JAPANESE ENCEPHALITIS VIRUS ISOLATED FROM SEVEN SPECIES OF MOSQUITOES COLLECTED AT SEMARANG REGENCY, CENTRAL JAVA
JAPANESE ENCEPHALITIS VIRUS ISOLATED FROM SEVEN
SPECIES OF MOSQUITOES COLLECTED
AT SEMARANG REGENCY, CENTRAL JAVA
Ratna k * , Sustriayu NalimX*, H. Suwasono** and G.B. Jennings*
Suatu penelitian telah dilakukan untuk mengisolasi arbovirus dan' nyamuk yang ditangkap
dari rumah penduduk dun kandang temak desa Susukan, kecamatan Ungaran, kabupaten
Semarang. Pada periode penelitian yang dilakukan selama 2 tahun, isolasi virus hanya didapatkan
pada bulan Januari dun Februari. Hrus Japanese encephalitis (JEV) adalah satu-satunya virus yang
dapat diisolasi. Mayoritas isolat JEV (72%) berasal dun nyamuk yang ditangkap di kandang temak.
Isolat JEV ini berasal dun 7 spesies nyamuk, dengan frekuensi Culex quinquefasciatus yang paling
tinggi (11.5%). Empat vektor JEV baru yang potensial di Indonesia adalah Cx.bitaeniorhynchu~
Cx.quinquefasciatus, Anopheles kochi dan Armigeres subalbatus Ini adalah laporan pertama
tentang isolasi JEV dari An.kochi dun Ar.subalbatus di Asia
INTRODUCTION
Arthropod-borne viruses (arboviruses)
are responsible for causing undiagnosed
symptomatic and asymptomatic infections in
human and animals1. In Indonesia, human and
animal serological surveys have detected
antibodies against several arboviruses. Olson
et aL2 used hemagglutination-inhibition (HI)
assays to demonstrate antibodies to Japanese
encephalitis virus (JEV), Zika virus,
Chikungunya virus (Chik) and Ross River
**
virus (RR), and Tesh et a13 demonstrated
neutralization antibody to Chik, RR, Getah,
Bebaru and Sindbiis viruses. The existence of
dengue virus4, Chi, (unpublished data) and
JEV' has been confirmed by viral isolation.
Previously, Suwasono et. al! conducted
a risk-assessment study in Ungaran in response
to historical reports about many patients with
fever of unknown origin at the local health
clinic. They reported the collection of 5 species
of Culex, 2 species of Aedes and 1 species of
Naval Medical Research Unit No. 2, Jakarta.
Vector Research Station, Health Ecology Research Center, Central Java.
Japanese encephalitis virus
Armigeres in cattle shelters and nearby houses
in the area, suggesting that these species might
be possible arbovirus vectors.
This concurrent portion of the study was
designed to determine if the collected
mosquitoes were possible arbovirus vectors.
This is a report of the identification of 4 new
potential JEV vectors in Indonesia and confirm
the existence of JEV circulation in Central Java.
MATERIALS AND METHODS
Mosquito collections.
Mosquitoes were collected once weekly
by mouth aspirator at the villages of Susukan,
Ungaran district, Semarang regency, between
September 1986 and August 1988~.Briefly,
resting collections were made during daylight
hours inside houses and during the evening in
cattle shelters. Female mosquitoes were held
for two days to permit digestion of blood meals,
then chilled, identified, and individually
separated into pools by species, collection site
and collection date with a maximum of 50
mosquitoes per pool.
Preparation of mosquito pools
Pools were ground in a chilled ten broeck
grinder in 3 ml. of chilled phosphate and tricine
(0.01 M. each), buffered saline (PBS) pH 7.8
containing 0.8 % bovine albumin, 100 units of
Penicillin and 100 mg of Streptomycin. The
resulting mosquito suspensions were
centrifuged at 3000 rpm for 20 minutes at 4OC.
..........Ratna Tan eta1
The supernatant was filtered, with 0.2 micron
filter, and stored at -60°C for virus isolation.
