitis, AIDS 118, 735

haemorrhagic diathesis 542 expression control 87–8

risk factors 93–5

history 540 HAART 116, 118

screening 107

IgG assay 543 healthcare workers 846–7

serology 106

IgM antibodies 543 hearing loss 98–9

sexual transmission 92

MAC ELISA 543 histopathology 102

shell vial assay 104

pathogenesis 542–3 HIV interaction 98

socioeconomic factors 91

pre-existing antibodies 543 host defences 96–7

specimen collection 101

severity 542 humoral immunity 96–7

suppression 115

Dependovirus 703, 704 IgG antibodies 96, 106

therapy monitoring 15

dermatitis IgM antibodies 96, 106

thymidine kinase 90

HSV 39–40 immediate early genes 88–9

topoisomerase II 90

HTLV-I 769 immune response detection 106

Towne vaccine 111, 112

detection of early antigen ﬂuorescent immunisation 111–12

transplant patients 13, 93, 113–15

foci (DEAFF) 104, 105 immunocompromised patients

congenital rubella 443 immunoﬂuorescence 102–3

valaciclovir 113

enteroviruses 481 immunoglobulin 113

valganciclovir 112

ketoacidosis, inﬂuenza 285 immunotherapy 112

virion RNAs 87

diagnosis in vitro growth 90

virus detection 101–5

disease 13–14 incubation periods 95–6

cytopathic eﬀect 1

infection 12–13 infection routes 92–3

cytosine arabinoside, PML treatment

diagnostic approaches 1–21 infectious mononucleosis 99

diagnostic assay evaluation 12–13 interferon-a 113

diarrhoea intranuclear inclusions 85, 102

DAPD, HIV 747–8

HHV-6 153 intrauterine infection 92, 96

Davidsohn–Henry test 136

HIV 736 JCV transactivation 683–4

deafness

SARS coronavirus 380 kissing disease 92

congenital rubella 442–3

toroviruses 380 late proteins 89

mumps 463

diarrhoeal disease 249–70 leaky-late genes 87

delaviridine, HIV 745

didanosine, HIV 745, 746 lung infection 118

demographics, emerging infections

diet, nasopharyngeal carcinoma 140 major immediate-early proteins 88

dilated cardiomyopathy, enteroviruses management 106–10

Denavir see penciclovir

dengue 535, 540–4

diagnosis 13–14 prediction 14

Dobrava virus 580, 582, 583 dolphin morbillivirus 400 drug resistance 16–18 drug susceptibility 16 Duck hepatitis

B virus (DHBV) 212 Dugbe virus 579 Duncan’s syndrome 142 Duvenhage virus 632, 637, 647, 655

Eastern equine encephalitis virus 511, 512 , 521–2 Ebola virus 611 amino acid sequences 614 animal models 621–2 biological warfare 620 cyanovirin-N 625 ecology 619 ELISA 624 epidemiology 616–17 genetic stability 614 glycoprotein gene 614 immunoﬂuorescent assay 624 immunology 624 incubation period 620 isolation 625 laboratory infections 620 lipids 614 mortality 621 non-human primate vectors 832 nucleotide sequences 614 past infection 623 pathology 623 persistence 623 Reston 618, 620, 622 risk factors 620 serology 618–19 therapy 625 transmission 620

echoviruses 474, 475 aseptic meningitis 477 cellular receptor sites 471 conjunctivitis 480 gastrointestinal 262 group O erythrocyte agglutination

483 isolation 483 neonatal infection 479 rashes 480 respiratory infections 480

eczema herpeticum 39 efavirenz, HIV 745, 748 elderly population, emerging infections

828 electrodiathermy, genital warts 672

electron microscopy 3 ELISA 3, 5

capture 4 competitive 4 indirect 4

emerging infections 606–7, 825–33 emtricitabine, hepatitis B 231 encephalitis

adenoviruses 352 alphaviruses 520–6 enteroviruses 477 herpes simplex 37–8, 49, 50 herpes zoster 70–1 inﬂuenza 284–5 measles 411–12, 415–16, 421 mumps 463 rabies

complications 644 histopathology 651 immune response 639–40 intravitam diagnosis 648–50 recovery 646–7

