Vaccine 2006 May 5; (Read article online)

Arya SC, Agarwal N

Investigations to assess episodes of any natural Japanese encephalitis (JE) infection following prior immunizations with JE inactivated vaccine in Japan are commendable. Employing differential production of nonstructural 1 protein NS1, during a natural infection and not by inactivated JE vaccine [Konishi E, Shoda M, Yamamoto S, Arai S, Tanaka-Taya K, Okabe N. Natural infection with Japanese encephalitis virus among inhabitants of Japan: a nationwide survey of antibodies against nonstructural 1 protein. Vaccine 2006;24:3054-956], natural infection was manifest in eight selected prefectures in Japan. Moreover, it would be possible to detect any foreign pathogenic JE virus (JEV) or any native strains associated with any major shift in JE clinical presentations. On the opposite, any NS1 antibody screen might be invalid among recipients of live JE vaccines.

Microbiol Immunol 2006; 50(5): 379-87 (Read article online)

Toriniwa H, Komiya T

We established a rapid, quantitative real-time reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay targeting the envelope gene of Japanese encephalitis virus. The RTLAMP enabled us to detect the target product within 1 hr by only reacting reverse transcriptase and Bst DNA polymerase in a single tube at an isothermal temperature. The detection sensitivity of the RT-LAMP for Japanese encephalitis virus was 1 PFU, similar to that of conventional reverse transcription-polymerase chain reaction (RT-PCR). Flaviviruses of the Japanese encephalitis virus group, such as Dengue virus and West Nile virus, could not be detected. This confirmed the specificity of the RT-LAMP assay for Japanese encephalitis virus. A standard curve was constructed by plotting viral titer against the time for virus detection by the RT-LAMP, validating the quantitative accuracy of the assay. In addition, the amount of virus estimated by RT-LAMP was strongly correlated (r = 0.902) with that determined by plaque assay, a conventional method for virus quantification. These results indicate that the RT-LAMP assay established in this study is specific for Japanese encephalitis virus, and allows more rapid detection and quantification of the virus.

Emerg Infect Dis 2006 Apr; 12(4): 549-55 (Read article online)

Gould EA

Arboviruses have evolved a number of strategies to survive environmental challenges. This review examines the factors that may determine arbovirus emergence, provides examples of arboviruses that have emerged into new habitats, reviews the arbovirus situation in western Europe in detail, discusses potential arthropod vectors, and attempts to predict the risk for arbovirus emergence in the United Kingdom. We conclude that climate change is probably the most important requirement for the emergence of arthropodborne diseases such as dengue fever, yellow fever, Rift Valley fever, Japanese encephalitis, Crimean-Congo hemorrhagic fever, bluetongue, and African horse sickness in the United Kingdom. While other arboviruses, such as West Nile virus, Sindbis virus, Tahyna virus, and Louping ill virus, apparently circulate in the United Kingdom, they do not appear to present an imminent threat to humans or animals.

AJNR Am J Neuroradiol 2006 May; 27(5): 1027-31 (Read article online)

Handique SK, Das RR, Barman K, Medhi N, Saharia B, Saikia P, Ahmed SA

BACKGROUND AND PURPOSE: On MR imaging and CT, Japanese encephalitis (JE) shows lesions in the thalami, substantia nigra, basal ganglia, cerebral cortex, cerebellum, brain stem, and white matter, whereas temporal lobe involvement is characteristically seen in Herpes simplex encephalitis (HSE). Temporal lobe involvement in JE may cause problems in differentiating it from HSE. We undertook this study to show the temporal lobe involvement pattern in JE and highlight differentiating features from temporal lobe involvement in HSE. METHODS: Sixty-two patients with JE underwent CT or MR imaging or both. MR imaging was done in 53 and CT in 53. The diagnosis of JE was confirmed by cerebrospinal fluid (CSF) IgM enzyme-linked immunosorbent assay. RESULTS: Eleven (17.7%) patients showed temporal lobe involvement with abnormal MR imaging in all. All the patients showed hippocampal involvement. Two patients showed extension of lesions into the amygdala and uncus with insular involvement in 1. The rest of the temporal lobe was spared. All patients had thalamic and substantia nigra involvement with basal ganglia involvement in 7. Six of 9 CT scans were abnormal and the temporal lesions were seen in 2. CONCLUSIONS: The temporal lobe involvement pattern is fairly characteristic and mostly involves the hippocampus, usually sparing the rest of the temporal lobe. This and the concurrent involvement of the thalami, substantia nigra (SN), and basal ganglia allow differentiation from HSE. However, if the temporal lobe involvement is more severe, laboratory tests may be the only way to differentiate it from HSE, and it may be prudent to start antiviral therapy in the interim period.