Virus Isolation
Mosquito-pool suspensions were
inoculated on to Toxorhynchites splendens
mosquito cells (TRA-284) obtained from CDC,
San Juan, Puerto Rico. Viral isolates were
identified as described previously7, using
alphavirus and flavivirus specific monoclonal
antibodies, and then confirmed with viral
specific monoclonal antibodies supplied by
CDC, Fort Collins, USA.
RESULTS
A total of 1385 mosquito pools from
resting collections in human dwellings and cattle
shelters were assayed with 25 (1.8%) positive
for virus. lhble 1shows the number of mosquito
species assayed and the results of virus isolation
attempts. JEV was the only arbovirus isolated
from the mosquitoes.The majority (72%) of the
JEV isolates were from mosquitoes collected
in the cattle shelters. Only one species collected
from the house, Cr. quinquefasciatus, was
positive (7 pools) for virus. In the cattleshelters,
JEV isolations were made from several species,
but Cx. quinquefasciatus had the highest
frequency (11.5%). Overall amongst all 4
genera studied, Culex species had the highest
frequency (1.23%). Although mosquitoes
were collected during the Zyear period, virus
isolations were made only from pools collected
during the wet months of January and
February 1987.
Japanese encephalitisvirus .......... Ratna Tan eta1
JW VIRUS ISOLATION
MOSQUlTO SPECIES
IN HOUSES
No of pools
tested
Cx. fuseocephalus
C x trilacniorhynchus
C x vkhnui
Cx. bilaeniorhynchus
Cx. p. quinquerasciatus
An.
An.
An.
An.
An.
An.
vqus
indelinitus
maculalus
annular&
kocbi
10
4
60
31
307
16
3
-
-
IN CATTLE SHELTERS
No of positive
pools
7
-
-
-
-
barbirostris
-
-
An. aconitus
Ae. aegypti
Ae. alboplctus
Ae. poicillius
Ae. caecus
Armigres subalbatus
87
3
-
-
-
-
DISCUSSIONS
35
-
No of pools
tested
175
161
93
54
26
77
2
54
35
28
9
1
12
14
9
79
No of positive
pools
3
3
1
3
3
-
-
2
-
-
3
throughout this study and were not affected
by the rainy season6. Isolated virus was from
Both the role of mosquitoes as the vector
mosquitoes
collected during the normally
and of vertebrates as the amplifying host, are
wet
months
of January and February. Six
critically important for the natural transmission
(24%) of the JEV isolates came from Cx.
of arboviruses. Mosquitoes of the genus culexs
bitaeniorhynchus a n d Cx. fuscocephalus,
and ~ n o ~ h e l e shad
' previous JEV isolates in
considered to be among the principal vectors
Indonesia. In this study, 4 new potential JEV
of JEV throughout the rice-growing areas of
vectors in Indonesia were identified : Cr.
~ s i aThis
~ . may identify the active transmission
bitaeniorhynchus, Cr. quinquefasciatus, An.
period, and the time period of greatest risk to
kochi and At: subalbatus. Additionally, An.
humans. Cjr. quinquefarciafus, the only JEV
kochi and Ar. subalbatus are reported as
infected mosquito found in both houses and
potential JEV vectors for the fvst time in ~ s i a ~ . cattle shelters, might pose the greatest threat
to man. Although not all mosquito species in
Percentages of all mosquitoes collected
were higher in cattle shelters than in houses
this study were found to contain virus, their
presence may indicate the potential for
increased risk of virus transmission in the area.
Several of the species examined are known
vectors to humans of other arboviruses, for
example Ae. aegypti and Ae. albopictus for
dengue and ~ i k a " " ~and Ae. aegypti .for
chikl1.
Although JEV was isolated, there have
been no reports of JEV clinical infection in
Indonesia. , ~ u h a r ~ o nhas
o l ~proposed several
reasons for the absence of JEV clinical cases
in Indonesia. One possible reason is the lack
of pigs in Indonesia as the amplifying host,
because the country is predominantly Moslem.