Rift Valley fever virus 573 tick-borne 535, 550–1 varicella 68

encephalomyelitis

Herpes simiae (B virus) 645 post-vaccinal 645 rabies 642–3, 650–1

encephalopathy

HIV 740 rubella 435

endocardial ﬁbroelastosis 464 endocrine disorders, congenital rubella

443 enfuvirtide, HIV 750 entecavir, hepatitis B 231 enterovirus 70 480, 481 enterovirus 71

aseptic meningitis 477 hand, foot and mouth disease 480

enteroviruses 467–89

antigenic structure 470–1 antigenic variation 475 antiviral therapy 486–7 aseptic meningitis 477

b barrel 469 biochemical structure 469 Bornholm disease 479 capsid 469 cell culture 483 cellular receptor sites 471–2 chronic fatigue syndrome 481–2 clinical aspects 474–82 CNS infections 476–7 complement ﬁxation 484 conjunctivitis 480 cross-reactivity 470–1

cytopathic eﬀect 483 diabetes 481 dilated cardiomyopathy 478–9 EIA 484 encephalitis 477 exanthemata 480 excretion 483 gastrointestinal 262 genome 469, 472 groups 474, 475 hand, foot and mouth disease 480 heart disease 477–9 herpangina 480 HEV species 473, 474 host range 475 hypogammaglobulinaemia 477 IgM response 484 incubation period 476 internal ribosomal entry sites

(IRES) 472 isolation 483 laboratory diagnosis 482–4 lipids 469 molecular diagnosis 484 molecular serotyping 484 myocarditis 477–8 myristic acid 469 neonatal infection 479 neurological complications 481 neutralisation tests 483 non-translated regions (NTRs) 472 nosocomial transmission 856 pancreatitis 481 pathobiology 474–82 PCR 484 physical characteristics 467–9 pleconaril 487 poliomyelitis 476 prevention 484–7 rashes 480 replication 472–3 respiratory infections 480 sequence comparisons 473–4 serology 483–4 treatment 484–7 vaccination 484–6 VP1–VP4 proteins 469

epidemic pleurodynia 479 epidemic polyarthritis 515, 516 epidemiology, viral genetic analysis 16 epidermodysplasia verruciformis

666–7 Epstein–Barr virus (EBV) 123–46 AIDS 143–4 associated disease 130–42 Burkitt’s lymphoma association 123,

131 , 137 capsid 124

864

INDEX

early genes 127 EBERS 125 EBNA-LP 126 envelope 124 epidemiology 130 episome 125 full latent gene expression 128 gastric carcinoma 131 genome 124–5 glycoproteins 127 growth 127–8 handling 128, 130 Hodgkin’s disease 131, 141 host range 127–8 human homologues 127 immediate-early genes 127 immortalisation 128 immunocompromised patients 142–4 infectious mononucleosis 123 late genes–viral capsid proteins 127 latent infection 125 latent membrane proteins 126–7 latent proteins 125–7 leiomyosarcoma 131 lytic cycle proteins 127 nasopharyngeal carcinoma 123, 131,

139 nuclear antigens (EBNAs) 125–6 primary eﬀusion lymphoma 131,

180 proteins 125–7 reactivated 142 spontaneously immortalized 130 strains 125 structure 123–4 transplant patients 142–3 tumours 123, 130, 131 types 1/2 (A/B) 125 vaccine 144 viral-coded proteins 125

erboviruses 467 erythema infectiosum (ﬁfth disease)

706, 709, 710 erythema multiforme 40 Erythrovirus 703, 704 European bat lyssavirus 632, 637,

647–8, 655 Everglades virus 524 exanthem subitum 153 exanthemata, enteroviruses 480 exposure-prone procedures (EPPs) 840 eye

adenoviruses 347, 350–1 congenital rubella 442 enterovirus conjunctivitis 480 herpes zoster 69, 71, 78, 736 HSV 35–6, 49 HTLV-I uveitis 769, 774

Rift Valley fever virus 573 see also retina

famciclovir (Famvir)

herpes zoster 77–8 HSV 45, 47, 48, 49

febrile seizures, HHV-6 and HHV-7

153–4 Feline coronavirus 380 fever blisters 35 ﬁfth disease (erythema infectiosum)

