Principles and Practice of Clinical Virology (Fifth Edition)

Clinical Virology F IFTH E DITION

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

F IFTH E DITION

Edited by

Arie J. Zuckerman

Royal Free and University College Medical School, London, UK

Jangu E. Banatvala

Guy’s, King’s and St Thomas’ School of Medicine, London, UK

John R. Pattison

Department of Health, London, UK

Paul D. Griffiths

Royal Free and University College Medical School, London, UK

Barry D. Schoub

National Institute for Communicable Diseases, Sandringham, South Africa

First published 1987; Second Edition published 1990; Third Edition published 1994; Fourth Edition published 2000 Copyright u 1987, 1990, 1994, 2000, 2004

John Wiley & Sons Ltd, The Atrium, Southern Gate, Chichester, West Sussex PO19 8SQ, England

Telephone (+44) 1243 779777

Email (for orders and customer service enquiries): cs-books@wiley.co.uk Visit our Home Page on www.wileyeurope.com or www.wiley.com

All Rights Reserved. No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, scanning or otherwise, except under the terms of the Copyright, Designs and Patents Act 1988 or under the terms of a licence issued by the Copyright Licensing Agency Ltd, 90 Tottenham Court Road, London W1T 4LP, UK, without the permission in writing of the Publisher. Requests to the Publisher should be addressed to the Permissions Department, John Wiley & Sons Ltd, The Atrium, Southern Gate, Chichester, West Sussex PO19 8SQ, England, or emailed to permreq@wiley.co.uk, or faxed to (+44) 1243 770620.

This publication is designed to provide accurate and authoritative information in regard to the subject matter covered. It is sold on the understanding that the Publisher is not engaged in rendering professional services. If professional advice or other expert assistance is required, the services of a competent professional should be sought.

Other Wiley Editorial Offices John Wiley & Sons Inc., 111 River Street, Hoboken, NJ 07030, USA Jossey-Bass, 989 Market Street, San Francisco, CA 94103-1741, USA Wiley-VCH Verlag GmbH, Boschstr. 12, D-69469 Weinheim, Germany John Wiley & Sons Australia Ltd, 33 Park Road, Milton, Queensland 4064, Australia John Wiley & Sons (Asia) Pte Ltd, 2 Clementi Loop #02-01, Jin Xing Distripark, Singapore 129809 John Wiley & Sons Canada Ltd, 22 Worcester Road, Etobicoke, Ontario, Canada M9W 1L1

Wiley also publishes its books in a variety of electronic formats. Some content that appears in print may not be available in electronic books.

British Library Cataloguing in Publication Data A catalogue record for this book is available from the British Library

ISBN 0-470-84338-1

Typeset in 9/11pt Times by Dobbie Typesetting Ltd, Tavistock, Devon. Printed and bound in Great Britain by Antony Rowe Ltd, Chippenham, Wilts. This book is printed on acid-free paper responsibly manufactured from sustainable forestry in which at least two trees are planted for each one used for paper production.

Contents

List of Contributors ................

vii

3 Hepatitis Viruses ............... 199 Tim J. Harrison, Geoffrey M. Dusheiko

Preface .........................

and Arie J. Zuckerman Preface to the Fourth Edition ..........

xi

xii

4 Viruses Associated with Acute Diarrhoeal Disease .............. 249

Preface to the Third Edition ........... xiii Ulrich Desselberger and Jim Gray Preface to the Second Edition .......... xiv

5 Influenza ..................... 271 Chris W. Potter

Preface to the First Edition ...........

xv

6 Parainfluenza Viruses ............ 299 Plates .......................... xvii

Stelios Psarras, Nikolaos G. Papadopoulos and Sebastian L. Johnston

1 Diagnostic Approaches ...........

Katie Jeffrey and Deenan Pillay

7 Respiratory Syncytial Virus ........ 323 Caroline Breese Hall

2 The Herpesviridae ..............

Graham M. Cleator and Paul E. Klapper

8 Adenovirus .................... 343 Marcela Echavarria

2A Herpes Simplex ................

Graham M. Cleator and Paul E. Klapper

9 Rhinoviruses .................. 361 Nikolaos G. Papadopoulos and

2B Varicella Zoster ................

53 Sebastian L. Johnston Judith Breuer

10 Coronaviruses and Toroviruses ...... 379 2C Cytomegalovirus ...............

85 David Cavanagh

Paul D. Griffiths

11 Measles ...................... 399 2D Epstein–Barr Virus ............. 123

Sibylle Schneider-Schaulies and Dorothy H. Crawford

Volker ter Meulen

2E Roseoloviruses: Human Herpesviruses

12 Rubella ...................... 427

6 and 7 ...................... 147 Jennifer M. Best and Jangu E. Banatvala Ursula A. Gompels

