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Geographic Distribution of Antibodies in Indigenous Human Title Populations in the Indo-Australian Archipelago

Kanamitsu, Masatsugu; Taniguchi, Koki; Urasawa, Shozo; Ogata, Author(s) Takayuki; Wada, Yoshito; Wada, Yoshitake; Saroso, J. Sulianti

American Journal of Tropical Medicine and Hygiene, 28(2), pp.351-363; Citation 1979

Issue Date 1979-03

URL http://hdl.handle.net/10069/21849

Copyright © 1979 by The American Society of Tropical Medicine and Right Hygiene

This document is downloaded at: 2019-01-13T23:55:15Z

http://naosite.lb.nagasaki-u.ac.jp Am. I. Trop. Med. Hyg., 28(2), 1979, pp. 351—363 Copyright ©1979 by The American Society of Tropical Medicine and Hygiene

GEOGRAPHIC DISTRIBUTION OF ARBOVIRUS ANTIBODIES IN INDIGENOUS HUMAN POPULATIONS IN THE INDO-AUSTRALIAN ARCHIPELAGO*

MASATSUGU KANAMITSU,t KOKI TANIGUCHI, SHOZO URASAWA, TAKAYUKI OGATA, YOSHITO WADA, YOSHITAKE WADA, AND J. SULIANTI SAROSO Department of Hygiene and Epidemiology, Sappora Medical College, Sapporo, Japan, Department of Virology and Rickettsiology, National Institute of Health, Tokyo, Japan, Department of Medical Zoology, Faculty of Medicine, Nagasaki University, Nagasaki, Japan, Department of Parasitology, Tokyo Women's Medical College, Tokyo, Japan, and National Institute of Health Research and Development, Ministry of Health, Jakarta, Indonesia

Abstract. Sera from lifetime residents in 16 localities of the Indo-Australian archipelago and adjacent areas were tested for hemagglutination-inhibition antibody against four alpha (Sindbis, Getah, , and Ross River) and for neutralizing antibody against six (dengue 2 and 3, Japanese , Murray Valley encephalitis, Kunjin, and Edge Hill) . collections were carried out in some of the localities to study vector relationships to distribution of the arbovirus antibodies. Antibodies specific to Sindbis and Getah viruses were rare except in north . Chikungunya -specific antibody was highly prevalent in localities of the Oriental zoogeographic region and Wallacea, rare in west New Guinea, and absent in north Australia. Age distribution of chikungunya antibody sug gested that the antibody last occurred in most Indonesian localities about 30 years ago. -specific antibody was confined to localities of the Australian zoogeographic region. Antibodies reacting to dengue 2 and 3 viruses occurred in high frequency in the entire area of the archipelago. Antibodies specific to Murray Valley encephalitis and Kunjin viruses were also widespread although at a very low frequency. virus-specific anti body was highly prevalent in areas west of Wallace's Line while it was absent in areas east of the line with the exception of Lombok. Antibody to Edge Hill virus was rare and confined to the Australian zoogeographic region. The distribution of arthropod vectors, vertebrate hosts, and of are discussed in relation to zoogeographic divisions.

The number of viruses known or suspected of Although a number of studies on arbovirus dis being arthropod-borne is now in excess of 300.' tribution in Southeast Asia and Australasia have A given arbovirus is usually confined to one or been reported,8'3 little is known about their two zoogeographic regions although antigenically distribution and prevalence in the Indo-Australian archipelago where the fauna of the Oriental and related arboviruses may have a wider distribu Australian regions meet.'4'7 Therefore, the Indo @ tion.2 It is highly probable that both the verte nesian islands provide an excellent area for the brate hosts and arthropod vectors play a role in ecologic study of arboviruses. the evolution and distribution of these viruses.47 Although isolation of virus from man and animals is an ideal way to determine arbovirus Accepted 1 July 1978. distribution, this was not possible due to lack of laboratory and logistical support in the area. Therefore, we attempted to study arbovirus dis * This study was reported in part at the conference on viral diseases which was held by the Japan-U.S. tribution by serologic survey of indigenous human Cooperative Medical Science Program in Washington, populations. Results of our preliminary survey D.C., in 1973, and also at the 21st general meeting demonstrated distinct regional differences in the of the Society of Japanese Virologists in Tokyo in the prevalence of some arbovirus antibodies.18 In same year. The study was supported in part by a grant in 1974 from the Ministry of Education, Japan. the present study, we extended the serologic sur Address reprint requests to : Dr. Masatsugu Kana vey to include four alpha- and six flaviviruses in mitsu, do JICA, Embassy of Japan, 24 Jalan Tham sera from the archipelago and adjacent areas. nfl, Jakarta, Indonesia. t Professor Emeritus. Collection of mosquitoes was also carried out at 351 352 KANAMITSU ET AL.

Viruses Four , Sindbis (SIN), Getah (GET), chikungunya (CHIK), and Ross River (RR), and six flaviviruses, dengue (D-2 and D-3), Japanese encephalitis (JE) , Murray Valley encephalitis (MVE), Kunjin (KUN), and Edge Hill (EH), were used in these studies. The Egypt Ar339 strain of SIN, AMM 202 1 strain of GET, JaGArOl strain of JE, Trinidad strain of D-2, and Phil H87 strain of D-3 viruses were supplied by Dr. A. Oya, Department of Virology and Rickettsiology, Na tional Institute of Health, Tokyo. The MVE virus strain was from the 406th Medical General Laboratory of U.S. Army, Japan. The African strain of CHIK was provided by Dr. A. Igarashi, FIGURE 1. Zoogeographic divisions of the Indo Australian archipelago, and localities at which human Institute of Microbial Diseases, Osaka University, serum samples were collected. 1, Vientiane ; 2, Chiang and T-48 strain of RR was provided by Dr. R. E. Mai ; 3, Bangkok ; 4, Pontianak ; 5, Samarinda ; 6, Shope, Yale Arbovirus Research Unit, New Haven, Balikpapan ; 7, Surabaya ; 8, Bali ; 9, Lombok ; 10, U.S.A. The MRM 16 strain of KUN and C281 Kupang; 11, Ujung Pandang; 12, Pomalaa; 13, Am bon ; 14, Jayapura ; 15, Pit River ; 16, Queensland. strain of EH were given by Dr. R. L. Doherty. The spotted areas indicate 200 m depth of the sea @ (continental shelves) Antibody Examination