706, 709, 710 ﬁloviruses 611–29

animal models 621–2 bats 619 clinical spectrum 620–1 contacts 626 containment 625–6 control 625–7 cross-reactivity 614 ecology 619–20 ELISA 625 epidemiology 614–19 genome 613 glycoprotein 614 hospital containment 626 immunology 622–4 isolation 625 laboratory diagnosis 624–5 laboratory infections 620 mortality 621 natural history 611 nucleoprotein 613 past infection 623–4 pathogenesis 622–4 patient management 625 persistence 623–4 polymerase 614 replication 613 risk factors 620 serology 618–19 specimen collection 624 therapy 625 transmission 620 vaccine 626–7 virology 611–14

ﬂaviviruses 531–54

antigenic classiﬁcation 533–4, 535 antigenic properties 533–4 biochemical properties 533 biophysical properties 533

C protein 533 cross-neutralisation tests 533 diagnosis 531–2

E protein 533 gene order 533 lipids 533 M protein 533

morphogenesis 532 morphology 532 non-structural proteins 533 outbreaks 532 pre-M protein 533

FLIP, KSHV 186 ﬂuorescence resonance energy transfer

9 ﬂuorescent antibody to membrane antigen (FAMA) 74 Fort Morgan virus 512, 523 Fort Sherman virus 564 foscarnet

aciclovir-resistant VZV 79 CMV 112, 113, 116, 118 HHV-6 and HHV-7 161 HSV 47 KSHV 190

fossil infections 721, 723 FTC, HIV 748 fusion inhibitors, HIV 750

G protein-coupled receptor 185–6, 188 Gammaherpesvirinae 24 ganciclovir

CMV 107–8, 112–13, 115, 116, 118 HHV-6 and HHV-7 161 HSV 47 KSHV 190

Ganjam virus 579 gastric carcinoma 131 gastroenteritis 249

adenoviruses 249, 257, 347, 351 bismuth subsalicylate 250 developing countries 251 diphenoxylate 251 epidemics 250 epidemiology 250 loperamide 251 oral rehydration ﬂuids 250 toroviruses 263, 391 treatment 250–1 viruses causing 249