Vaccine 2006 Apr 24; (Read article online)

Liu W, Clemens JD, Yang JY, Xu ZY

This study examined the impact of Japanese encephalitis (JE) immunization policies on disease trends in China. Through a document review and key source interviews in eight provinces, the study found, JE immunizations were provided via a combination of fee-for-service and government funding. Unequal government funding and unbalanced economic development led to variation in JE incidence levels between provinces. Government support of low-fee JE immunization, has led to nationwide decline of JE incidence by >90%; however, greater reliance on user fees and market mechanisms in rural areas has limited the provision of JE and other childhood immunizations to poor rural children.

Mem Inst Oswaldo Cruz 2006 Feb; 101(1): 57-63 (Read article online)

Santos CL, Sallum MA, Franco HM, Oshiro FM, Rocco IM

The molecular characterization of SPH253157, a new strain of St. Louis encephalitis virus (SLEV), isolated in 2004 from the first case of human infection recognized in the state of São Paulo, Brazil, is reported. The patient, presenting a febrile illness without neurological involvement, was hospitalized as a probable case of dengue fever. Genomic RNA was isolated from the supernatant of C6/36 cells infected with acute phase-serum specimen of the patient and the envelope gene was amplified by reverse-transcription-polymerase chain reaction. The complete nucleotide sequence of the envelope gene of this isolate was directly sequenced from the amplified products and compared with other Brazilian and American SLEV strains. Phylogenetic analyses were carried out under maximum likelihood criterion with outgroups both included and excluded. Outgroups comprised four flavivirus of the Japanese encephalitis group. Phylogeny also included Bayesian analysis. The results indicated that the new SLEV isolate belongs to lineage III, being closely related to an Argentinean strain recovered from Culex sp. in 1979. It is concluded that there are at least 3 lineages of SLEV in Brazil.

Comp Immunol Microbiol Infect Dis 2006 Mar-May; 29(2-3): 166-75 (Read article online)

Pant GR, Lunt RA, Rootes CL, Daniels PW

A regional survey was conducted in Nepal for antibody to Japanese encephalitis virus (JEV) in domestic animals. Sera from pigs, and limited numbers of ducks and horses were collected from 16 districts in 2002-2003 and subjected to three serological tests. Of 270 porcine sera tested by C-ELISA, 55% were found positive for the presence of antibodies against Japanese encephalitis virus. Additional testing for IgM antibody to JEV revealed less than 2% of C-ELISA positive sera had evidence of recent JEV infection. Plaque reduction neutralisation tests (PRNT) using JEV, Murray Valley encephalitis (MVEV) and Kunjin (KUNV) viruses implicated JEV as the flavivirus associated with the observed antibody response in most sero-positive pigs. However, eight porcine sera with predominant neutralising antibody for KUNV (an Australasian subtype of West Nile Virus) provided evidence for the circulation of West Nile virus in Nepal.

J Virol 2006 Jun; 80(11): 5451-64 (Read article online)

Uchil PD, Kumar AV, Satchidanandam V

Flaviviral replication is believed to be exclusively cytoplasmic, occurring within virus-induced membrane-bound replication complexes in the host cytoplasm. Here we show that a significant proportion (20%) of the total RNA-dependent RNA polymerase (RdRp) activity from cells infected with West Nile virus, Japanese encephalitis virus (JEV), and dengue virus is resident within the nucleus. Consistent with this, the major replicase proteins NS3 and NS5 of JEV also localized within the nucleus. NS5 was found distributed throughout the nucleoplasm, but NS3 was present at sites of active flaviviral RNA synthesis, colocalizing with NS5, and visible as distinct foci along the inner periphery of the nucleus by confocal and immunoelectron microscopy. Both these viral replicase proteins were also present in the nuclear matrix, colocalizing with the peripheral lamina, and revealed a well-entrenched nuclear location for the viral replication complex. In keeping with this observation, antibodies to either NS3 or NS5 coimmunoprecipitated the other protein from isolated nuclei along with newly synthesized viral RNA. Taken together these data suggest an absolute requirement for both of the replicase proteins for nucleus-localized synthesis of flavivirus RNA. Thus, we conclusively demonstrate for the first time that the host cell nucleus functions as an additional site for the presence of functionally active flaviviral replicase complex.