Antibodies to the alphaviruses were tested by limited localities to study vector relationships to the hemagglutination-inhibition (HI) method. virus distribution. Antigens were prepared from virus-infected BHK 2 1 cell cultures according to the method of Muss MATERIALS AND METHODS gay and Rott,19 except for SIN virus hemagglu Sera tinins which were prepared from virus-infected suckling mouse brains according to the method Human serum samples were collected at 16 of Clarke and Casals.2°Survey sera were screened localities between Laos and north Australia, with at a dilution of 1 :10 using eight units of antigen, particular emphasis on the Indo-Australian achi and positive sera were tested further at higher pelago (Fig. 1). Blood donors were carefully dilutions. questioned about their life histories since birth, Antibodies to the flaviviruses were tested by and blood samples were taken only from lifetime the neutralization method using a microplate. The residents in the respective localities. Thus, about method of De Madrid and Porterfield2' was 2,500 serum samples were obtained since 1972. modified for mass examination of neutralizing The Laosian sera were collected through coopera antibody using minimal amounts of serum as tion of Dr. S. Yamaguchi, Tha Ngone Medical follows :@ 0.025 ml of PS cell suspension, ad Center, Vientiane. Siamese sera were given by justed to contain 2.0 to 2.5 X l0@ cells per ml, Dr. P. Chiowanich, Chiang Mai University School were poured into each well of a microplate. Con of Medicine and Dr. P. Tuchinda, Department of fluent cell sheets were formed after incubation Medical Science, Ministry of Health, Thailand. at 37°C for 24 hours. Twofold serial dilutions, Sera of New Guinean aborigines in Pit River were beginning from 1 :4, 0.025 ml each, of serum supplied by Dr. M. J. Simon, WHO Immunology were mixed with same volume of virus (32—100 Research and Training Center, Singapore, and TCID,0) . The serum-virus mixtures were trans those of natives in north Australia were provided by Dr. R. L. Doherty, Queensland Institute of ferred to the microplate with cell sheets after Medical Science, Brisbane. All sera were heat replacement of growth medium with 0.02 5 ml of inactivated at 56°C for 30 ruin and stored at Earle's MEM supplemented with 10% triptose —75°Cuntil use. phosphate broth, 8% heat-inactivated bovine ARBOVIRUS ANTIBODIES IN INDONESIA 353 serum, 2% newborn calf serum and antibiotics. TABLE 1 The plate was kept at 37°C for an additional Comparisons between arbovirus antibody titers in hour, then 0.075 ml of overlay medium consisting hyperimmune rabbit sera tested by the microneu tralization test (MNT) and 50% plaque reduction of 1.5% carboxy-methyl-cellulose (CMC) in neutralization test (PRNT) Eagle's MEM was poured into the wells. After incubation at 37°C for 4—6days, the overlay MNTJapaneseVirusStrainCellPRNTMNTPRNT/ medium was decanted, the cell sheets were fixed with Zenker's solution and stained with 0.1% trypan blue. Neutralization tests with D-2 virus encephalitisJaGArOlJaGArOl BHK-2 1 8,000 480 16/1 12/1Dengue-2TrinidadBHK-211,0006416/1Negishi*Exp.NakayamaPS PS8,000 2,000800 16010/1 were carried out in the same manner using BHK-2 1 cells to which this virus demonstrated more distinct cytopathic effects. The reciprocal 1 of the highest dilution of serum where more than 12/1* Exp. 2PS PS1,6001,60016012810/1

50% of the cell sheets remained intact was read Data from Hashimoto et al.@ as antibody titer. Antibodies tested by this method were 10- to 16-fold lower in titer than those obtained from the standard method of 50% adults and the aged. The subjects in both cities plaque reduction test (Table 1). Both PS and were considered together in the same group to BHK-2 1 cells disintegrated by D-3 virus did not cover a wide range of age, and their locality was exfoliate from the well. This made reading of called “Thailand―in the present study. Of persons results difficult. The plaque reduction test in a from whom serum samples were collected in microplate with Vero cell monolayers was used Surabaya, only those under 20 years of age were for D-3 virus.as Dilution of serum started at 1 :16 chosen as the subjects because life histories of because most survey sera were short in amounts older persons were ambiguous. All subjects from owing to the preceding antibody examinations. Australia were natives of north Queensland. The Sera which reduced plaques 90% and more was number of subjects in each age group differed in taken as positive. several localities. Therefore, antibody rates over all ages were standardized using a population of Collection of Mosquitoes 25 persons in each 10-year age group, and the standardized antibody rate (Rst) was compared Mosquitoes were collected in February and between localities. The results are summarized March of 1975 at the following seven Indonesian in Tables 2 and 3. localities surrounding Wallace's Line : Sarnarinda, Sindbis and GET virus antibodies were rare in Balikpapan, Surabaya, Bali, Lombok, Ujung Pan every locality except for Queensland, where 11% dang, and Pomalaa. Adult mosquitoes were and 5% of sera reacted with the viruses, respec sampled by outdoor collections using cattle as bait tively. CHIK virus antibody was widely dis or using a double mosquito-net for 1 or 2 hours tributed in the study area, but the highest preva after sunset. Mosquitoes resting indoors were also lence was observed in sera from Samarinda and sampled at few localities. Mosquito larvae and Balikpapan. It will be noted that there were few pupae were sampled mainly at paddy fields, positive reactors to CHIK in the two localities swamps, ditches, and water containers. After the of the Australian zoogeographic region and also taxonomic examinations, some of the mosquitoes in Bali and Lombok. RR virus antibody was were sent to Department of Entomology, National confined largely to the Australian region. New Museum of Natural History, Smithsonian Institu Guinean aborigines in Pit River, which is at an tion, Washington, D.C., U.S.A., to confirm our altitude of 2,600 m, were devoid of the four identification. group A arbovirus antibodies. Neutralization tests with the six group B arbo RESULTS viruses were carried out for all survey sera except those from Queensland which were cytotoxic at Antibody Prevalence a dilution 1 :100 or more even after dialysis. D-2 All subjects in Chiang Mai were children and and D-3 virus antibodies were widespread in the young adults while those in Bangkok were mostly study area, although the survey of D-3 antibody 354 KANAMITSU ET AL.