GB virus (GBV) 813, 830 animals 814 genome 814–15 tissue culture 815

GBV-A

animals 814 genome 814 representational diﬀerence analysis

(RDA) 829 GBV-B

animals 814 genome 814 representational diﬀerence analysis

(RDA) 829 tissue culture 815

INDEX

865

normal human immunoglobulin blood transfusions 817

haemorrhagic cystitis 351

(NHIG) 206 clinical signiﬁcance 242–3

haemorrhagic fever with renal

pathogenesis 205 E2 region 815

syndrome (HFRS) 561, 579,

prevention 206 genome 814–15

prophylaxis 206, 207 HCV co-infection 817

hand, foot and mouth disease 480

recurrent 206 healthy population 815–16

Hantaan virus 579, 582

seasonal patterns 203 hepatic disease link 815

Hantavirus 579–85

spread 202 history 813–14

distributions 582

vaccines 206–7 HIV 817

emerging infections 831

hepatitis A virus (HAV) 199, 200 mother-to-child transmission 817

investigations 584–5

biology 203 non A–E hepatitis 816

pathogenesis 584

CD8 + 205 patients 816–17

serology 580

cell culture 204–5 phylogenetic clustering 815

hantavirus pulmonary syndrome

genome 203–4 replication site 815

(HPS) 561, 579, 580, 584, 831

inactivation 203 representational diﬀerence analysis

HBcAg 212, 215

stability 203 (RDA) 829

HBeAg 212, 215

hepatitis B 210–34 tissue culture 815

HBsAg 210, 211, 212–214, 217, 224–5

acute 219, 226 gene therapy, HHV-6 and HHV-7 150

HDAg 235

adefovir 227, 228, 230–1 genetic analysis, transmission events

healthcare workers

anti-HBc 217–18 and epidemiology 16

blood-borne viruses 16, 837–9, 840

antiviral therapy 226–33 geniculate herpes 70

cytomegalovirus 846–7

monitoring 15 genital cancer, HPV 668–70

exposure-prone procedures 840

biological response modiﬁers 231–2 genital herpes 34, 35, 37, 50

HBeAg-negative 16, 840

children 211 genital HPV 667

HBV 16, 837

chronic 200, 211–12, 215–17, 219– genital warts 668, 670

cirrhosis 220–1 genome

measles 847–8

clevudine 231 qualitative detection 13

VZV 843–6

control 222–5 sequencing, transmission events and

hearing loss

diagnostic assays 217–19 epidemiology 16

CMV 98–9

emtricitabine 231 genotypic assays, drug resistance 16,

congenital rubella 442–3

entecavir 231 17–18

mumps 463

epidemiology 210–11 genotyping 14

heart disease

fulminant 219, 226 German measles see rubella

enteroviruses 477–9

genotyping 14 Germiston virus 564

HHV-6 and HHV-7 156–7

HBeAg-negative patients 220, 221 Gertsmann–Stra¨ussler–Scheinker dis-

Henderson–Paterson bodies 502

HBIG 222 ease (GSS) 779, 798

Hendra virus 831

HBV infection 215–17 Getah virus 512, 515, 526

Hepacare 225

hepatitis C and 221 GIGA-HAART 752

Hepadnaviridae 212

IL12 231–2 glandular fever see infectious mono-

hepatitis

immunisation nucleosis

acute 199–200, 201

active 222 GM-CSF, HIV 753

autoimmune 206

indications 226 Ground squirrel hepatitis virus (GSHV)

immunoprophylaxis 225 Guama virus 567

liver function tests 201

immunotherapy 232–3 Guanarito virus 607

non-A–E 816, 820

in utero infection 211 Guaroa virus 567

non-A, non-B 236–7

interferon-a 227–8 Guillain–Barre´ syndrome

pathology 199–201

lamivudine 227, 228, 229–30 inﬂuenza 284–5

pregnancy 201

LdT 231 parainﬂuenza viruses 312

hepatitis A 201–7

age incidence 203

liver biopsy 220

nucleoside analogues 229–31 HAART 752

autoimmune chronic hepatitis 206

nucleotide analogues 229–231 CMV 116, 118

cholestatic hepatitis 206

occult 221 GIGA-HAART 752

clinical course 206

pathogenesis 221–2 HIV-related PML 700

control 206

pathology 200 Haemagglutinating encephalomyelitis

fatality 206

pegylated interferon 228–9 virus (HEV) 380

incubation period 202

laboratory diagnosis 205–6

perinatal transmission 211, 221–2 perinatal transmission 211, 221–2

HBIG 222 transplant patients 227

serology 219 sexual transmission 211 spontaneous remission 220 tenofovir 227 thymosin 232 vaccination

anti-HBs kinetics 223–4 boosters 223–4 Hepacare 225 injection site 223 non-response 223 third generation 225 triple antigen 225

hepatitis B core antigen (HBcAg) 212, 215 hepatitis B immunoglobulin (HBIG) 222 hepatitis B surface antigen (HBsAg) 210, 211, 212–214, 217, 224–5 hepatitis B virus (HBV) 199, 200 anti-HBc 217–18 biology 212 chronic hepatitis B 215–17 core 212 extra-hepatic tissue 217 genome 212 genotypes 220 HBcAg 212, 215 HBeAg 212, 215 HBsAg 210, 211, 212–214, 217,

224–5 HDV co-infection 234 healthcare workers

screening 840 transmission to patients 16, 837

HIV co-infection 227, 736 KSHV association 177 liver cancer 233–4 morphology 212 oncogenesis 234 post-exposure prophylaxis 839–40 pre-exposure measures 839 replication 215 structure 212 transmission prediction 15 white blood cells 217