J Am Mosq Control Assoc 2006 Mar; 22(1): 15-21 (Read article online)

van den Hurk AE, Montgomery BL, Zborowski P, Beebe NW, Cooper RD, Ritchie SA

The responses of Japanese encephalitis virus (JEV) mosquito vectors to 1-octen-3-ol (octenol) and CO2 were evaluated using Centers for Disease Control (CDC) light traps at 3 sites in northern Australia. There was no significant difference between the number of Culex sitiens subgroup mosquitoes or Cx. gelidus collected in CDC light traps baited with either CO2 alone or CO2 + octenol on Badu Island. At both mainland locations, using octenol in combination with CO2 significantly increased collections of Cx. sitiens subgroup mosquitoes. Collections of nontarget species, such as Ochlerotatus spp., Anopheles spp., and Verrallina spp. were also significantly increased with the addition of octenol. At all 3 locations, reducing collections of nontarget mosquitoes by not using octenol increased the proportion of Culex spp. collected, thus potentially reducing the time and resources required to sort and process collections for JEV detection. Our results also indicate that trials into the efficacy of using octenol as an attractant should be carried out in each area prior to the implementation of a mosquito-based arbovirus surveillance system.

J Vet Med Sci 2006 Mar; 68(3): 293-5 (Read article online)

Yamanaka T, Tsujimura K, Kondo T, Yasuda W, Okada A, Noda K, Okumura T, Matsumura T

Japanese encephalitis (JE) developed in an unvaccinated half-bred horse kept in Tottori Prefecture, Japan. The animal showed ataxia with pyrexia and low appetite, and ultimately died. A viral strain was isolated from the cerebrum of the horse and was identified as JE virus (JEV) by RT-PCR using JEV specific primers. The isolated JEV was classified into genotype I by nucleotide sequence analysis of the viral envelope gene. We believe that this is the first report of the genotype I strain being isolated from a horse.

Curr Pharm Des 2006; 12(11): 1315-38 (Read article online)

Frick DN, Lam AM

Helicases are promising antiviral drug targets because their enzymatic activities are essential for viral genome replication, transcription, and translation. Numerous potent inhibitors of helicases encoded by herpes simplex virus, severe acute respiratory syndrome coronavirus, hepatitis C virus, Japanese encephalitis virus, West Nile virus, and human papillomavirus have been recently reported in the scientific literature. Some inhibitors have also been shown to decrease viral replication in cell culture and animal models. This review discusses recent progress in understanding the structure and function of viral helicases to help clarify how these potential antiviral compounds function and to facilitate the design of better inhibitors. The above helicases and all related viral proteins are classified here based on their evolutionary and functional similarities, and the key mechanistic features of each group are noted. All helicases share a common motor function fueled by ATP hydrolysis, but differ in exactly how the motor moves the protein and its cargo on a nucleic acid chain. The helicase inhibitors discussed here influence rates of helicase-catalyzed DNA (or RNA) unwinding by preventing ATP hydrolysis, nucleic acid binding, nucleic acid release, or by disrupting the interaction of a helicase with a required cofactor.

Nature 2006 Apr 9; (Read article online)

Kato H, Takeuchi O, Sato S, Yoneyama M, Yamamoto M, Matsui K, Uematsu S, Jung A, Kawai T, Ishii KJ, Yamaguchi O, Otsu K, Tsujimura T, Koh CS, Reis E Sousa C, Matsuura Y, Fujita T, Akira S