TABLE 2 Prevalence of hemagglutination-inhibiting antibody to four alphaviruses, Sindbis (SIN), Getah (GET), chikun gunya (CHIK), and Ross River (RR), in human sera

Antibody Number Zoogeographical region Locality tested SIN GET CHIK RR

Continental Southeast-Asia (1) Vientiane 200 2 (1)* 0 58(30) 3 (2) (2, 3) Thailand 142 3 (2) 0 37(24) 0 Borneo (4) Pontianak 351 0 0 14(11) 0 Oriental region (5) Samarinda 150 0 0 65(42) 1 (1) (6) Balikpapan 191 0 1 (1) 72(40) 1 (1) Java (7) Surabaya 54t 0 0 0 0 The LesserSundas (8) Bali 107 0 0 2 (2) 0

WALLACE'S LINE (9) Lombok 140 0 0 6(4) 0 (10) Kupang 121 0 0 15(15) 0 Wallacea Celebes (11) Ujung Pandang 147 2 (1) 0 33(28) 1 (1) (12) Pomalaa 151 0 0 22 (16) 0 WEBER'S LINE The Moluccas (13) Ambon 321 4 (1) 0 37(22) 0

WzsT EDGE OF SAHUL SHELF New Guinea (14) Jayapura 176 5 (3) 0 7 (5) 11 (7) Australian region (15) Pit Rivert 130 0 0 0 0 Australia (16) Queensland 132 15(11) 7 (5) 2 (1) 41(31)

* Number positive (@1 : 10) . The numeral in the parentheses indicates the standardized antibody rate (Rat) over all ages. t All subjects were less than 20 years old. * Locatedat an altitudeof 2,600m. was limited in locality. JE virus antibody was positive to other viruses were rare suggests that prevalent with different frequencies in the Oriental CHIK antibody was specific among antibodies in zoogeographic region, but was relatively low in the multi-positive sera. The reverse was true for Lombok. It will be noted that there was an abrupt antibody titers in the three Queensland sera. decline of JE antibody prevalence to negligible Specific antibodies only to RR and SIN viruses levels in Wallacea and Australian region. Anti were found in these sera. Sera singly-positive to bodies to MVE and KUN viruses were widespread SIN virus were scattered in Thailand, Ujung and occurred at a moderate frequency. EH anti Pandang, Arnbon, Jayapura, and Queensland. body, on the other hand, had a limited distribution None of the survey sera reacted singly with GET. and was confined mostly to Jayapura. There were eight sera multi-positive to GET and other viruses, but they were always lower in titer Specificity of Antibodies against GET. Multi-reactors occurred with higher frequency Although the great majority of survey sera to group B arboviruses, especially to D-2, JE, reacted singly with one of the four group A arbo MVE, and KUN. This could be due to antigenic viruses in the HI test, those remaining reacted crossings between or sequential infections with with two or more of the viruses. Some of the the viruses. We attempted to identify virus multi-positive sera are presented in Table 4. Seven specific antibody as follows : sera tested for the sera from Vientiane, Samarinda, Balikpapan, and four viruses were grouped by highest antibody Ujung Pandang were distinctly higher in titer to titer to a given virus. Similarly, sera singly CHIK virus. The fact that many sera from these positive to the same virus were grouped, and sera localities reacted only with CHIK and those of both groups were compared for their prevalence @@@@@@@@@@@ .@n@‘,@I :@.@ec — ‘@@0—.. 2a .@

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TABLE 4 indicate homologous virus infection. D-2 virus Comparisons between group A arbovirus antibody is antigenically related to other types of dengue titers in multi-positive sera viruses,1 and, therefore, the D-2 antibody in the survey sera is thought to be an indication of infection with any of the dengue viruses. Anti CHIKRRVientianeL-120LocalitySerum no.Age (yrs)SINAntibody'GET bodies ascribable to MVE and KUN virus infec 80 tions were few in every locality. This would mean L-180 67 0 0 80 10 10Samarinda5-1405400L-21649 20Ot 00 0 4020 low prevalence of both viruses, but the possibility that high-titered heterologous virus antibody in 8020BalikpapanB-122 the serum masked the presence of virus-specific 160 antibody must also be taken into consideration. 20Ujung B-9615 530 010 0 800 Serum singly-positive to EH virus was absent. 8010QueenslandA-3455PandangU-l9660400 Ten sera were multi-positive to EH and other viruses (Table 6). Of the ten, only one from 40 Jayapura, JH-107, had a higher titer to EH. How A-967 39 0 20 0 80 A-147332 4380 8020 0 2080 20 ever, considering the low cross-reactivity between EH and other group B arboviruses in the neu ‘SIN, Sindbis; GET. Getah; CHIK, chikungunya; RR, Ross River. tralization test,24 EH antibody in another serum t<1:10. from Jayapura, JH-l01, might also be specific. in localities. Sera multi-positive to the viruses Age Distribution of Antibodies with same titer were excluded from the study. The results are shown in Table 5. Of the four group A arbovirus antibodies, only The numbers of sera in the paired groups were those to CHIK and RR viruses were high enough proportional to each other in almost every locality. in prevalence to study age distribution. The data The results suggested that sera with the highest are shown in Table 7. The age distribution of antibody titer in multi-positive specimens would CHIK antibody in Vientiane would mean that

TABLE 5 Geographic distribution of sera singly-positive to a given group B arbovirus and those multi-positive with highest titer to the same virus*