X gene 234 hepatitis C 236–42 acute 239–40 alcohol link 240 chronic 200–1, 240

diagnosis 238 EIA 238 epidemiology 238 immunisation 242 interferon-a 239, 240–1 mother-to-child transmission 242 occult hepatitis B 221 pathology 200–1 pegylated interferon 241 persistence 239 prevention 242 ribavirin 240–1 RT-PCR 238 serology 239 sexual transmission 242 staging 14 surgical transmission 242 therapy monitoring 15 transmission prediction 16 treatment 239–42

hepatitis C virus (HCV) 199, 200, 532

GBV-C co-infection 817 genome 237 genotypes 239 genotyping 14 healthcare worker-to-patient trans-

mission 837–8 HIV 736 identiﬁcation 828–9 non-A, non-B hepatitis 236–7 post-exposure prophylaxis 840 quasi-species eﬀect 237

hepatitis D 234–6

clinical features 236 fulminant 236 IFN-a 236 immunisation 236 laboratory diagnosis 235 pathogenesis 236 treatment 236

hepatitis D virus (HDV) 199, 200

epidemiology 235 genome 234, 235 genotypes 235 HBV co-infection 234 HDAg 235 infection modes 234 replication 235 structure 235 transplantation patients 235

hepatitis E 208–10

clinical features 209 diagnosis 209 fulminant 209 immunisation 209–10 pathogenesis 209 PCR 209 pregnancy 209

zoonotic infection 210 hepatitis E virus (HEV) 199, 200 biology 208 genome 208–9 identiﬁcation 828

hepatitis G virus (HGV) 242–3, 532, 813, 814, 830 hepatocellular carcinoma, HBV 233–4 herbal medicines, nasopharyngeal car-

cinoma 140 herpangina, enteroviruses 480 herpes febrilis 35 herpes gladiatorum 39 herpes keratitis 35–6, 49 herpes labialis 32, 35, 40 Herpes simiae (B virus), encephalo-

myelitis 645 herpes simplex virus (HSV-1 and HSV-2) 27–51 aciclovir 44, 45, 46, 47, 48, 49, 50 antiviral chemotherapy 44–7 capsid 27 CD4 + /CD8 + cells 32 cell-mediated immunity 32 cidofovir 47 clinical features 34–40 core 27 CSF 44 CTL response 32 culture 40–1 dermatitis 39–40 diagnosis 40–4 drug resistance 47 early genes 29 electron microscopy 40 ELISA 41, 43 encephalitis 37–8, 49, 50 endogenous reinfection 30, 34 envelope 28 epidemiology 33–4 exogenous reinfection 30, 34 famciclovir 45, 47, 48, 49 foscarnet 47 ganciclovir 47 gastrointestinal 263 gene expression 29 genital infection 34, 35, 37, 50 glycoproteins 28–9, 32 HIV 39, 736 HLA associations 31 humoral immunity 31–2 IgA antibodies 31 IgG antibodies 31 IgM antibodies 31, 43 immediate early genes 29 immune response 31

virus-induced modulation 32 immunisation 47–8

INDEX

867

herpes simplex virus (HSV-1 and HSV-2) (cont.) immunoassay 41 immunocompromised patients 32–3,

38–9, 49 in situ hybridisation 43 in situ PCR 43 initial infection 29, 48–9 isolation 41 late genes 29 latency 30–1 latency-associated transcripts

(LATs) 30–1 latent infection 29 light microscopy 40 management 44–50 meningitis 39, 49 morphology 27–8 neonatal infection 36–7, 50 nosocomial spread 843 nucleic acid detection 41–3 occupationally-acquired 34 ocular infection 35–6, 49 orofacial infection 34–5 oropharyngeal infection 34–5 pathogenesis 29–33 PCR 41–3 penciclovir 45, 46–7, 48 peripheral blood 43 pregnancy 37, 50 primary infection 29, 33–4, 48–9 reactivation triggers 31 recurrent infection 30, 34, 39, 49 reinfection 30, 34 replication 28–9 serology 43–4 sexual transmission 33 socioeconomic factors 33 steroids 49 tegument 27–8, 29 triggers 31 UV microscopy 40 valaciclovir 45, 46, 48, 49 valganciclovir 47 VZV

common ancestry with 55 cross-reaction 60

zosteriform 40 herpes zoster aciclovir 77, 78 amitriptyline 78 antiviral drugs 77–9 brivudin 78 common aetiology with varicella 53 complications 70–1 disseminated 70 encephalitis 70–1 epidemiology 65

eye 69, 71, 78, 736 famciclovir 77–8 HIV infection 71 idoxuridine 77 immunocompromised patients 70,