13 Mumps ...................... 459 2F Kaposi’s sarcoma-associated

Pauli Leinikki

Herpesvirus (Human Herpesvirus 8) .. 169 Abel Viejo-Borbolla, Cornelia

14 Enteroviruses .................. 467 Henke-Gendo and Thomas F. Schulz

Philip D. Minor and Peter Muir Philip D. Minor and Peter Muir

CONTENTS

15 Poxviruses .................... 491

24 Human Parvoviruses ............. 703 Inger Damon, Peter Jahrling and

Kevin E. Brown

James LeDuc

25 Human Immunodeficiency Viruses ... 721

16 Alphaviruses .................. 509 Robin A. Weiss, Angus G. Dalgleish, Graham Lloyd

Clive Loveday and Deenan Pillay

17 Flaviviruses ................... 531 25A The Human T Cell Lymphotropic Barry D. Schoub and Nigel K.

Viruses ...................... 759 Blackburn

Graham P. Taylor

26 Human Prion Diseases ........... 779

18 Bunyaviridae .................. 555

John Collinge

Robert Swanepoel

27 GBV-C and TTV ............... 813

19 Arenaviruses .................. 589

Shigeo Hino

Colin R. Howard

28 Emerging Virus Infections ......... 825

20 Filoviruses .................... 611

Brian W. J. Mahy

Susan P. Fisher-Hoch

29 Hospital-acquired Infections ........ 835

21 Rabies and Other Lyssavirus 29A Infections Acquired via the Infections .................... 631

Blood-borne Route .............. 837 Mary J. Warrell

Anthea Tilzey

22 Papillomaviruses ................ 661 29B Infections Acquired via Other Dennis McCance

Routes ...................... 843 Philip Rice

23 Human Polyomaviruses ........... 675 Kristina Do¨rries

Index ........................... 859

Contributors

Jangu E. Banatvala Emeritus Professor of Clinical Inger Damon Chief, Poxvirus Section, Division of Virology, Guy’s, King’s and St Thomas’ School of

Viral and Rickettsial Diseases, National Center for Medicine, Lambeth Palace Road, London SE1 7EH,

Infectious Diseases, Centers for Disease Control and UK

Prevention, 1600 Clifton Rd NE, Mailstop G-18, Jennifer M. Best Reader in Virology, Guy’s, King’s

Atlanta, GA 30333, USA

and St Thomas’ School of Medicine, Lambeth Palace Ulrich Desselberger Consultant Virologist and Road, London SE1 7EH, UK Director, Clinical Microbiology and Public Health

Nigel K. Blackburn Senior Consultant Virologist, Laboratory, Addenbrooke’s Hospital, Hills Road, National Institute for Communicable Diseases,

Cambridge CB2 2QW, UK

Sandringham, South Africa Kristina Do¨rries Senior Scientist and Group Leader,

Judith Breuer Reader and Consultant in Virology, Institute for Virology and Immunobiology, Julius- Skin Virus Laboratory, St Bartholomew’s and the

Maximilians-University Wu¨rzburg, Versbacher Strasse Royal London School of Medicine and Dentistry,

7, D-97078 Wu¨rzburg, Germany 25–29 Ashfield Street, London E1 1BB, UK

Kevin E. Brown Senior Investigator, Virus Discovery Geoffrey M. Dusheiko Professor of Medicine, Group, Hematology Branch, National Heart, Lung and

Department of Medicine, Royal Free and University Blood Institute, Bethesda, MD, USA

College Medical School, Rowland Hill Street, London NW3 2PF, UK

David Cavanagh Principal Scientist, Institute for Animal Health, Compton Laboratory, Compton,

Marcela Echavarria Assistant Professor of Newbury, Berks RG20 7NN, UK

Microbiology, Centro de Educacio´n Me´dica e Graham M. Cleator Reader in Medical Virology,

Investigaciones Clı´nicas, CEMIC University Hospital, Laboratory Medicine Academic Group, Department of

Galvan 4102, (C1431FWO), Buenos Aires, Argentina Virology, 3rd floor, Clinical Sciences Building,

Manchester Royal Infirmary, Oxford Road, Manchester Susan P. Fisher-Hoch Professor of Biological M13 9WL, UK

Sciences, University of Texas, Houston School of Public Health at Brownsville, 80 Fort Brown, Brownsville, TX

John Collinge Head, Department of

78520, USA

Neurodenegerative Disease and Director, MRC Prion Unit, Institute of Neurology, University College

Ursula A. Gompels Senior Lecturer in Molecular London, London, UK

Virology, Pathogen Molecular Biology Unit, Dorothy H. Crawford

Professor of Medical Department of Infectious and Tropical Diseases, London Microbiology and Head of the School of Biomedical and

School of Hygiene and Tropical Medicine, University of Clinical Laboratory Sciences, The University of

London, Keppel Street, London WC1E 7HT, UK Edinburgh, Hugh Robson Building, George Square,

Jim Gray Head, Enteric Virus Unit, Enteric, Edinburgh EH8 9XD, UK Respiratory and Neurological Virus Laboratory,

Angus G. Dalgleish Professor of Oncology, St Specialist and Reference Microbiology Division, Health George’s Hospital Medical School, Cranmer Terrace,

Protection Agency, 61 Colindale Avenue, London NW9 London SW17 0RE, UK

5DF, UK 5DF, UK

CONTRIBUTORS

Paul D. Griffiths Professor of Virology, Royal Free Clive Loveday Clinical Director, International Clinical and University College Medical School, Rowland Hill

Virology Centre, Great Missenden, UK Street, London NW3 2PF, UK

Brian W. J. Mahy Senior Scientific Research Advisor, Caroline Breese Hall Professor of Pediatrics and

National Center for Infectious Diseases, CDC, 1600 Medicine in Infectious Diseases, University of Rochester

Clifton Road, Mailstop C12, Atlanta, GA 30333, USA School of Medicine, 601 Elmwood Avenue, Box 689, Rochester, NY 14642, USA

Dennis McCance Department of Microbiology and Immunology, Head, Virology Unit, University of

Tim J. Harrison Reader in Molecular Virology, Rochester, Box 672, 601 Elmwood Avenue, Rochester, Royal Free and University College Medical School,

NY 14642, USA

Royal Free Campus, Rowland Hill Street, London NW3 2PF, UK

Philip D. Minor Head, Division of Virology, National Institute for Biological Standards and Control, Potters

Cornelia Henke-Gendo Clinical Virologist,

Bar, Hertfordshire, UK

Department of Virology, Hannover Medical School, Carl-Neuberg Strasse 1, 30625 Hannover, Germany

Peter Muir Health Protection Agency South West, Myrtle Road, Bristol BS2 8EL, UK

Shigeo Hino Department of Virology, Faculty of Medicine, Tottori University, 86 Nishi, Yonago 683-