RegionLocalityAntibodytD-2JEMvEKUNVientiane37

(17)0(0)0(0)Pontianak18(27)0(2)113 (2)0(2)1(1)Oriental regionSamarinda (30) (0) Balikpapan 55(36) 0 (0) 4 (1) 0 (0) Surabaya@ 4(16) 0 (0) 1 (0) 0 (0) Bali40(1)WALLACE'S 22 (3)1 3(19)2 1(1)(1)0 0(2)

LINE Lombok (0) (1)WallaceaUjung Kupang13(50) 20(24)5 0 (0)1 0(0)(0)0 0(0) Pandang (45) (0) Pomalaa 14(70) 0 (0) 1 (1) 0 (3) WEBER'S LINE (1)Australian Ambon39 25(49)0 0 (0)0 0(0)(1)0 0(0)

regionI.SAmn SHELF -*TheresultsonserafromThailandJayapura28(17)0wereexcludedasthestartingdilutionofthe(0)0(0)0(5)seradifferedwithtestsforD-2antibodyand three other virus antibodies. t D-2, dengue 2; JE, Japaneseencephalitisvirus; MVE, Murray Valley encephalitisvirus; KUN, Kunjin. :j:Number of multi-positive sera with highest titer to respective viruses. The number of sera singly-positive to the same virus is shown inparentheses. §Allsubjectswerelessthan 20 yearsold. ARBOVIRUS ANTIBODIES IN INDONESIA 357

TABLE 6 Comparisons between antibody titers of sera multi-positive to Edge Hill (EH) and other group B arboviruses*

(yrs)AntibodyEHD-2JEMVEKUNVientianeL-18131432>32Ot8PontianakP-26198>32N.D400SamarindaS-12263816880KupangT-28LocalitySerum numberAge

0JayapuraJH-101 T-2437 504 416 160 00 40

JH-103 50 4 32 0 0 8 JH-107 50 >32 32 0 8 8 JH-111 43 4 32 4 8 8 JH-12039 3216 832 160 168 0>32 8

S D-2, dengue 2 ; JE, Japanese encephalitis; MVE, Murray Valley encephalitis; KUN, Kunjin. t<1:4. :1:ND., notdetermined. this virus has been active there for many years. 20 years of age. These results suggest that CHIK A similar age pattern in distribution of CHIK virus has been inactive for about 30 years, before antibody was observed for Thailand sera. In which it was highly active. Age distribution of Balikpapan, CHIK antibody rates were remark RR antibody suggests high RR virus activity in ably high over all ages, and the same was true Queensland. SIN antibody, which was the next in Samarinda, suggesting epidemic spread of highest in prevalence there, showed an age pattern CHIK virus among residents in recent years. In similar to RR antibody. The data suggest low Pontianak, Kupang, Pomalaa, and Ambon, CHIK RR virus activity in Jayapura. antibody was rare or absent in persons aged under Of the six group B arbovirus antibodies, the 30, but beyond 30 years of age it increased pre age distribution of antibodies to D-2, JE, MVE, cipitously in prevalence with increasing age. A and KUN viruses were studied. In view of the similar age pattern in distribution of the antibody possibility of the presence in multi-positive sera could be seen for Ujung Pandang and Lombok. of virus-specific antibody masked by high-titered In Surabaya, no CHIK antibody was observed, heterologous virus antibody, all sera positive to but all of the sera were from persons less than the respective viruses were included regardless of

TABLE 7 Age distribution of antibodies to chikungunya (CHIK) and Ross River (RR) viruses in selected localities

AntibodyLocalityAge0—910—1920—2930—3940—4950+CHIKVientiane

(7)* (11) (15) (35) (50) (47) Balikpapan 10/28(36) 21/43 (49) 13/48(27) 10/24(42) 8/24 (33) 10/24(42) Pontianak 0/104 (0) 0/107 (0) 0/65 (0) 5/42 (12) 5/20 (25) 4/13 (31) Kupang 0/18 (0) 1/23 (4) 0/28 (0) 4/24(17) 3/10(30) 7/18(39) Pomalaa 0/25 (0) 0/32 (0) 0/24 (0) 3/24(12) 10/23(43) 9/23(39) Ambon 0/96 (0) 5/106(5) 2/51 (4) 12/34 (35) 4/12 (33) 12/22 (54) Jayapura 0/23 (0) 0/49 (0) 3/31 (10) 0/20 (0) 0/19 (0) 0/24 (0) (0)RRJayapuraQueensland3/42 0/20 (0)4/360/23 (0)4/270/23 (0)9/260/21 (0)11/220/22 (0)22/470/23

(3) (0) (5) (43)* Queensland1/33 1/20 (5)0/495/23 (22)3/31(10)7/23 (30)2/20(10)10/21 (48)1/198/22 (36)4/24(17)10/23

Numberpositive/number tested(percent positive). 358 KANAMITSU ET AL.

Vector Relationships and Geographic LOCALITYD-2 % E % 60I 6090J 30 60 90MVE7 3060KUNz 30 Distribution of Arbovirus Antibodies

ENTI AF4E Thousands of mosquito samples were collected in the seven Indonesian localities surrounding THALAND:@PONTI Wallace's Line. Entomologic study of the mos quitoes will be reported in a separate paper. IANAK@@1 —I@:3SAMAR Distribution in the localities of the mosquitoes

ItWA, assumed as vectors of the ten group A and B I—@:iBALIKPAPAN@,i@=@=BALI:@i@;@,:@LOMBOKarboviruses is shown in Table 8. Numbers of mosquitoes varied in different localities with the