78 management 77–9 meningitis 70–1 motor neuropathies 71 myelitis 70–1 neuralgia 70 pain 70 penciclovir 78 prednisolone 78 pregnancy 70 recurrent 69–70 secondary bacterial infection 70 sorivudine 78 steroids 78 sympathetic nerve blocks 78 vaccination 81 valaciclovir 77–8

Herpesviridae 23–6

biological properties 24 classiﬁcation 24–6 DNA 23 genome 23 latent infection 23 oncogenic associations 23–4 reactivation 23 subfamilies 25

herpetic whitlows 39 HERV-K 723, 724 HERV-W 724 heterophil antibody test 136 Highlands J virus 512, 523 histology 4 HIV infection

abacavir 745, 747 adenoviruses 352 amdoxovir 747–8 amprenavir 745, 749 antiretroviral therapy 734, 743–52

initiation 14 monitoring 15

apoptosis 729 aseptic meningoencephalitis 733 asymptomatic phase 729, 734 atazanavir 750 breast-feeding 726, 739 Burkitt’s lymphoma 143 Caesarean section 739 candidiasis 735 capravirine 748 CCR5 inhibitors 751 CD4:CD8 ratio 733 CD4 count 733 , 734

therapeutic range 744

cervical cancer 739 children 726, 739–40 classiﬁcation 732 clinical disease 732–43 Cryptococcus neoformans 735 cytokines 752–3 DAPD 747–8 delaviridine 745 diagnosis 740–3 diarrohea 736 didanosine 745, 746 diﬀerential diagnosis 734 drug resistance

GIGA-HAART 752 phenotyping/genotyping 17,

742–3 transmission 751–2 treatment interruption 752

efavirenz 745, 748 ELISA 740 encephalopathy 740 enfuvirtide 750 epidemiology 725–6 failure to thrive 740 FTC 748 fungal infections 735–6 fusion inhibitors 750 GBV-C 817 GIGA-HAART 752 global burden 725 GM-CSF 753 HAART 752 HBV 227, 736 HCV 736 healthcare workers 837, 838 herpes zoster 71 HHV-6 and HHV-7 152, 158–9 HHV-8 primary infusion lymphoma

143 HLA status 731 Hodgkin’s disease 143 HSV 39, 736 HTLV-I/II co-infection 774–5 human papillomavirus 736, 738 immunotherapy 752 indinavir 745, 749 integrase inhibitors 751 interleukin-2 752–3 lamivudine 745, 746–7 latent phase 734 lopinavir 745, 750 lymphadenopathy 733 lymphocyte depletion 729 lymphoma 143 management 732–43 measles vaccination 424 molluscum contagiosum 736 monitoring 734, 740–3

868

INDEX

Mycobacterium infections 735 nelﬁnavir 745, 749 neonates

diagnosis 741–2 therapy 744

nevirapine 745, 748 labour 739 non-Hodgkin’s lymphoma 143, 737–8 non-nucleoside reverse transcriptase inhibitors (NNRTIs) 744, 745, 748–9

nucleoside analogues 744–8 cross-resistance 747 nucleotide analogues 744–8 ophthalmic zoster 736 opportunistic infections 734–6 oral hairy leukoplakia 143–4, 734 oral lesions 734 p24/antibody assays 5 parvovirus B19 anaemia 714 passive immunisation 752 pathogenesis 729–32 PCR 740–2 plasma viral loads 734 Pneumocystis carinii pneumonia

735 prediction 14 preditor–prey model 733–4 pregnancy 739

therapy 744 primary 729, 732–4 primary central nervous system

lymphoma (PCNSL) 143 prognostic indicators 734 protease inhibitors 744, 745, 749–50 REMUNE 753 respiratory syncytial virus 331 ritonavir 745, 750 rubella vaccination 449–50 saquinavir 745, 749 serology 740–2 stavudine 745, 746 symptomatic disease 734 T-20 750 T-1249 750 tenofovir 745, 747 therapeutic vaccination 752–3 tipranavir 750 TMC120 748 TMC125 748 Toxoplasma gondii 735 vaccines 753–4 vertical transmission 739 viral load assays 742 VZV 736 zalcitabine 745, 746 zidovudine 739, 743, 745–6