In this study, we found that cattle are potential
amplifying hosts since numerous JEV infected
mosquitoes were found in the cattle shelter.
Chen et al.14 recently identified a new JEV
genotype in Indonesia. Whether this genotype
has decreased virulence is unknown, but if true,
could account for the lack of clinical cases.
REFERENCES
1.
Haktead SB, (1988). Arboviruses of the Pacific and
Southeast Asia. Textboak of Pediatric Inlectious
Dkases Part III:1502- 1510.
2.
Olson JG, Ksiazek TG, Gubler DJ, Lubis SI
Simanjuntak G, Lee VH, Nalim S, Juslis K, and
y arboviral antibodies in
Seer. (1983). A s u ~ e for
sera of humans animals in Lombok, Republic of
Indonesia. Ann Trop Med & Par ?7 (2):131-137
3.
Tesh RB, Gajdusek DC, Garruto RM,Cross JH
and Rosen L. (1975). The distribution and
prevalence of group A arbovirus neutralizing
antibodies among human populations in Southeas
Asia and the Pacific islands. Am J Trop Med Hyg
24 (4):664-675.
4.
Suharyono W, Gubler DJ, Lubis I, Abidin M, Sie
A. and Sarosa S. (1979). Dengue virus isolation
in Indonesia, 1975-1978.Asian J Infect Dis 3:27-32
5.
Van Peenen PF9, and Irsiana R, (1974). Firs
isolation of Japanese Encephalitis virus from Java
MUitnry Medicine 139 (10):821-823.
6.
Suwasono H, Widiarti, Sumardi and Tri Suwaryono
(1990). Hasil penangkapan nyamuk cu licinae di
kecamatan Ungaran, Kabupaten Semarang. B d
PeneUiian Kesehatan 18 (2):16-20.
7.
Gubler DJ, Kuno G, Sather GE, Velez M, and Oliver
A. (1984). Mosquito cell cultures and specific
monoclonal antibodies in surveillance for dengue
viruses. Am J Trop Med and Hyg 33:158- 165.
8.
Olson TG, Ksiazek TG, Lee VH, Tan R, and Shope
RE. (1985) Isolation of Japanese Encephalitis virus
from Anopheles annularis and Annopheles vagus
in Lombok, Indonesia. Trans Roy Soc Trop Med
~ y 19
g (6):~s-847.
9.
Burke DS, and Leake CI, (1988). Japanese
encephalitis. The Arboviruses: Epidemiolop~
- and
Ecol0gy:Vol III:63-92.
ACKNOWLEDGMENT
This study was supported by the Naval
Medical Research and Development
Command, Navy Department, for Work Unit
62787A3M162787 AB70. The opinion and
assertions contained herein are those of the
and do not purport to
those of
the U.S. Navy, U.S. Department of Defense or
the Indonesian Ministry of Health.
Reprint requests to Publications Ofice,
U.S. Naval Medical Research Unit No. 2.
Kompleks LITBANGKES, J1. Percetakan
Negara No. 29 Jakarta.
4
BuL Penelit Kesehat 21 (1) 1993
10.
11.
Gubler DJ, Suharyono W, Lubis I, Eram S, and
S a m o S. ( l m ) . midernic dengue hemorrhagic
fever in rural Indonesia. I. Virological and
epidemiological Studies. Am J Trop Med Hyg 28
(4):ml-710.
Jupp PG, and Mcintosh BM. (1988). Chikungunya
virus disease. The Arboviruses:Epidemiology and
Ekology vol. Ik137-157.
12.
Olson JG, KsiazekTG, Suhandimanand Triwibowo.
(1981). Zika virus, a cause of fever in Central Java,
Indonesia. Trans Roy Soc Trop Med Hyg 75
(3):389-393.
13.
Suharyono W, (1990). Japanese Encephalitis in
Indonesia. Virus Inform Exchange Newsl 7(3).
14.