______time and manpower used for the collection. Limitations of some Aedes mosquitoes to few

______localities may be explained by the collection hay ing been carried out mainly outdoors. Aedes and 3@= Culex mosquitoes are broadly distributed on both KUPANG@R ‘I sides of Wallace's Line. Despite detailed exam /@I 11=PANDANO inations, Aedes vigilax and , I@[email protected] ‘ which are believed to be main vectors of many Australian arboviruses,25 were absent in our col ‘@ lections. ._@ Even though the mosquito data are limited, a PMBON—1@' I' ]@::@JAYAPURA—: basic understanding of the vector relationships @ •@, _____maymosquitobe obtainedsurveybytoTablescomparing2andthe 4resultsforgroupof theA arboviruses, and Tables 3 and 5 for group B. arbovirus . FIGURE 2 . Age distribution of group B Potential vectors of SIN and GET viruses had antibodies. Each histogram indicates percentage posi tive in 10-year age groups from 0—9 (top) to 40 and a wide distribution, but antibodies to the two over (bottom). viruses were rare. CHIK antibody prevalence in the area differed with age ; however, if age groups their antibody levels. The results are shown in 30 and older were taken, the antibody was con Figure 2. sistent in distribution with vectors. The failure Dengue-2 antibody rates were very high even to collect potential vectors of RR and EH viruses in the age group under 10 years in almost every was also consistent with the absence of antibodies locality, indicating high activity. specific to the viruses. D-2 and D-3 antibodies Similarly, the data suggest high JE virus activity had a high prevalence even though the distribu in areas west of Wallace's Line. The age pattern tion of Aedes aegypti and Aedes albopictus was of JE antibody distribution in Lombok indicates limited, an indication that our mosquito collec that this virus is not endemic there. JE antibody tions were not thorough. Both of these mosquito in sera from other localities was not specific. It species are known to have a wide distribution in will be noted that D-2 antibody rates were lower Indonesia and the Pacific. over all ages in localities with high JE antibody Potential vectors of JE virus were also wide rates, and vice versa. As mentioned above, iden spread, but virus-specific antibody was almost tification of antibody ascribable to MVE and confined to localities west of Wallace's Line, KUN virus infections were usually difficult. How suggesting that Oriental animals may be important ever, remarkably high MVE antibody rates were in distribution of the virus. Potential vectors of observed in persons under 20 years of age in KUN virus had a widespread distribution which Ambon, suggesting that this virus was active in was consistent with the presence of virus-specific recent years. Similarly, the paucity of KUN antibody although at a very low frequency. Culex positives over all ages in Bali and Lombok prob bitaeniorhynchus, a potential vector of MVE ably means that this virus has been almost in virus, was widespread in every locality where active for many years. virus-specific antibody was scattered. ARBOVIRUS ANTIBODIES IN INDONESIA 359

TABLE 8 Distribution of potential vectors collected in seven localities surrounding Wallace's Line

of Wallace's LineEast of Line POMSAMBLPSURBALAnaphelesPotential vectorLocality'West LOMWallaceUP‘a barbirastris group (JE)t — — — — — Aedes aegypti (CHIK, dengue) + + — — — A. albopictus (CHIK, dengue) + + + + — — — — — — A. vexans(GET, JE) + A.vigilax(RR,EH) ------Culex annulus(JE) + + + + + + + C. bitaeniarhynchus (SIN, GET, MVE) + + + + + + + C. tritaeniorhynchus (SIN, GET, CHIK, JE) + + + + + + + - - - C. gelidus (GET, CHIK, JE, KUN) + + + + — — — C. pipiens fatigans (SIN, CHIK, JE) + + + + — - — C. pseudavishnui (SIN, JE, KUN) + + + + C. annulirostris (SIN, RR, MVE, KUN, EH)+ -+ -— -+ -+ -— -+ -

‘SAM, Samarinda; BLP, Balikpapan; SUR, Surabaya; BAL, Bali; LOM, Lombok; UP. Ujung Pandang; POM, Pomalaa. t Arbovirus presumably transmitted by the mosquito. JE, Japanese encephalitis; CHIK, chikungunya; GET, Getah; RR, Ross River; EH, Edge Hill; SIN, Sindbis; MVE, Murray Valley encephalitis.

DISCUSSION is useful to study the distribution and prevalence of the virus. The present survey suggests that Although the HI test is quite sensitive for CHIK virus has been widely distributed in the detecting arbovirus antibodies, cross-reactions Oriental region and Wallacea, but rare in west often occur among the viruses of the same groups.2 New Guinea and absent in north Australia. The For this reason the neutralization test has been presence in the Australian zoogeographic region recommended for serologic surveys for viruses.'3 @ of vectors of CHIK virus is sug However, a great majority of sera used for the gesting the absence in the region of vertebrate present study reacted with only one of the four hosts of the virus. Monkeys are believed to be alphaviruses by the HI test. This greatly facili the main vertebrate of CHIK virus in Africa.2 tated interpretation of the results. Multi-positive Although various kinds of monkeys live in Su sera can also be useful if antibody highest in titer matra, Borneo, and Java, monkeys in Celebes are is taken. SIN antibody was found at a rate of limited to two genera of Macaca and Cynofñthe about 11% of sera from Queensland where re cus.'5 Only Cynopithecus monkeys are recognized peated isolation of the virus from mosquitoes has in Moluccas, and Cynomolgus in the Lesser Sun been reported.25 However, SIN antibody was rare das. No wild monkeys are said to live in the or absent in many other localities where vectors Australian zoogeographic region.― From these are known to 627 The absence in human facts, CHIK virus seems to be very close in serum of SIN antibody has also been reported in distribution to that of monkeys. Two pieces of Manila, Sarawak, and Australasia where wild and serologic evidence contradict the hypothesis in domestic birds have high antibody rates.283' We criminating monkeys as a vertebrate host of failed to detect GET virus-specific antibody in CHIK virus in this area. One is the paucity of the survey sera. A high GET antibody prevalence positive reactors to CHIK in Bali and Lombok has been reported in New Guinean aborigines,32 where habitation of wild monkeys is recognized. but this finding must be accepted with caution The paucity of CHIK-positives in Lombok has because the present survey indicated the presence of RR virus which is antigenically related to GET. been reported previously.9 The other is the At any rate, SIN and GET antibodies in human peculiar age pattern of CHIK antibody distribu serum do not portray the activity of the viruses. tion in many Indonesian localities; that is, the Presumably, they survive mainly in a bird-mos antibody was rare or absent in residents under quito cycle. age 30, but beyond 30 years of age it increased In contrast, CHIK antibody in human serum precipitously in prevalence with increasing age. 360 KANAMITSU ET AL.