Hodgkin Reed–Sternberg (HR-S) cells

141 Hodgkin’s disease 131, 141–2, 143

HIV 143 homosexual men, KSHV 177 horses

Eastern equine encephalitis virus

521 Venezuelan equine encephalitis virus

524, 525, 526 Western equine encephalitis virus

523, 524 hospital-acquired infections see noso-

comial infection HTLV-I associated myelopathy

(HAM) 760, 768, 769, 772–3 Human adenoviruses A–F 344 human anti-varicella zoster immuno-

globulin 79 human behaviour, emerging infections

828 human enteroviruses (HEV) 473, 474 Human herpesvirus 1 24 , 25 see also

herpes simplex virus Human herpesvirus 2 24 , 25 see also

herpes simplex virus Human herpesvirus 3 24 , 25 see also

varicella zoster virus Human herpesvirus 4 24 , 25 see also

Epstein–Barr virus Human herpesvirus 5 24 , 25 see also

cytomegalovirus Human herpesvirus 6 (HHV-6) 147–67

aciclovir 161 adults 154 AIDS 158–9 biology 147–8 brain commensal 157 bystander eﬀect 151 CD3 and CD4 surface expression

151 CD46 receptor 148 chemokine receptor homologues 152 cidofovir 161 clinical features 153–9 complications 153–4 congenital infection 152 conserved herpesvirus genes 149 cytokines 151 diagnosis 159–60 diarrhoea 153 DNAaemia 159 drug-resistant 161 early genes 150 electron microscopy 159 epidemiology 152–3 exanthem subitum 153 fatalities 155–6

febrile seizures 153–4 fever 153 foscarnet 161 ganciclovir 161 gene regulation 150 gene therapy 150 genome 149 glycoproteins 150 heart disease 156–7 heparin/heparin sulphate receptors

148 HHV-7 reactivation 149 HIV interaction 152, 158–9 IM association 154 immediate-early genes 150 immunity 150–2 immunoﬂuorescence 159–60 immunomodulation 151–2 infection routes 148 JC virus transactivation 684 late genes 150 latency 149 lytic replication 148, 150 MHC class I molecules 151 molecular biology 149–50 monocyte dysfunction 151–2 multiple sclerosis 157–8 neontal infection 152 neurological disease 157 PCR 160 phosphonoacetic acid 161 primary infection 153–4 protein encoding 152 quantitative detection 13 rash 153 reactivation 154–5 serology 160 T lymphocytes 148 transplantation 154–5 treatment 160–1 variant A (HHV-6A) 24, 25, 147,

148 , 153 variant B (HHV-6B) 24, 25, 147, 148 , 153 vascular disease 156–7 xenotransplantation 159

Human herpesvirus 7 (HHV-7) 24, 25, 147–67 aciclovir 161 adults 154 AIDS 158–9 biology 147–8 brain commensal 157 bystander eﬀect 151 CD3 and CD4 surface expression

151 CD4 receptor 148 chemokine receptor homologues 152

INDEX

869

Human herpesvirus 7 (HHV-7) (cont.) cidofovir 161 clinical features 153–9 complications 153–4 congenital infection 152 conserved herpesvirus genes 149 diagnosis 159–60 DNAaemia 159 drug-resistant 161 early genes 150 electron microscopy 159 epidemiology 152–3 exanthem subitum 153 fatalities 155–6 febrile seizures 154 fever 153 foscarnet 161 ganciclovir 161 gene regulation 150 gene therapy 150 genome 149 glycoproteins 150 heart disease 156–7 heparin/heparin sulphate receptors

148 HHV-6 reactivation 149 HIV interaction 152, 158–9 IM association 154 immediate-early genes 150 immunity 150–2 immunoﬂuorescence 159–60 immunomodulation 151–2 infection routes 148 JI 147 late genes 150 latency 149 lytic replication 148, 150 MHC class I molecules 151 molecular biology 149–50 multiple sclerosis 157–8 neontal infection 152 neurological disease 157 PCR 160 phosphonoacetic acid 161 primary infection 153–4 protein encoding 152 quantitative detection 13 rash 153 reactivation 154–5 RK 147 serology 160 strains 147–8 T lymphocytes 148 transplantation 154–5 treatment 160–1 vascular disease 156–7 xenotransplantation 159