Nikolaos G. Papadopoulos Lecturer, Allergy Unit, 8503, Japan

2nd Department of Pediatrics, University of Athens, Greece

Colin R. Howard Vice-Principal for Strategic Development and Professor of Microbiology, Royal

Deenan Pillay Reader in Virology, Royal Free and Veterinary College, University of London, Royal

University College Medical School, Windeyer Building, College Street, London NW1 0TU, UK

46c Cleveland Street, London W1P 6DB, UK Chris W. Potter Emeritus Professor, University of

Peter Jahrling USAMRIID, Fort Detrick, Frederick, Sheffield, Division of Genomic Medicine, School of MD 21702-5001, USA Medicine and Biomedical Sciences, F Floor, Beech Hill

Katie Jeffery Consultant Virologist, Department of Road, Sheffield S10 2RX, UK Microbiology, John Radcliffe Hospital, Headington,

Stelios Psarras Research Associate, Allergy Unit, 2nd Oxford OX3 9DU, UK Pediatric Clinic, University of Athens, Greece

Sebastian Johnston Professor, Department of Philip Rice Consultant Virologist, St George’s Respiratory Medicine, National Heart and Lung Hospital Medical School, Cranmer Terrace, London Institute, Imperial College, London, UK

SW17 0RE, UK

Paul E. Klapper Consultant Clinical Scientist and Sibylle Schneider-Shaulies Institute for Virology and Honorary Senior Lecturer, Health Protection Agency,

Leeds Laboratory, Bridle Path, York Road, Leeds LS15 Immunobiology, University of Wu¨rzburg, Versbacher

7TR, UK Strasse 7, D-97078, Wu¨rzburg, Germany

Barry D. Schoub Executive Director, National James LeDuc Director, Division of Viral and

Institute for Communicable Diseases, Sandringham, Rickettsial Diseases, National Center for Infectious

South Africa

Diseases, Centers for Disease Control and Prevention, 1600 Clifton Road, Mail-stop A-30, Atlanta, GA 30333,

Thomas F. Schulz Head of the Department of USA

Virology, Hannover Medical School, Carl-Neuberg Strasse 1, 30625 Hannover, Germany

Pauli Leinikki Professor, Department of Infectious Diseases Epidemiology, National Public Health Institute

Robert Swanepoel National Institute for (KTL), Mannerheimintie 166, FIN-00300 Helsinki,

Communicable Diseases, Sandringham, South Africa Finland

Graham P. Taylor Senior Lecturer/Honorary Graham Lloyd Head, Special Pathogens, Centre for

Consultant, Department of Genito-urinary Medicine and Applied Microbiology and Research, Porton Down,

Communicable Diseases, Faculty of Medicine, Imperial Salisbury, Wiltshire SP4 0JG, UK

College, London, UK College, London, UK

CONTRIBUTORS

Mary J. Warrell Research Associate, Centre for Immunology, University of Wu¨rzburg, Versbacher

Tropical Medicine, John Radcliffe Hospital, Oxford Strasse 7, D97078 Wu¨rzburg, Germany

OX3 9DU, UK

Anthea Tilzey Clinical Senior Lecturer, Virology Section, Department of Infectious Diseases, Guy’s,

Robin A. Weiss Professor of Viral Oncology, King’s and St Thomas’ School of Medicine, St Thomas’

University College, London, UK Campus, UK

Arie J. Zuckerman Professor of Medical Abel Viejo-Borbolla Postdoctoral Fellow, Department

Microbiology, Royal Free and University College of Virology, Hannover Medical School, Carl-Neuberg

Medical School, Rowland Hill Street, London NW3 Strasse 1, 30625 Hannover, Germany

2PF, UK

Preface

The knowledge and practice of clinical virology epidemiological patterns of infection. Between this new continues to expand. The first edition of Principles

edition and the last, much concern has been focused on and Practice of Clinical Virology , published in 1987,

the global threat posed by new viruses. Consequently, a contained 16 chapters and 590 pages. Each of the

new chapter on ‘Emerging Infections’ is included. subsequent editions became progressively larger. This

There is also a new chapter on ‘Hospital-acquired edition has 902 pages and 38 chapters, including seven

Infections’, which will be of benefit to those who have within the section on the Herpesviridae, each of which

to deal with the day-to-day management of patients in is comprehensive.

hospital. This chapter also includes some advice Rapid progress in the field has occurred between the

relating to SARS. However, fresh knowledge about fourth and fifth editions. There are now two new editors

SARS continued to accumulate as the fifth edition was and a number of new authors, which will increase

in preparation, and further information is included, international representation. In addition, each of the

not only in the chapter on ‘Emerging Infections’ but remaining chapters has been extensively revised or

also in the one on ‘Coronaviruses’. rewritten, taking into account knowledge accumulated

In comparison with the fourth edition, additional in molecular biology with its applications for laboratory

colour plates have been included, and, as in previous diagnosis, immunisation and antiviral chemotherapy.

editions, an attempt has been made to limit references Each chapter also highlights the clinical features and

to key publications.