This would mean that CHIK virus has been Sahul shelf and Wallacean islands has remained inactive for about 30 years, before which it was open despite the violent geological activity and highly active. Chronologically, residents who repeated glaciations during the Cenozoic period were at ages 30 and more at the time of bleeding when mammals and birds evolved in large part.@° were born in or before 1945 when the Second These facts would explain why CHIK and RR World War ended in this area. Many foreign virus antibodies were limited in distribution by troops nonimmune against CHIK came to and the same zoogeographic line. stayed in Indonesia after stationing in continental The microneutralization test was 10- to 16-fold Southeast Asia where CHIK was endemic. It is less in sensitivity than the 50% plaque reduction possible that the troops could have brought neutralization test for detecting group B arbovirus CHIK virus into the country to spread it among antibodies. However, it is known that plaque residents, and the virus faded out there with reducing antibody rises in titer usually to 1 :40 or repatriation of the troops after the end of the more to the causative virus, and maintains a high war. According to this assumption, CHIK virus titer by subsequent infections with homologous may have been absent in most Indonesian local and heterologous viruses. Arbovirus antibody ities before the war, and monkeys would not be with such levels was also detected with high natural hosts of the virus in the area. Reasons reproducibility by the microneutralization test. why CHIK virus thus introduced into the area Moreover, the relatively low sensitivity of the was not maintained, however, are obscure. test had the advantage of reducing cross-reactions Very high CHIK antibody prevalence over all with heterologous viruses which make interpreta ages in Samarinda and Balikpapan suggests epi tion of results difficult. demic spread of the virus among residents in The serologic data indicate that dengue viruses recent years. In fact, a severe epidemic of febrile are highly prevalent in areas between Laos and disease occurred there in 1973 (Dr. Djamas E. west New Guinea. Vector mosquitoes are also @ Harahap, Center for Biomedical Research, Mm widespread in the Besides man, mon istry of Health, Indonesia, personal communica keys are suspected as a possible host of the tion). We diagnosed this disease as CHIK by viruses.7 However, epidemic outbreaks of dengue examination of antibody in sera from patients and have been reported in north Australia where contacts just after the epidemic.18 A large amount monkeys are believed to be absent. The main of timber had been brought by truck from remote host of dengue viruses in the study area is prob forests to both cities for shipment. It is con ably man, whose habitation is unrestricted by ceivable that the trucks also brought CHIK virus the zoogeographic divisions. infected mosquitoes from the forests ; alterna Although JE virus has been isolated from van tively, the virus may have been introduced from ous countries of Southeast Asia and adjacent abroad. ea2 its distribution in the Indo-Australian Ross River virus-specific antibody was found archipelago is almost unknown. The present sun only in Jayapura and Queensland where presumed vey demonstrated serologically that JE virus has vectors are prevalent. Marsupials, believed to be been active in areas west of Wallace's Line. the main vertebrate host of RR virus, are also About 16% of sera from Lombok contained JE confined to the Australian zoogeographic region antibody, but the age pattern of the antibody except for few species of Phalanger.'5 Distribu distribution differed from that in endemic areas tion in areas west of Weber's Line of Australian of JE. A significant proportion of residents in murid rodents of the subfamily, Pseudomys nova Lombok are Hindu, and it may be that much of hollandiae, and of Australian fruit bats, Pteropus the antibody in Lombok sera were acquired in poliocephalus, which are both thought to be Bali which is a holy place for Hindus. The potential hosts of RR virus, is still obscure.34 failure to detect JE virus-specific antibody in From a serologic viewpoint, westward extension areas east of Wallace's Line despite the presence of RR virus is limited by the western marginal of vectors may mean the absence of vertebrate line of Sahul shelf which also forms a barrier hosts. Domestic animals which act as a host of against eastward extension of CHIK virus. Sahul JE virus such as swine are widely distributed in land was separated from Sundaland at the late the area although their density may differ with Cretaceous period.@ The water gap between the localities. Of the wild animals, particular interest ARBOVIRUSANTIBODIES IN INDONESIA 361