Chen WR, Rico-Hesse R,and Tesh RB,(1992). A
new genotype of Japanese encephalitis virus from
Indonesia. Am Trop Med Hyg 47 (1):61-69.
SPECIES OF MOSQUITOES COLLECTED
AT SEMARANG REGENCY, CENTRAL JAVA
Ratna k * , Sustriayu NalimX*, H. Suwasono** and G.B. Jennings*
Suatu penelitian telah dilakukan untuk mengisolasi arbovirus dan' nyamuk yang ditangkap
dari rumah penduduk dun kandang temak desa Susukan, kecamatan Ungaran, kabupaten
Semarang. Pada periode penelitian yang dilakukan selama 2 tahun, isolasi virus hanya didapatkan
pada bulan Januari dun Februari. Hrus Japanese encephalitis (JEV) adalah satu-satunya virus yang
dapat diisolasi. Mayoritas isolat JEV (72%) berasal dun nyamuk yang ditangkap di kandang temak.
Isolat JEV ini berasal dun 7 spesies nyamuk, dengan frekuensi Culex quinquefasciatus yang paling
tinggi (11.5%). Empat vektor JEV baru yang potensial di Indonesia adalah Cx.bitaeniorhynchu~
Cx.quinquefasciatus, Anopheles kochi dan Armigeres subalbatus Ini adalah laporan pertama
tentang isolasi JEV dari An.kochi dun Ar.subalbatus di Asia
INTRODUCTION
Arthropod-borne viruses (arboviruses)
are responsible for causing undiagnosed
symptomatic and asymptomatic infections in
human and animals1. In Indonesia, human and
animal serological surveys have detected
antibodies against several arboviruses. Olson
et aL2 used hemagglutination-inhibition (HI)
assays to demonstrate antibodies to Japanese
encephalitis virus (JEV), Zika virus,
Chikungunya virus (Chik) and Ross River
**
virus (RR), and Tesh et a13 demonstrated
neutralization antibody to Chik, RR, Getah,
Bebaru and Sindbiis viruses. The existence of
dengue virus4, Chi, (unpublished data) and
JEV' has been confirmed by viral isolation.
Previously, Suwasono et. al! conducted
a risk-assessment study in Ungaran in response
to historical reports about many patients with
fever of unknown origin at the local health
clinic. They reported the collection of 5 species
of Culex, 2 species of Aedes and 1 species of
Naval Medical Research Unit No. 2, Jakarta.
Vector Research Station, Health Ecology Research Center, Central Java.
Japanese encephalitis virus
Armigeres in cattle shelters and nearby houses
in the area, suggesting that these species might
be possible arbovirus vectors.
This concurrent portion of the study was
designed to determine if the collected
mosquitoes were possible arbovirus vectors.
This is a report of the identification of 4 new
potential JEV vectors in Indonesia and confirm
the existence of JEV circulation in Central Java.
MATERIALS AND METHODS
Mosquito collections.
Mosquitoes were collected once weekly
by mouth aspirator at the villages of Susukan,
Ungaran district, Semarang regency, between
September 1986 and August 1988~.Briefly,
resting collections were made during daylight
hours inside houses and during the evening in
cattle shelters. Female mosquitoes were held
for two days to permit digestion of blood meals,
then chilled, identified, and individually
separated into pools by species, collection site
and collection date with a maximum of 50
mosquitoes per pool.
Preparation of mosquito pools
Pools were ground in a chilled ten broeck
grinder in 3 ml. of chilled phosphate and tricine
(0.01 M. each), buffered saline (PBS) pH 7.8
containing 0.8 % bovine albumin, 100 units of
Penicillin and 100 mg of Streptomycin. The
resulting mosquito suspensions were
centrifuged at 3000 rpm for 20 minutes at 4OC.
..........Ratna Tan eta1
The supernatant was filtered, with 0.2 micron
filter, and stored at -60°C for virus isolation.