The innate immune system senses viral infection by recognizing a variety of viral components (including double-stranded (ds)RNA) and triggers antiviral responses. The cytoplasmic helicase proteins RIG-I (retinoic-acid-inducible protein I, also known as Ddx58) and MDA5 (melanoma-differentiation-associated gene 5, also known as Ifih1 or Helicard) have been implicated in viral dsRNA recognition. In vitro studies suggest that both RIG-I and MDA5 detect RNA viruses and polyinosine-polycytidylic acid (poly(I:C)), a synthetic dsRNA analogue. Although a critical role for RIG-I in the recognition of several RNA viruses has been clarified, the functional role of MDA5 and the relationship between these dsRNA detectors in vivo are yet to be determined. Here we use mice deficient in MDA5 (MDA5(-/-)) to show that MDA5 and RIG-I recognize different types of dsRNAs: MDA5 recognizes poly(I:C), and RIG-I detects in vitro transcribed dsRNAs. RNA viruses are also differentially recognized by RIG-I and MDA5. We find that RIG-I is essential for the production of interferons in response to RNA viruses including paramyxoviruses, influenza virus and Japanese encephalitis virus, whereas MDA5 is critical for picornavirus detection. Furthermore, RIG-I(-/-) and MDA5(-/-) mice are highly susceptible to infection with these respective RNA viruses compared to control mice. Together, our data show that RIG-I and MDA5 distinguish different RNA viruses and are critical for host antiviral responses.

J Vet Med B Infect Dis Vet Public Health 2006 May; 53(4): 171-5 (Read article online)

Chvala S, Bakonyi T, Hackl R, Hess M, Nowotny N, Weissenböck H

Usutu virus (USUV) is a mosquito-borne flavivirus of the Japanese encephalitis virus group, which has been associated with avian mortality in Austria since 2001. The affected birds are predominantly Eurasian blackbirds (Turdus merula). In the present study, the pathogenicity of USUV for domestic geese (Anser anser f. domestica) was investigated. Eleven 2-week-old geese were inoculated intramuscularly with 5 x 10(4) 50% tissue culture infectious dose of USUV strain Vienna-2001 blackbird. No clinical signs were seen during the observation period. Four inoculated and one in-contact geese died without preceding clinical signs. Two of the deaths could be attributed to bacterial septicaemia and strangulation, respectively. The cause of death of two experimental and one in-contact animals remained unclear, but lack of evidence for viral lesions and viral antigen in their tissues argued against association with the USUV infection. Although in organs of the majority of inoculated geese (9/11) USUV was detected by reverse transcriptase-polymerase chain reaction, immunohistochemistry for USUV antigen was negative in all tissues of all geese. Evidence of plasma viraemia or viral excretion was found in one goose only. Seroconversion was detected in three inoculated geese 10 days post-inoculation. Geese placed in contact with inoculated geese and control animals did not exhibit USUV in their internal organs or plasma and lacked USUV-specific antibodies. This experiment shows that USUV is able to replicate in geese, but does not induce clinical disease, is unlikely to induce mortality, and only infrequently leads to viraemia or virus shedding.

Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi 2006 Mar; 20(1): 61-5 (Read article online)

Wang JW, Fu SH, Wang HY, Mao XY, Liu WB, He Y, Sun XH, Cai ZL, Wu LP, Zhao XF, Han RH, Jing Y, Liang GD

BACKGROUND: To study arboviruses carried by mosquitoes in Liaoning Province in 2002. METHODS: Totally 4927 mosquitoes were collected from Liaoning Province in July 2002. Virus strains were isolated by inoculating BHK-21, C6/36 and Vero cells. The newly isolated strains were identified by serological (ELISA and IFA) and molecular methods (Real-Time PCR and RT-PCR). RESULTS: Two strains were isolated from mosquitoes causing cytopathogenic effect (CPE) on cells and were fatal to suckling mice. Serological tests showed that both were positive for the antibody to JEV. The PrM and E gene were cloned and sequenced. The phylogenetic analysis showed that the new isolates belonged to genotype I, JEV. Sequence analysis showed that the homology of nucleotide sequences and amino acid (AA) sequences between the two strains was 100%. Compared with the nucleotide sequences between the two strains and the standard JEV vaccine strain SA14-14-2, the difference was up to 4.11%,and the difference of AA was 0.6%. CONCLUSION: Two strains of JEV were isolated and identified in Liaoning province, both belonged to genotype I JEV.

Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi 2006 Mar; 20(1): 47-51 (Read article online)

Xu LH, Cao YX, He LF, Wang HQ, He Y, Fu SH, Sun XH, Wang HY, Liu WB, Wang LH, Liang GD

BACKGROUND: To develop a rapid and more sensitive TaqMan RT-PCR assay specific for Bannavirus. METHODS: Total RNA of strains of Bannavirus and other arboviruses were extracted and reverse transcribed to cDNAs. With the cDNAs as templates, the TaqMan RT-PCR assay was developed and optimized on ABI 7300 apparatus for specific-detection of Bannavirus. The sensitivity, specificity and the possibility of this assay to detect Bannavirus in pools of mosquitoes. Meanwhile, human sera samples added with different dilutions of Bannavirus culture medium supernatant were evaluated. RESULTS: All the collected 12 Bannavirus strains in our country were detected as positive, while the results of other arboviruses such as Japanese encephalitis virus (JEV), Batai virus, Sindbis virus and Orbiviruses were negative. This assay was more than 100 times sensitive than that of traditional PCR. About 0.5 CCID-50 of Bannavirus in 50 microliter samples could be detected with this assay. The sensitivity decreased by about 10 times when the dsRNA was denatured in 65 degrees instead of 99 degrees, and also when the virus seeds were kept at room temperature for 1 day or 1 week at 4 degrees. There was no significant difference in the results of Bannavirus detection between artificial human serum samples and positive control virus. Six were detected as positive for Bannavirus-specific nucleic acids in 112 pools of mosquitoes, while 3 was positive with virus isolation. CONCLUSION: The Bannavirus-specific TaqMan RT-PCR assay was proved to be highly sensitive, specific and showed a good reproducibility; the assay may be applied for surveillance of this virus in clinical samples and pools of mosquitoes sceening.

J Vector Borne Dis 2006 Mar; 43(1): 21-8 (Read article online)

Devi NP, Jauhari RK

BACKGROUND & OBJECTIVES: Mosquito-borne diseases particularly malaria and Japanese encephalitis (JE) are becoming most dreaded health problems in Dehradun district. Keeping in view that the climatic factors particularly temperature and rainfall may alter the distribution of vector species--increasing or decreasing the ranges, depending on weather conditions that are favourable or unfavourable for mosquito breeding, it is aimed to find out the effect of climatic factors on malaria incidence with particular emphasis to capture the essential events as a result of climatic variability. METHODS: Mosquito sampling and identification was done using WHO entomological methods and follow-up of recognised keys and catalogues. Data on malaria incidence and meteorological information were gathered in a collaborative study with the District Malaria Office, and the Forest Research Institute, Dehradun respectively. Pearson's correlation analysis was applied for establishing relationship between climate variables and malaria transmission. RESULTS: Higher positive correlation of association was found between monthly parasite incidence and climatic variables (temperature, rainfall and humidity). However, highest significant correlation was found between rainfall and malaria incidence (r = 0.718, p < 0.0001) when the data were staggered to allow a lag of one-month. INTERPRETATION & CONCLUSION: Climatic variables that predict the presence or absence of malaria are likely to be the best suited for forecasting the distribution of this disease at the edges of its range.

Virus Res 2006 Apr 16; (Read article online)

Abraham S, Manjunath R

Infection with Flaviviruses upregulates the cell surface expression of MHC-I, MHC-II, ICAM-1 (CD54), VCAM-1 (CD106) and TAP proteins. Although all these studies have been confirmed using West Nile virus and other Flaviviruses, there are few reports that have examined the effects of Japanese encephalitis virus (JEV) infection directly on nonclassical and classical MHC expression in astrocytes. We show in this report that JEV infection of mouse brain astrocytes results in induction of the nonclassical MHC Class Ib genes, H-2T23, H-2Q4 and H-2T10 in addition to MHC-I, Type I (alpha/beta) IFNs, TAP-1, TAP-2, Tapasin, LMP-2, LMP-7 and LMP-10 but not IFNgamma, CD80, CD86 and MHC-II genes. The increased cell surface expression of these antigens as well as induction of the genes mentioned above as measured by RT-PCR suggests that JEV infection may lead to the induction of classical MHC Class Ia as well as nonclassical MHC Class Ib molecules.