has been directed to ardeids since they have high Australia, which is consistent with the limited sensitivity to JE virus and are highly mobile.37 distribution of its main vertebrate hosts (mar They have a widespreaddistribution from tropical supials) and vectors (Aedes vigilax and Culex to temperate Asia, and migrate between summer annulirostris) . Thus, our failure to detect EH and winter residences. Considering their wide virus-specific antibody in sera from the Oriental sphere of action, neither the Macassar nor Lom region and Wallacea was not unexpected. How bok straits, where Wallace's Line runs, appear to ever, EH antibody was detected in a few sera be strong barriers against their movement. Most from Jayapura (west New Guinea) , similar to Oriental animals without activity to fly would some Australian localities where animals have high have become extinct from Wallacea during the antibody rates.45 Presumably, this virus survives Tertiary period of Cenozoic era.'4 38,39 On the mainly in a marsupial-mosquito cycle. assumption that some of the animals would be New Guinean aborigines in Pitt River were hosts of JE virus, the limited distribution of JE devoid of antibodies to the seven group A and B antibody could be understood. arboviruses. This village is at an altitude of 2,600 In tropical areas where dengue is highly en m and is said to be free from malaria (Dr. M. L. demic, severe diseases due to other group B Simons, Director of WHO Immunology Research arboviruses rarely occur. The reverse is the case and Training Center, Singapore, personal com in temperate areas. The rare occurrence of munication). Climatic conditions at such a high epidemic dengue in temperate areas can be ex altitude make it less likely that the mosquitoes, plained on the absence of winter carry-over if they did occur, would be able to transmit mechanisms of the virus.@°However, we found arboviruses. a tendency that dengue antibody prevalence was low in localities with high JE antibody prevalence, ACKNOWLEDGMENTS and vice versa. This would mean that epidemic The authors express thanks to Dr. Pien Chio dengue is also rare in tropical areas where the wanich and Dr. Prakorb Tuchinda for supplying mechanisms operate year-round, if JE is highly Siamese sera ; Dr. Shingo Yamaguchi for Laosian endemic there. sera ; Dr. M. J. Simons for sera of New Guinean Antibody ascribable to MVE virus infection aborigines ; Dr. Ralph L. Doherty for sera of was found in 17 sera from the Oriental region and natives in Queensland ; and Dr. Djamas E. Hara Wallacea where isolation of the virus has not yet hap, Dr. Pohan Bosman, and many other Indo been reported. Culex annulirostris, the main vec nesian colleagues for collecting Indonesian sera. tor of MVE virus in Australia, was absent in our We are also indebted to Dr. Akira Oya, Dr. Akira collections ; however, Culex bitaeniorhynchus, Igarashi, Dr. Robert E. Shope, and Dr. Ralph L. another possible vector, is widespread in the Doherty for providing arbovirus strains. Partic area.' 26.41 Although Wallace's and Weber's lines ular thanks are expressed to Dr. Y. M. Huang, interfere with the westward extension of Aus Mr. E. L. Peyton and Dr. S. Sirivanakarn, Smith tralian birds,14 it is unlikely that both lines form sonian Institution, for identifying some of our a complete barrier to viremic birds which trans mosquito samples ; staffs of our laboratories for port MVE virus to susceptible vectors and verte brate hosts in the Oriental region and Wallacea. technical assistance in serologic tests ; and Dr. Isolation of virus from these animals in the areas Duane J. Gubler, NAMRU-2, Jakarta, for giving would verify the MVE antibody distribution. helpful advice on preparation of this report. Kunjin virus was first isolated in Australia, and @ subsequently in Sarawak.42 Our serologic cvi REFERENCES dence suggests widespread distribution of KUN 1. Berge, T. 0., 1975. International Catalogue of virus in the Indo-Australian archipelago. This is Arboviruses. U.S. Department of Health, Edu supported by the wide distribution in the area of cation, and Welfare, Washington, D.C. Culex pseudovishnui, the main vector of KUN 2. Theiler, M., and Downs, W. G., 1973. The virus in Sarawak, and also of domestic fowl and Arthropod-borne Viruses of Vertebrates. Yale University Press, New Haven. cattle which are both assumed as a vertebrate host 3. Sabin, A. B., 1959. Survey of knowledge and of the virus.4245 problems in the field of arthropod-borne virus Edge Hill virus has been isolated only in infections. Arch. Ges. Virusforsch., 9: 1-10. 362 KANAMITSU ET AL.

4. Mattingly, P. F., 1960. Origin and evolution of of the geographic distribution of arbovirus viruses. II. Ecological aspects of the evolution antibodies among residents in Southeast Asia. of mosquito-borne virus diseases. Trans. R. Virus (Proc. 21st Annu. Meetings, Soc. Jap. Soc. Trap. Med. Hyg., 54: 97—112. Viral.), 23: 353. 5. Smith, C. E. G., 1960. Origin and evolution of 19. Mussgay, M., and Rott, R., 1964. Studies on the viruses. III. Factors in the past and future structure of a hemagglutinating component of evolution of the arboviruses. Trans. R. Soc. a group A arbovirus (Sindbis). Virology, 23: Trop. Med. Hyg., 54: 113—129. 573—581. 6. Lvov, D. K., and Lebedev, A. D., 1970. On 20. Clarke, D. H., and Casals, J., 1958. Techniques evolution of arboviruses. Vop. Virusol., 15: for hemagglutination and hemagglutination 3 72—376. inhibition with arthropod-borne viruses. Am. 7. Miles, J. A. R., 1964. Some ecological aspects J. Trap. Med. Hyg., 7: 561—573. of the problem of arthropod-borne animal 21. Dc Madrid, A. T., and Porterfield, J. S., 1969. viruses in the western Pacific and South-east A simple micro-culture method for the study Asia regions. Bull. WHO., 30: 197—210. of group B arboviruses. Bull. W.H.O., 40: 8. Pond, W. L., 1963. Arthropod-borne virus anti 113—121. bodies in sera from residents of South-east 22. Hashimoto, M., Yamada, K., and Kanamitsu, M., Asia. Trans. R. Soc. Trap. Med. Hyg., 57: 1971. A microtiter method for assay of neu 364—371. tralizing antibodies against group B arboviruses. 9. Hotta, S., Aoki, H., Samoto, S., Yasui, T., and Virus, 21: 55—59. Kawabe, M., 1970. Virologic-epidemiological 23. Earley, E., Peralta, P. H., and Johnson, K. M., studies on Indonesia: II. Measurement of anti 1967. A plaque neutralization method for arbovirus antibodies in sera from residents of arboviruses. Proc. Soc. Exp. Biol. Med., 125: Lombok, South Sumatra and West Java, in 741—747. comparison with results concerning sera from 24. Westaway, E. G., 1965. The neutralization of residents of Japanese main islands. Kobe J. arboviruses. II. Neutralization in heterologous Med. Sci., 16: 215—234. virus-serum mixtures with four group B arbo 10. Hotta, S., Aoki, H., Samoto, S., Yasui, T., and viruses. Virology, 26: 528—537. Noerjasin, B., 1970. Virologic-epidemiological 25. Doherty, R. L., 1972. Arboviruses in Australia. studies on Indonesia: III. HI antibodies Aust. Vet. J., 48: 172—180. against selected arboviruses (groups A and B) 26. Bonne-Wepster, J., 1954. Synopsis of a hundred in human and animal sera collected in Sura common non-anopheine mosquitoes of the baya, East Java, in 1968. Kobe J. Med. Sci., Greater and Lesser Sundas, the Moluccas and 16: 235—250. New Guinea. Spec. PubI. R. Trap. Inst. Am 11. Rao, T. R., 1971. Immunological surveys of sterdam, 111: 1—147. arbovirus infections in South-east Asia, with 27. Sirivanakarn, S., 1972. Contributions to the special reference to dengue, chikungunya and mosquito fauna of Southeast Asia. XIII. The . Bull. WHO., 44: genus Culex, subgenus Eumelanomyia Theobald 585—591. in Southeast Asia and adjacent areas. Contr. 12. Doherty, R. L., 1974. Arthropod-borne viruses Am. Entomol. Inst., 8: 1—188. in Australia and their relation to infection and 28. Venzon, E. L., 1965. A study of antibody pat disease. Progr. Med. Viral., 17: 136—192. terns to Australian arboviruses in human sera 13. Tesh, R. B., Gajdusek, D. C., Garruto, R. M., collected in Manila. Philipp J. Sci., 94: 171— Cross, J. H., and Rosen, L., 1975. The dis 178. tribution and prevalence of group A arbo@irus 29. Simpson, D. I. H., Smith, C. E. G., Bowen, E. neutralizing antibodies among human popu T. W., Platt, G. S., and Macdonald, W. W., lations in Southeast Asia and the Pacific 1970. Arbovirus infections in Sarawak: Virus Islands. Am. J. Trap. Med. Hyg., 24: 664—675. isolations from mosquitoes. Ann. Trap. Med. 14. Mayr, E., 1944. Wallace's Line in the light of Parasitol., 64: 137—151. recent zoogeographic studies. Q. Rev. Biol., 30. Smith, C. E. G., Simpson, D. I. H., Peto, S., 19: 1—14. Bowen, E. T. W., McMahon, D., Platt, G. S., 15. Darlington, P. J., Jr., 1957. Zoogeography. John Way, H., Bright, W. F., and Maidment, B., Wiley & Sons, New York. 1974. Arbovirus infections in Sarawak: Sero 16. Wilma, G., 1962. Animal Geography. Heine logical studies in man. Trans. R. Soc. Trap. mann, London. Med. Hyg., 68: 96—104. 17. Zeuner, F. E., 1941. The biogeographic division 31. Doherty, R. L., Whitehead, R. H., Wetters, E. J., of the Indo-Australian archipelago: 7. The and Gorman, B. M., 1968. Studies of the epi divisions as indicated by the distribution of demiology of arthropod-borne virus infections insects in relation to geology. Proc. Linnean at Mitchell River Mission, Cape York Penin Soc. London, 154th session, 157—163. sula, North Queensland. II. Arbovirus infec 18. Kanamitsu, M., Fukuhara, M., Tamura, H., tions of mosquitoes, man and domestic fowls, Ishizawa, F., Ogata, T., Yoshida, I., Yoshioka, 1963—1966. Trans. R. Soc. Trap. Med. Hyg., I., and Saroso, J. S., 1973. Serologic survey 62: 430—438. ARBOVIRUS ANTIBODIES IN INDONESIA 363