Virus Isolation
Mosquito-pool suspensions were
inoculated on to Toxorhynchites splendens
mosquito cells (TRA-284) obtained from CDC,
San Juan, Puerto Rico. Viral isolates were
identified as described previously7, using
alphavirus and flavivirus specific monoclonal
antibodies, and then confirmed with viral
specific monoclonal antibodies supplied by
CDC, Fort Collins, USA.
RESULTS
A total of 1385 mosquito pools from
resting collections in human dwellings and cattle
shelters were assayed with 25 (1.8%) positive
for virus. lhble 1shows the number of mosquito
species assayed and the results of virus isolation
attempts. JEV was the only arbovirus isolated
from the mosquitoes.The majority (72%) of the
JEV isolates were from mosquitoes collected
in the cattle shelters. Only one species collected
from the house, Cr. quinquefasciatus, was
positive (7 pools) for virus. In the cattleshelters,
JEV isolations were made from several species,
but Cx. quinquefasciatus had the highest
frequency (11.5%). Overall amongst all 4
genera studied, Culex species had the highest
frequency (1.23%). Although mosquitoes
were collected during the Zyear period, virus
isolations were made only from pools collected
during the wet months of January and
February 1987.
Japanese encephalitisvirus .......... Ratna Tan eta1
JW VIRUS ISOLATION
MOSQUlTO SPECIES
IN HOUSES
No of pools
tested
Cx. fuseocephalus
C x trilacniorhynchus
C x vkhnui
Cx. bilaeniorhynchus
Cx. p. quinquerasciatus
An.
An.
An.
An.
An.
An.
vqus
indelinitus
maculalus
annular&
kocbi
10
4
60
31
307
16
3
-
-
IN CATTLE SHELTERS
No of positive
pools
7
-
-
-
-
barbirostris
-
-
An. aconitus
Ae. aegypti
Ae. alboplctus
Ae. poicillius
Ae. caecus
Armigres subalbatus
87
3
-
-
-
-
DISCUSSIONS
35
-
No of pools
tested
175
161
93
54
26
77
2
54
35
28
9
1
12
14
9
79
No of positive
pools
3
3
1
3
3
-
-
2
-
-
3
throughout this study and were not affected
by the rainy season6. Isolated virus was from
Both the role of mosquitoes as the vector
mosquitoes
collected during the normally
and of vertebrates as the amplifying host, are
wet
months
of January and February. Six
critically important for the natural transmission
(24%) of the JEV isolates came from Cx.
of arboviruses. Mosquitoes of the genus culexs
bitaeniorhynchus a n d Cx. fuscocephalus,
and ~ n o ~ h e l e shad
' previous JEV isolates in
considered to be among the principal vectors
Indonesia. In this study, 4 new potential JEV
of JEV throughout the rice-growing areas of
vectors in Indonesia were identified : Cr.
~ s i aThis
~ . may identify the active transmission
bitaeniorhynchus, Cr. quinquefasciatus, An.
period, and the time period of greatest risk to
kochi and At: subalbatus. Additionally, An.
humans. Cjr. quinquefarciafus, the only JEV
kochi and Ar. subalbatus are reported as
infected mosquito found in both houses and
potential JEV vectors for the fvst time in ~ s i a ~ . cattle shelters, might pose the greatest threat
to man. Although not all mosquito species in
Percentages of all mosquitoes collected
were higher in cattle shelters than in houses
this study were found to contain virus, their
presence may indicate the potential for
increased risk of virus transmission in the area.
Several of the species examined are known
vectors to humans of other arboviruses, for
example Ae. aegypti and Ae. albopictus for
dengue and ~ i k a " " ~and Ae. aegypti .for
chikl1.
Although JEV was isolated, there have
been no reports of JEV clinical infection in
Indonesia. , ~ u h a r ~ o nhas
o l ~proposed several
reasons for the absence of JEV clinical cases
in Indonesia. One possible reason is the lack
of pigs in Indonesia as the amplifying host,
because the country is predominantly Moslem.
In this study, we found that cattle are potential
amplifying hosts since numerous JEV infected
mosquitoes were found in the cattle shelter.