A. J. Zuckerman J. E. Banatvala J. R. Pattison P. D. Griffiths

B. D. Schoub

Preface to the Fourth Edition

It is now 13 years since the first edition of Principles Advances in diagnostic methods, particularly mo- and Practice of Clinical Virology was published. A

lecular biological techniques and their application to comparison of the first and fourth editions testifies to

clinical problems, are reflected in a new chapter on the rapid expansion in virology during the intervening

‘Diagnostic Approaches’, which presents an overview years, including major developments in technology, the

of the value and limitations of established and more application of these to clinical practice and advances in

recently developed techniques. Advances in serological the treatment of viral infections with an increasing

techniques as well as in virus identification are number of antiviral drugs. Indeed, such has been the

emphasised. This chapter also includes an important progress in the field of clinical virology even within the

section on assays for determining antiviral drug period between the third and fourth editions that we

resistance.

have asked a number of new authors to contribute Most of the chapters reflect advances in patient chapters. These include the chapters on rhinoviruses,

management, including—where appropriate—antiviral viruses associated with acute diarrhoeal disease, and

chemotherapy. As expected, the chapters on hepatitis human polyomaviruses. Of the remaining chapters,

viruses and human retroviruses have been expanded virtually all have been revised substantially. The

considerably in the light of continuing and rapid chapter on the Herpesviridae has been expanded

advances in these fields. Both chapters now include a considerably, particularly in relation to Human her-

component by authors with everyday practical experi- pesviruses 6 , 7 and 8; new authors have contributed to

ence in the management and treatment of patients with these sections. The chapter on hepatitis viruses reflects

these infections.

the considerable expansion of information relating to In comparison with the third edition, more coloured hepatitis E and C as well as the role of such newly

plates have been included, which we hope our readers recognised agents as GB and the new human virus,

will appreciate. As in previous editions, we have TTV.

attempted to limit the reference lists to key publications.

A. J. Zuckerman J. E. Banatvala J. R. Pattison

Preface to the Third Edition

Principles and Practice of Clinical Virology was first As expected, rapid developments continue to occur published in 1987; a third edition within 7 years of the

in the field of human retroviruses, particularly the first attests to the continuing and rapid progress in the

human immunodeficiency viruses, and this chapter field of clinical virology.

reflects the accumulation of new and important data in All the chapters have been revised and some completely

this area. The chapter on human prion disease has rewritten, reflecting the increasing knowledge of viruses

been almost entirely rewritten to reflect many of the or groups of viruses included in the various chapters.

substantial advances in prion research. Thus, the chapter on hepatitis viruses now contains

Most of the authors stress the developments and sections on hepatitis A and E viruses as well as individual

application of molecular biological techniques which contributions on hepatitis B, D and C. The previous

are leading not only to improved methods of diagnosis edition contained two chapters on viral haemorrhagic

but also to an increased understanding of viral fevers but this section now includes separate chapters for

pathogenesis.

the flaviviruses, alphaviruses, Bunyaviridae, arenaviruses Rather than burden readers with a large number of and filoviruses. The chapters on arenaviruses and

references, we have aimed to include most of the key filoviruses have been contributed by new authors.

ones.

New authors have also contributed the chapter on Finally, we are grateful for the helpful comments herpes simplex virus infections and the chapter on the

which we received from many of our readers; some of more newly-recognised herpesviruses now includes a

their suggestions have been incorporated into the third section on Human herpes virus 7.

edition.

A. J. Zuckerman J. E. Banatvala J. R. Pattison

Preface to the Second Edition

In the preface to the first edition of Principles and more information on human immunodeficiency viruses Practice of Clinical Virology we stated that it was our

but also on HTLV-1 and -2. The chapter on hepatitis intention, with new editions, to remain up-to-date as

has been separated into two sections: the first on the subject advanced. Such is the pace of development

hepatitis A and the viruses causing non-A, non-B in clinical virology that plans were laid for a new

hepatitis, two of which have been identified as hepatitis edition within a few months of the first being

C and E, and the second on hepatitis B and D (delta published, and this second edition is appearing only

agent).

two and a half years after the first. Each chapter has As before, we were aware when organising the book been revised, many extensively, to take account of

that no single arrangement is entirely satisfactory. We progress in the understanding of the epidemiology,

have chosen to arrange the chapters on the basis of pathogenesis, diagnosis, management and prevention

individual viruses or groups of viruses. General of virus infections. Perhaps the greatest single recent

chapters on virus structure, taxonomy and patho- contribution to the subject has been made by the

genesis are not included, but the information on these application of molecular biological techniques, and

aspects necessary for an understanding of the practice each of our authors has highlighted the contribution of

of clinical virology is included in the individual this rapidly developing discipline to clinical virology.

chapters.

Human herpesvirus 6 is now the subject of a new Such factors as the likely discovery of yet new chapter, and we have also added a new chapter on

viruses, improvements in the rapidity and sensitivity of haemorrhagic fevers to include much new information

diagnostic techniques, and the development of new on their pathogenesis. As expected, the extensive

vaccines, which are likely to involve recombinant accumulation of new information relating to infection

techniques and progress in antiviral therapy, will by human retroviruses has resulted in extensive

ensure that thought will be given to revision and updating of this chapter, not only to include much

preparation of another edition.

A. J. Zuckerman J. E. Banatvala J. R. Pattison

Preface to the First Edition

There has been a spectacular increase during the last 30 and practice of clinical virology are concerned with years in our knowledge of virology. This has taken

rapid laboratory diagnosis leading to appropriate place to such an extent that virology can now be

patient management which might involve specific regarded as an umbrella term encompassing a variety

therapy and/or infection control measures at a of distinct but related disciplines. There are funda-

hospital, a national and occasionally at an inter- mental connections with biochemistry, genetics and

national level.

molecular biology, and each of these aspects would be In organising the book we were aware that there is no worth a treatise in itself. Clinical virology is that aspect

single arrangement that is entirely satisfactory. We have which is concerned with the cause, diagnosis, treatment

chosen to arrange the chapters on the basis of individual and prevention of virus infections of man. It too

viruses or groups of viruses. General chapters on virus has acquired a substantial body of knowledge and

structure, taxonomy and pathogenesis are not included accumulated experience over the past 30 years and

but the information on these aspects necessary for an this book is intended to be an authoritative account of

understanding of the practice of clinical virology is the present situation. Formerly virological diagnosis

included in the individual chapters. was time consuming, retrospective and rarely influ-