Human herpesvirus 8 24 , 25, 829 see

also Kaposi’s sarcoma-associated herpesvirus

human immunodeﬁciency virus

(HIV) breast milk 739 CCR5 731 CD4 731 cell tropism 731 CEM-15 731 classiﬁcation 724 CMV interaction 98 co-receptors 731 CSF 733 culture 726–7 cytopathic eﬀect 727 dynamics 729–31 ELISA 727, 728 gag 727 gastrointestinal 263 genome 727–8 host factors and replication 731 isolation 726–7 load 728–9 Lv1 731 mass screening 728 nef 728 neutralising antibodies 728 occupational exposure 837, 838 p17 728 p24 capsid antigen 728 post-exposure prophylaxis 840 previous names 722 proteins 727–8 quantitative PCR 8 receptors 731 resistance 728–9 rev 728 RT-PCR 729 serology 728 subpopulations in body compart-

ments 731 tat 728 transmission prediction 15–16 vif 728 vpr 728 vpu 728 Western blot 728 see also HIV infection

Human immunodeﬁciency virus type 1

(HIV-1) 722 children, clinical categories 733 clades 726 gag 727 genome 727–8 isolation 724 JC virus transactivation 683 nef 728

non-human primate vectors 832 NSI substrain 724 p17 728 p24 capsid antigen 728 phenotypic classiﬁcation 724 proteins 727–8 R5 viruses 724 rev 728 SI substrain 724 tat 728 vif 728 vpr 728 vpu 728 X4 viruses 724

Human immunodeﬁciency virus type 2 (HIV-2) 722 gag 727 genome 727–8 isolation 724 nef 728 non-human primate vectors 832 p17 728 p24 capsid antigen 728 phenotypic classiﬁcation 724 proteins 727–8 rev 728 tat 728 transmission rate 726 vif 728 vpr 728 vpx 727

human papillomavirus (HPV) ASCUS 671 cervix 668–70 condylomata acuminata 668 genital 667 genital cancer 668–70 genital warts 668, 670

treatment 671–2 genome 661–2 genotyping 14 high-grade squamous intraepithelial

lesions (HSILs) 669 HIV/AIDS 736, 738 HPV-6 669 HPV-11 665, 669 HPV-16 662–5, 669, 670 HPV-18 669, 670 HPV-30 669 HPV-31 665, 669 HPV-33 669 HPV-34 669 HPV-35 669 hybrid capture 671 intraepithelial neoplasia 668 laryngeal infections 667 low-grade squamous intraepithelial

lesions (LSILs) 668–9

870

INDEX

871 natural history of infections 666–7

INDEX

p30 763 neonatal infection 667

heterologous transactivation of

pathogenesis 770–3 oncogenic potential 667–8

virus transcription 683–4

peptide-based assays 763 pathogenesis 667–70

humoral immune response 698

pol 762 PCR 671

late proteins 677–8, 679

prevention of disease 774 penile cancer 670

life cycle 676–7

pX 762 persistent infection 670

oncogenicity 694–6

receptors 760 serology 671

origin of DNA replication (ORI)

recombinant surface membrane gly- vaccine 672–3

coprotein 763 viral coded proteins 662

PCR 697

respiratory infections 770 human parainﬂuenza virus type 1

persistent infection 688–90

Rex 762–3 (HPIV1) 299

pregnancy 691

Schistosoma mansoni 769 croup 308–10

primary infection 686–8

serology 763 epidemiology 311

structure 675–6

sexual transmission 768 host range 302

TAg 676–7, 678, 681, 682

Sjo¨gren’s syndrome 770 pathogenesis 305

tAg 677

strongyloidiasis 679 receptors 301

transcriptional control elements

tax 762 replication 304

(TCR) 677, 679–80

Tax 762, 763, 771 human parainﬂuenza virus type 2

transcriptional expression 679–83

transmission 768 (HPIV2) 299

treatment 699–701

treatment 773–4 croup 308

VP1–3 675, 677

tuberculin hypersensitivity 769 epidemiology 311

YB-1 678, 681–2, 683

urinary infections 770 interferon-mediated immune

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