32. Wisseman, C. L., Jr., Gajdusek, D. C., Schofield, arc tectonics in eastern Indonesia. Nature, F. D., and Rosenzweig, E. C., 1964. Arthro 241: 197—198. pod-borne virus infections of aborigines in 39. Hooijer, D. A., 1975. Quaternary mammals west digenous to Australasia. A preliminary report. and east of Wallace's Line. Netherl. J. Zool., Bull. W.H.O., 30: 211—219. 25: 46—56. 33. Marks, E. N., 1973. An Atlas of Common 40. Hammon, W. McD., 1969. Observations on den Queensland Mosquitoes (With a Guide to gue , benign protector and killer: A Dr. Common Queensland Biting Midges). Univer Jekyll and Mr. Hyde. Am. J. Trop. Med. Hyg., sity of Queensland Bookshop, Queensland. 18: 159—165. 34. Gard, G., Marshall, I. D., and Woodroofe, G. 41. Bram, R. A., 1967. Contributions to the mos M., 1973. Annually recurrent epidemic poly quito fauna of Southeast Asia. II. The genus arthritis and Ross River virus activity in a Culex in Thailand (Diptera: Culicidae) . Contr. coastal area of New South Wales. II. Mos Am. Entomol. Inst., 2: 1—296. quitoes, viruses and wildlife. Am. J. Trop. 42. Doherty, R. L., Carley, J. G., Mackerras, M. J., Med. Hyg., 22: 55 1—560. and Marks, E. N., 1963. Studies of arthropod 35. Scrivenor, J. B., 1941. A discussion on the bio borne virus infections in Queensland. III. Iso geographic division of the Indo-Australian lation and characterization of virus strains archipelago, with criticism of the Wallace and from wild-caught mosquitoes in North Queens Weber Lines and of any other dividing lines, land. Aust. J. Exp. Biol. Med. Sci., 41: 17—30. and with an attempt to obtain the names used 43. Bowen, E. T. W., Simpson, D. I. H., Platt, G. S., for the divisions. I. Geological and climatic Way, H. J., and Smith, C. E. G., 1970. Arbo factors affecting the distribution of life in the virus infections in Sarawak: The isolation of archipelago. Proc. Linnean Soc., 154th session, Kunjin virus from mosquitoes of the Culex 120—126. pseudovishnui group. Ann. Trop. Med. Para 36. Huang, Y. M., 1972. Contributions to the mos sitol., 64: 263—268. quito fauna of Southeast Asia. XIV. The sub 44. Sanderson, C. J., 1969. A serologic survey of genus Stegomyia of Aedes in Southeast Asia. I. Queensland cattle for evidence of arbovirus The scutellaris group of species. Contr. Am. infection. Am. J. Trop. Med. Hyg., 18: 433— Entomol. Inst., 9: 1—109. 439. 37. Buescher, W. F., Buescher, E. L., and McLure, 45. Doherty, R. L., Carley, J. G., and Gorman, B. H. E., 1959. Ecologic studies of Japanese en M., 1964. Studies of arthropod-borne virus cephalitis virus in Japan. V. Avian factors. infections in Queensland. IV. Further sero Am. J. Trop. Med. Hyg., 8: 689—697. logical investigations of antibodies to group B 38. Audley-Charles, M. G., 1973. Relation of Pleisto arboviruses in man and animals. Aust. J. Exp. cene migrations of pygmy stegodonts to isolated Biol. Med. Sci., 42: 149—164.