Clinical virology is a subject which continues to enced the management of the patient. During the past

evolve. This is usually for one of two reasons, either 10–15 years the picture has changed dramatically.

the need to apply new technology or the need to study Newly recognised diseases such as AIDS and some

new diseases or epidemiological situations. We have haemorrhagic fevers, which have very serious con-

therefore invited authors who are specialist investiga- sequences for individuals and populations, have been

tors into each of the viruses to contribute up-to-date, shown to be due to viruses. In the clinical virology

stimulating accounts of the practice of clinical virology laboratory there has been a change in emphasis

and provide a framework for the assimilation of towards rapid diagnostic techniques. Finally, effective

imminent advances. One chapter has already had to antiviral chemotherapy is a reality at least for some

be significantly updated during the time of preparation virus infections and there has been an expansion in the

of the book and it is our intention, with new editions, use of immunoprophylaxis. Thus the current principles

to remain up-to-date as the subject advances.

A. J. Zuckerman J. E. Banatvala J. R. Pattison

PLATE I

xvii

Figure 2A.2 M ‘Cold sores’ at the pustular and crusting stages Figure 2A.3 M Rose Bengal staining of herpes simplex virus dendritic ulceration in a grafted cornea. (Kindly provided by R. E. Bonshek and A. B. Tullo)

Figure 2C.4 M‘ Proteins of CMV which have been mapped to date. UL = unique long region; US = unique short region; TR = terminal repeat; IR = inverted repeat. The genome is linear within the virus but has been circularized for convenience. Open reading frames of known function are coloured according to the following code: orange = transactivators; pink = DNA replication; green = capsid and/or assembly; red = tegument; pale blue = envelope; dark green = immune evasion; dark blue = miscellaneous

PLATE II

xviii

Figure 2C.8 M‘ Schematic representation of the ways viruses can interfere with presentation of HLA-peptide complexes at the plasma membrane. Rib, ribosome; ER, endoplasmic reticulum; V, virus-encoded protein; PRO, proteosome; PM, plasma membrane; TAP, transporter associated with antigen presentation. Peptides derived from virus-infected cells are generated in the proteosome and actively transported by TAP into the lumen of the ER. A ribosome is shown producing a protein with a signal peptide, which folds in the ER to produce the HLA Class I chain. This should normally associate with peptide and be transported to the plasma membrane. Misfolded HLA molecules can be re-exported from the lumen of the ER back into the cytosol where they are degraded by the proteosome. Virus-encoded genes interfere with this process as follows: the proteins may

be inherently insusceptible to proteosome digestion (EBNA of EBV) or may be modified to reduce their digestion (pp65 acts on MIE protein of HCMV). Proteins may block the function of TAP (ICP47 of HSV; US6 of HCMV). Proteins may bind mature class I molecules within the ER and so sequester them (E3-19K protein of adenoviruses; US3 protein of HCMV; m152 protein of MCMV). Two proteins of HCMV (US2 and US11) facilitate the re-export from the ER to the cytosol of mature HLA class I molecules. If all of these mechanisms are completely successful, the level of HLA display at the PM will be insufficient to prevent NK cells or macrophages recognising the cell as being abnormal and so destroying it. Proteins/peptides encoded within HCMV (UL18) or MCMV (m144) are presented at the plasma membrane to act as a decoy for NK cells by providing a negative signal. In addition, HCMV UL16 blocks transmission of a positive signal to another group of NK cells

Figure 2D.7 M‘ Photomicrograph of a May–Grunwald–Giemsa-stained peripheral blood film from acute infectious mononucleosis. An atypical mononuclear cell is illustrated (x 1000)

PLATE III

xix

Figure 2F.5 M‘ Genome diagram of KSHV. Open boxes with Roman numbers denote groups of structural or metabolic genes which are conserved among ȍherpesviruses and also many other herpesviruses. The solid line represents the long unique (coding) region, open and filled rectangles internal or terminal repeat regions. Solid circles represent originas of lytic relication [ori-(L), ori-(R)]. The position and transcriptional orientation of viral genes discussed in the text is indicated by pointed boxes. A red shading indicates genes known to be expressed in latently infected spindle cells and PEL cells (see text), light blue, dark blue and green shading refers to genes expressed in the different stages of the lytic cycle (see text). Colour coding of the names of individual viral genes refers to their presumed function, as indicated (see also text)

Figure 2F.6 M‘ Expression of vIL-6 in B cells of Multicentric Castleman’s Disease. vIL-6 is expressed in a small number of KSHV-infected

B cells, but may affect others through paracrine action (see text). Photograph kindly provided by Drs Y. Chang and P. Moore

PLATE IV

xx

Figure 2F.7 M‘ Overview of some KSHV–encoded proteins that may contribute pathogenesis. Vital proteins are coloured in red, interacting cellular proteins in grey. See text for a detailed explanation of the pathways and receptors engaged by KSHV proteins

Figure 2F.8 M‘ KSHV–encoded proteins involved in the control of apoptosis. Two apoptosis pathways exist in human cells and are regulated by a number of cellular and viral components. The extrinsic pathway is initiated by Fas-L or tumour necrosis factor (TNF) and assembly of the procaspase

8 complex is inhibited by FLIP (FLICE–inhibitory protein; see text). A KSHV–encloded FLIP homologue, vFLIP, acts in a similar manner. The intrinsic pathway is triggered by DNA damage, cytokine withdrawal, cytotoxic drugs and regulated by the Bcl/Bad/Bax group of proteins. A viral bcl-2 homologue, vbcl-2, acts at this stage. Finally, vIAP acts in the intrinsic pathway and on procaspase 3, as discussed in the text