Expert Rev Anti Infect Ther 2006 Apr; 4(2): 313-24 (Read article online)

Bharati K, Vrati S

Japanese encephalitis (JE) is the most common form of viral encephalitis that appears in the form of frequent epidemics of brain fever throughout Southeast Asia, China and India. The disease is caused by a Flavivirus named Japanese encephalitis virus that is spread to humans by mosquitoes. An internationally approved mouse brain-derived inactivated vaccine has been available that is relatively expensive, gives immunity of uncertain duration and is not completely safe. Cell culture-derived inactivated and attenuated JE vaccines are in use in China, but these are not produced as per the norms acceptable in most countries. Several new promising JE vaccine candidates have been developed, some of which are under different stages of clinical evaluation. These new candidate JE vaccines have the potential to generate long-lasting immunity at low cost.

J Clin Microbiol 2006 Apr; 44(4): 1295-304 (Read article online)

Chien LJ, Liao TL, Shu PY, Huang JH, Gubler DJ, Chang GJ

Serotyping dengue virus (DENV) from suspect human specimens is crucial for developing sound epidemiological control measurements early in the transmission season and for effective patient management. We modified DENV consensus D1 (mD1) and serotype-specific TS2 (mTS2) and redesigned serotype-specific TS1 (rTS1) and TS4 (rTS4) as described previously in the conventional capsid and premembrane gene (C-prM) protocol (R. S. Lanciotti, C. H. Calisher, D. J. Gubler, G.-J. Chang, A. V. Vorndam, J. Clin. Microbiol. 30:545-551, 1992). In addition, we designed two new sets of amplimers and probes, located at nonstructural protein 5 (NS5) and the 3' noncoding region (3'NC) of DENV. The NS5 protocol utilizes two flaviviral consensus outer amplimers (mFU1 and CFD2) and four dengue virus serotype-specific TaqMan fluorogenic probes. The 3'NC protocol uses two DENV consensus amplimers, DC10418 and CDC10564. The conventional gel-based, heminested detection method was adapted for the C-prM protocol for detecting and serotyping dengue viruses. In addition, we developed the real-time SYBR green I and postamplification melting temperature curve analysis for the mD1/TS and 3'NC protocols using identical amplification conditions. The NS5 amplimer/probe set was formulated as a one-tube, multiplex, real-time reverse transcriptase PCR for serotype identification. Three sets of amplimers and probes were verified for their specificity in tests with yellow fever, Japanese encephalitis, St. Louis encephalitis, and West Nile viruses; optimized against 109 DENV strains; and validated for detection of the virus in sera from two different panels of acute-phase human dengue serum specimens and one panel of virus isolates from dengue patients' serum specimens. Clinical evaluation by two separate laboratories indicated that the C-prM was more sensitive (100%) than the NS5 (91%) or the 3'NC (91%) protocol.

BMC Med 2006 Apr 7; 4(1): 8 (Read article online)

Kari K, Liu W, Gautama K, Mammen MP, Clemens JD, Nisalak A, Subrata K, Kim HK, Xu ZY

ABSTRACT: BACKGROUND: Japanese encephalitis (JE) is presumed to be endemic throughout Asia, yet only a few cases have been reported in tropical Asian countries such as Indonesia, Malaysia and the Philippines. To estimate the true disease burden due to JE in this region, we conducted a prospective, hospital-based surveillance with a catchment population of 599,120 children less than 12 years of age in Bali, Indonesia, from July 2001 through December 2003. METHODS: Balinese children presenting to any health care facility with acute viral encephalitis or aseptic meningitis were enrolled. A confirmed diagnosis of JE required the detection of JE virus (JEV)-specific IgM in cerebrospinal fluid, whereas a diagnosis of probable JE was assigned to those cases in which JEV-specific IgM was detected only in serum. RESULTS: In all, 86 confirmed and 4 probable JE cases were identified. The annualized JE incidence rate was 7.1 and adjusted to 8.2 per 100,000 for children less than 10 years of age over the 2.5 consecutive years of study. Only one JE case was found among 96,920 children 10-11 years old (0.4 per 100,000). Nine children (10%) died and 33 (37%) of the survivors had neurological sequelae at discharge. JEV was transmitted in Bali year-round with 70% of cases in the rainy season. CONCLUSIONS: JE incidence and case-fatality rates in Bali were comparable to those of other JE-endemic countries of Asia. Our findings contradict the common wisdom that JE is rare in tropical Asia. Hence, the geographical range of endemic JE is broader than previously described. The results of the study support the need to introduce JE vaccination into Bali.