Chen et al.14 recently identified a new JEV
genotype in Indonesia. Whether this genotype
has decreased virulence is unknown, but if true,
could account for the lack of clinical cases.
REFERENCES
1.
Haktead SB, (1988). Arboviruses of the Pacific and
Southeast Asia. Textboak of Pediatric Inlectious
Dkases Part III:1502- 1510.
2.
Olson JG, Ksiazek TG, Gubler DJ, Lubis SI
Simanjuntak G, Lee VH, Nalim S, Juslis K, and
y arboviral antibodies in
Seer. (1983). A s u ~ e for
sera of humans animals in Lombok, Republic of
Indonesia. Ann Trop Med & Par ?7 (2):131-137
3.
Tesh RB, Gajdusek DC, Garruto RM,Cross JH
and Rosen L. (1975). The distribution and
prevalence of group A arbovirus neutralizing
antibodies among human populations in Southeas
Asia and the Pacific islands. Am J Trop Med Hyg
24 (4):664-675.
4.
Suharyono W, Gubler DJ, Lubis I, Abidin M, Sie
A. and Sarosa S. (1979). Dengue virus isolation
in Indonesia, 1975-1978.Asian J Infect Dis 3:27-32
5.
Van Peenen PF9, and Irsiana R, (1974). Firs
isolation of Japanese Encephalitis virus from Java
MUitnry Medicine 139 (10):821-823.
6.
Suwasono H, Widiarti, Sumardi and Tri Suwaryono
(1990). Hasil penangkapan nyamuk cu licinae di
kecamatan Ungaran, Kabupaten Semarang. B d
PeneUiian Kesehatan 18 (2):16-20.
7.
Gubler DJ, Kuno G, Sather GE, Velez M, and Oliver
A. (1984). Mosquito cell cultures and specific
monoclonal antibodies in surveillance for dengue
viruses. Am J Trop Med and Hyg 33:158- 165.
8.
Olson TG, Ksiazek TG, Lee VH, Tan R, and Shope
RE. (1985) Isolation of Japanese Encephalitis virus
from Anopheles annularis and Annopheles vagus
in Lombok, Indonesia. Trans Roy Soc Trop Med
~ y 19
g (6):~s-847.
9.
Burke DS, and Leake CI, (1988). Japanese
encephalitis. The Arboviruses: Epidemiolop~
- and
Ecol0gy:Vol III:63-92.
ACKNOWLEDGMENT
This study was supported by the Naval
Medical Research and Development
Command, Navy Department, for Work Unit
62787A3M162787 AB70. The opinion and
assertions contained herein are those of the
and do not purport to
those of
the U.S. Navy, U.S. Department of Defense or
the Indonesian Ministry of Health.
Reprint requests to Publications Ofice,
U.S. Naval Medical Research Unit No. 2.
Kompleks LITBANGKES, J1. Percetakan
Negara No. 29 Jakarta.
4
BuL Penelit Kesehat 21 (1) 1993
10.
11.
Gubler DJ, Suharyono W, Lubis I, Eram S, and
S a m o S. ( l m ) . midernic dengue hemorrhagic
fever in rural Indonesia. I. Virological and
epidemiological Studies. Am J Trop Med Hyg 28
(4):ml-710.
Jupp PG, and Mcintosh BM. (1988). Chikungunya
virus disease. The Arboviruses:Epidemiology and
Ekology vol. Ik137-157.
12.
Olson JG, KsiazekTG, Suhandimanand Triwibowo.
(1981). Zika virus, a cause of fever in Central Java,
Indonesia. Trans Roy Soc Trop Med Hyg 75
(3):389-393.
13.
Suharyono W, (1990). Japanese Encephalitis in
Indonesia. Virus Inform Exchange Newsl 7(3).
14.
Chen WR, Rico-Hesse R,and Tesh RB,(1992). A
new genotype of Japanese encephalitis virus from
Indonesia. Am Trop Med Hyg 47 (1):61-69.