PLATE V

xxi

Figure 8.1 M‘ Schematic graphic of adenovirus particle

PLA TE VI

Figure 11.3 M‘

A schematic representation of CD46 (MCP, membrane cofactor protein) (left), the major protein receptor for attenuated MV strains. MV binding sites are located within the short consensus repeat (SCR) domains 1 and 2, whereas complement components C3b/C4b bind to SCR 3 and 4, respectively. Proximal to the transmembrane domain, oligo-saccharide-rich serin/threonine/proline (STP) domains are located. CD150, a member of the lg superfamily, (right) is the receptor of all MV

xi strains tested as yet. MV binding occurs at the membrane distal domain (the V domain). Glycosylation sites in the extracellular domains are indicated as are residues in the

cytoplasmic domain identified as important for signaling

PLATE VII

xxiii

Figure 12.2 M Schematic representation of the replication and translation of rubella virus structural and non-structural proteins. (Reproduced from Best, Cooray & Banatvala, Topley and Wilson, 10th edition)

PLATE VIII

xxiv

Figure 21.3 M‘ World distribution of rabies. Rabies free areas are white; red indicates terrestrial rabies with or without bat rabies, and countries with only bat lyssaviruses are green

Figure 24.4 M‘ Giant pronormoblast

PLATE IX

xxv

Figure 24.5 M‘ Children with characteristic slapped cheek appearance and reticular lacy rash of fifth disease

Figure 25A.8 M‘ Perivascular lymphocytic infiltration in the central nervous system. (Courtesy of Dr Margaret Esiri)

PLATE X

xxvi

Figure 25A.9 M‘ Using unstimulated, uncultured peripheral blood lymphocytes from a patient with HTLV-I associated myelopathy the accumulation of viral proteins at the cell-cell junction and subsequent transfer of gag proteins and HTLV-I nucleic acids to a CD4 lymphocyte from an uninfected donor is demonstrated (courtesy of Professor Charles Bangham)

PLATE XI

xxvii

Figure 28.3 M‘ Recent Emergence and Reemergence of Human Viral Diseases, Examples

Figure 28.4 M‘ World Distribution of Dengue – 2002

PLATE XII

xxviii

Figure 28.5 M‘ New World Hantaviruses

1 Diagnostic Approaches

1 Katie Jeffery 2 and Deenan Pillay

1 John Radcliffe Hospital, Oxford, and

2 Royal Free and University College Medical School, London, UK

INTRODUCTION laboratory. Historically, viruses were propagated in laboratory animals and embryonated eggs, although

If clinical virology in the 1980s was characterised by most virus isolation techniques now rely on cultured the widespread use of enzyme-linked immunosorbent

cells. With appropriate specimens and optimal cell assay (ELISA) technology, then there is no doubt that

lines, this technique can be highly sensitive and the 1990s will be seen as the time when molecular

specific, and a presumptive diagnosis made on the methods of virus detection entered routine diagnostic

basis of a characteristic cytopathic effect (CPE), use. Following on from this development, the first few

confirmed by immunostaining. The judicious use of years of the twenty-first century will be seen as the

two or three cell lines, such as a monkey kidney line, a period when real-time PCR and virus quantitation

human continuous cell line and a human fibroblast came of age, along with increasing automation of

line, will allow the detection of the majority of molecular diagnostics. Concurrently, the emphasis and

cultivatable viruses of clinical importance, such as priorities of diagnostic virology laboratories have

herpes simplex virus (HSV), varicella zoster virus (VZV), shifted in response to: the availability of rapid

cytomegalovirus (CMV), enteroviruses, respiratory diagnostic methods; the identification of new viruses,

syncytial virus (RSV), adenovirus, parainfluenza many of which are non- or poorly cultivatable; the

viruses, influenza viruses and rhinoviruses. In addition, increasing availability of effective antiviral agents; the

the ability to grow virus from a clinical specimen emergence of antiviral resistance; the increasing

demonstrates the presence of viable virus (albeit viable number of immunocompromised patients, in whom

within the chosen cell line)—this is not necessarily the opportunistic viral infections are life-threatening; and

case with detection of viral antigen or genome. For new cost pressures on pathology services.

example, following initiation of antiviral therapy for This chapter provides an overview of diagnostic

genital herpes, HSV antigen can be detected from serial techniques against this background, and highlights

genital swabs for longer than by virus propagation in those clinical scenarios of particular importance to

cell culture. This infers that antigen persists in the virologists in a diagnostic setting.

absence of viral replication and underlines the impor- tance of correct interpretation of laboratory results. Nevertheless, virus isolation has now been shown to be

TECHNIQUES—AN OVERVIEW less sensitive than molecular amplification methods for this and other viruses (see later).

Virus Isolation The advantages of virus isolation include: the ability to undertake further examination of the isolate, such Many of the advances in clinical virology have come

as drug susceptibility assays (see later) or typing (Table about because of the ability to propagate viruses in the

1.1); the provision of epidemiological information on

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

2 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

Table 1.1 Virus isolation undertaken with polyclonal antisera, and then sub- sequently with pools of monoclonal antibodies, this

Advantages Disadvantages method uses either indicator-labelled antibody or a Sensitive

Slow (conventional cell culture) labelled antispecies antibody (indirect) to directly ‘Catch-all’

Labour-intensive visualise viral antigens in clinical specimens. Usually, Generates isolate for

Multiple cell lines required the label used is fluorescein. The indirect method is further study

more sensitive, since more label can be bound to an Detects ‘viable’ virus

infected cell. Results can be available with 1–2 h of Adaptation for rapid result specimen receipt. The most common use of this

technique is for the diagnosis of respiratory viral viruses of public health importance; and the culture

infections whereby a panel of reagents are utilised to and identification of previously unrecognised viruses,

detect RSV, parainfluenza viruses, influenza A and B

e.g. human metapneumovirus (van den Hoogen et al., and adenovirus in multiple wells of a microscope slide. 2001) and SARS-associated coronaviruses (Drosten et

This technique is sensitive compared to cell culture, al. , 2003). However, routine cell culture techniques

especially for the detection of RSV. The ideal specimen available in most laboratories will not detect a number

for such testing is a nasopharyngeal aspirate, most of clinically important viruses such as gastroenteritis

usually obtained from infants with suspected bronch- viruses, hepatitis viruses, Epstein–Barr virus (EBV),

iolitis, for whom a rapid result is essential for correct Human herpesvirus 6 , 7 and 8 (HHV-6, -7, -8), and/or

clinical management and implementation of infection human immunodeficiency virus (HIV). Other than

control measures. However, detection can also be HSV, for which most isolates will grow in human

made from a well-taken throat/nasal swab. There is fibroblast cells within 3 days, the time for CPE (or, for

increasing evidence that community or nosocomial example, haemadsorption) to develop for most clinical

acquired respiratory viruses lead to severe disease in viral isolates is between 7 and 21 days. For this reason,

immunocompromised patients (for review, see Ison

a number of modifications to conventional cell culture and Hayden, 2002), and it is important that broncho- have been reported, to provide more rapid results.

alveolar lavage specimens from such patients with These include centrifugation of specimens on to cell

respiratory disease are also tested for these viruses in monolayers, often on cover slips, and immunostaining

addition to the more common pathogens, such as with viral protein-specific antibodies at 48–72 h follow-

CMV. Respiratory virus antigens are expressed within ing inoculation (Shell Vial Assay) (e.g. Stirk and

the epithelial cells, and the success of the technique Griffiths, 1988). Such techniques can also be under-

depends on an adequate collection of cells. A taken in microtitre plates (O’Neill et al., 1996).

particular advantage of IF, compared to the commer- The role of conventional cell culture for routine

cial rapid antigen tests available for RSV and diagnosis of viral infections is diminishing and is a

influenza, is that microscopic examination of the subject of active debate within the virology community

fixed cells can determine the presence of adequate (Carman, 2001; Ogilvie, 2001). Many laboratories are

cell numbers for analysis (Table 1.2). IF has been used discontinuing or downgrading virus isolation methods

widely for the direct detection of HSV and VZV in in favour of antigen or genome detection for the rapid

vesicle fluid, and has advantages over electron micro- diagnosis of key viral infections (usually those that are

scopy in both sensitivity and specificity. IF methods treatable, such as CMV and VZV). Nevertheless, it is

have also been used to detect more unusual viruses, important for large laboratories to maintain the ability

such as Lassa fever (Wulff and Lange, 1975). An to employ this methodology for the reasons given

important limitation of IF is that well-trained micro- above. Where primary diagnosis is undertaken by cell

scopists are required for interpretation, which remains culture, there will be increasing pressure to generate

subjective.

quicker results by use of the many rapid techniques that have been reported.

Table 1.2 Antigen detection by immunofluorescence

Antigen Detection

Advantages

Disadvantages

Immunofluorescence

Rapid

Requires skilled staff

Variable sensitivity One of the most effective rapid diagnostic tests is

Sensitive for some viruses

Dependent on high-quality indirect or direct immunofluorescence (IF). Initially

(e.g. RSV)

specimen

Detection and semi-quantitation of CMV antigen- containing cells in blood can also be undertaken by direct IF (CMV/pp65 antigenaemia assay). This technique involves separation of peripheral blood mononuclear cells (PBMCs) and fixing on a slide, followed by staining with a monoclonal antibody directed against the matrix protein pp65. The fre- quency of positive cells can predict CMV disease in the immunocompromised patient (van der Bij et al., 1989), and is used in a number of laboratories. However, it is labour-intensive, needs large numbers of PBMCs (making it unsuitable for all patient populations) and requires a rapid processing of blood specimens if a reduction in sensitivity of detection is to be avoided (Boeckh et al., 1994). Therefore, PCR is rapidly replacing antigenaemia as the method of choice for qualitative and quantitative detection of CMV.

ELISA/Latex Agglutination for Antigen Detection

Solid phase systems for antigen detection are now used widely. ELISAs are based on the capture of antigen in

a clinical specimen to a solid phase via a capture antibody, and subsequent detection using an enzyme- linked specific antibody. Variation in capture and detector antibody species has increased the sensitivity of these assays, which are widely used for hepatitis B virus (HBV) surface antigen detection (HBsAg) and, more recently, for hepatitis C core antigen in donor blood-testing laboratories (Peterson et al., 2000). ELISA-based systems for the diagnosis of, for exam- ple, RSV, influenza and HSV may be appropriate in some contexts for point-of-care testing, although often at the expense of sensitivity when compared to IF and/ or virus culture.

Small latex particles coated with specific antibody can be agglutinated in the presence of antigen, which can then be observed with the naked eye. This rapid assay is used for rotavirus diagnosis, with an equiva- lent sensitivity to electron microscopy. Capture of antibody, rather than antigen, can also be undertaken, although latex assays for CMV and VZV antibodies may lack sensitivity and specificity compared to ELISA systems (see below).

Electron Microscopy Electron microscopy (EM) is the only technique

available for directly visualising viruses, and therefore

has many applications beyond purely diagnostic purposes. The major role of EM in a clinical setting is in the diagnosis of viral gastroenteritis, for which many of the aetiological agents are non-cultivatable, and analysis of skin lesions for herpes, pox and papillomaviruses.

Preparation of specimens and the technique of negative staining are straightforward and quick, and the method is a ‘catch-all’ approach to detecting viruses. Nevertheless, it has a limit of sensitivity of

approximately 10 6 viral particles per millilitre of fluid. Vast numbers of virions are present during acute skin and gastrointestinal disease, and a diagnosis is easily made. This becomes more difficult later in the course of infection, when viral shedding is reduced below the level of detection. Sensitivity can be enhanced by antibody-induced clumping of virus (immune EM) or ultracentrifugation; however, it is unrealistic to under- take these methods routinely. The advantages and disadvantages of electron microscopy are summarised in Table 1.3.

The survival of EM within the routine clinical virology laboratory hinges on the availability of alternative, more sensitive methods of diagnosis. Many centres already use latex agglutination for rotavirus diagnosis, and polymerase chain reaction (PCR) is more sensitive for the detection of herpes- viruses in vesicular fluid (Beards et al., 1998). Currently, EM in diagnostic virology laboratories is used primarily for outbreak investigation. Now that PCR-based methods of Norovirus detection are established (Green et al., 1995), show increased sensitivity with respect to EM (O’Neill et al., 2001) and can be adapted for real-time PCR detection (Miller et al., 2002), the future of EM in clinical virology is in doubt. One of the first indications for electron microscopy was for the rapid diagnosis of smallpox; in the era of bioterrorism, EM will continue to play a role in specialist centres in the event of a bioterrorist attack, such as confirming VZV infection in cases of vesicular rash.

DIAGNOSTIC APPROACHES

Table 1.3 Electron microscopy Advantages

Disadvantages ‘Catch-all’

Requires skilled staff Economical running costs

Poor sensitivity Detects unculturable viruses

Large capital outlay Adaptable, e.g. immunoelectron microscopy confirms cytopathic effect

4 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY

or Mycoplasma), since there are few alternative serological methods. Other serological techniques include haemagglutination inhibition, latex agglutina- tion and immunofluorescence (used most widely for EBV diagnosis). Serum is the specimen of choice for most serological assays, but oral fluid can be used as a non-invasive alternative for the detection of a number of different antibodies, which may be useful for surveillance studies or in children (Perry et al., 1993; Parry et al., 1989). In patients with viral central nervous system infections, the cerebrospinal fluid (CSF) may be tested for virus antibodies, and the antibody ratio compared with serum to confirm

Figure 1.1 Typical evolution

intrathecal antibody synthesis. following an acute viral infection

Increasingly, solid-phase ELISAs are used in diag- nostic laboratories. Recent technological advances, Histology/Cytology

e.g. using synthetic peptides or recombinant antigens instead of whole viral lysates, and improvements in Direct microscopic examination of stained histology or

signal detection have led to more sensitive, specific and cytology specimens can on occasion provide the first

rapid methods for measuring virus specific antibody indication that a virus may be responsible for a

levels. The ELISA format is extremely versatile, and pathological process; e.g. the intranuclear (early) or

new assays can be designed quickly to cope with basophilic (late) inclusions seen in interstitial nephritis

clinical demands, e.g. the investigation of new viruses, in renal transplant biopsies due to BK polyoma virus;

such as severe acute respiratory syndrome (SARS)- changes in cervical cytology seen in association with

associated coronaviruses. Many of these assays are human papillomavirus (HPV); and the nuclear inclu-

available commercially, and can be automated. They sions seen in erythroid precursor cells in parvovirus

are essentially of three types (Figure 1.2): B19 infection. . Indirect assays . Viral antigen is immobilised onto a solid phase, specific antibody in the patient serum Serology

sample binds to this antigen and, after a washing step, this antibody is detected by an enzyme-

All viral infections generate a humoral response, and labelled antihuman immunoglobulin. In this way, this can be used for diagnostic purposes. The classical

either specific IgG or IgM can be detected, pattern of response following an acute infection is

depending on the indicator immunoglobulin (Fig- illustrated in Figure 1.1. The functional nature of this

ure 1.2a,b). Clearly, detection of IgM species is response is extremely variable. In some instances, these

dependent on the prevailing level of IgG, such that antibodies are neutralising and can be assessed for this

a high level of specific IgG reduces the sensitivity of activity (e.g. polioviruses). However, many infections

an IgM assay for the same virus. If rheumatoid are controlled more effectively by T cell responses, and

factor is present in the clinical sample, it may lead antibody detection is used as a surrogate of infection.

to false-positive IgM results (Figure 1.2c). Traditionally, methods of antibody detection did not

. Capture assays . IgG or IgM species are captured distinguish between IgG and IgM responses, and

onto the solid phase by antihuman immunoglobu- diagnosis was based on seroconversion or a significant

lin, followed by addition of antigen and then rise in antibody titre between acute and convalescent

labelled antibody. With regard to IgM assays, this samples (10–14 days apart). The complement fixation

method reduces the potential interference of test was used widely in this respect; however, assay

rheumatoid factor, and is used increasingly for a insensitivity and the cross-reactivity of many antigens

number of IgM species (Figure 1.2d). used within the assay limited its clinical usefulness.

. Competitive assays . In this case, a labelled antibody Most importantly, a diagnosis could only be made

in the ELISA system competes for binding to after the time of acute illness. Currently, the major use

immobilised antigen with antibody in the clinical of this assay is for the diagnosis of ‘atypical’

sample. This assay improves both the specificity pneumonia (Chlamydia psittaci/pneumoniae, Coxiella

and sensitivity of the assay (Figure 1.2e).

DIAGNOSTIC APPROACHES

Figure 1.2 ELISA formats: (a) indirect IgG assay; (b) indirect IgM assay; (c) rheumatoid factor interference in IgM assay (indirect); (d) IgM capture assay; (e) competitive assay. Note that the solid horizontal line represents the solid phase

Serological diagnosis of acute infection is best suited identified by non-viral antigenic epitopes. Immunoblot to situations in which detection of the virus itself is