Japanese Encephalitis Virus Strains Research Articles

Molecular characterization of full-length genome of Japanese encephalitis virus (KV1899) isolated from pigs in Korea

We have determined the complete nucleotide and deduced amino acid sequences of the Japanese encephalitis virus (JEV) strain KV1899, isolated from a fattening pig in Korea. In comparison with 22 fully sequenced JEV genomes currently available, we found that the 10,963-nucleotide RNA genome of KV1899 has a 13-nucleotide deletion in the 3' non-translated variable region and 53 unique nucleotide sequences including 3' non-translated region (NTR). Its single open reading frame has a total of 28 amino acid substitutions. Comparison of the KV1899 genomic sequence with those of the 21 fully sequenced JEV strains in published databases showed nucleotide homology ranging from 97.4% (Ishikawa strain) to 87.0% (CH2195 strain). Amino acid homology with KV1899 strain ranged from 96.4% (K94P05) to 91.0% (GP78). The KV1899 showed the highest nucleotide homology with Ishikawa strain and the highest amino acid homology with K94P05. We performed an extensive E gene based phylogenetic analysis on a selection of 41 JEV isolates available from the GenBank. Compared with Anyang strain, isolated from a pig in 1969, that is current live vaccine strain for swine in Korea, the homology of nucleotide sequence in envelope gene was only 87.1%. The prM gene of the isolate was closely related with those of Ishikawa and K94P05 strains, which were grouped into genotype I of JEV.

Neurovirulence and host factors in flavivirus encephalitis--evidence from clinical epidemiology.

Japanese encephalitis virus (JEV) and West Nile virus (WNV) provide some of the most important examples of emerging zoonotic viral encephalitides. For these flaviviruses, only a small proportion of those infected develop clinical features, and these may range from a non-specific flu-like illness to a severe fatal meningoencephalitis, often with Parkinsonian features, or a poliomyelitis-like flaccid paralysis. The factors governing the clinical presentations, and outcome of flavivirus infections are poorly understood, but studies have looked at viral virulence determinants and the host immune response. Previous studies on JEV have suggested that the distribution of the four genotypes across Asia may relate to the differing clinical epidemiology (epidemic disease in the north, endemic disease in the south). However, new data based on the complete nucleotide sequence of a virus representing one of the oldest lineages, and phylogenetic analyses of all JEV strains for which genetic data are available, suggest that the distribution is best explained in terms of the virus' origin in the Indonesia-Malaysia region (where all genotypes have been found), and the spread of the more recent genotypes to new geographical areas. Clinical studies have shown that innate immunity, as manifested by interferon alpha levels, is important in JEV and other flaviviruses, but treatment with interferon alpha did not improve the outcome. A failure of the humoral immune response, is associated with death from encephalitis caused by JEV and WNV. Cellular immunity has been less well characterized, but CD8+ and CD4+ T cells are thought to be important.

SHORT REPORT: DETECTION OF JAPANESE ENCEPHALITIS VIRUS IN MOUSE PERIPHERAL BLOOD MONONUCLEAR CELLS USING AN IN SITU REVERSE TRANSCRIPTASE–POLYMERASE CHAIN REACTION

Japanese encephalitis (JE) is an important mosquito-borne viral disease in Southeast Asia. Isolation of JE virus from peripheral blood is usually difficult because of transient and low titer of viremia. An in situ reverse transcriptase-polymerase chain reaction (RT-PCR) method was designed to amplify gene (envelope) fragments of JE virus residing in peripheral blood mononuclear cells (PBMCs) without extraction of RNA. Baby hamster kidney-21 cells infected with the T1P1 strain of JE virus (an isolate from Armigeres subalbatus collected in Taiwan) were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. The RT-PCR was then performed in microtubes using digoxigenin-labeled primers. Virus-positive PBMCs were detected in mice at day 1 and day 3, but not day 5, after intravenous inoculation with JE virus, suggesting that detectable virus circulating in the blood of mice is present for only 2-3 days. On examination of mouse brain tissues, viral RNAs were absent until day 3 post-inoculation. This implied that virus migration from the peripheral blood into the central nervous system occurs at or after day 3 post-inoculation. This method is unique in that the reactions can be conducted in tubes; this makes it convenient, accurate, and efficient compared with the conventional in situ RT-PCR on slides.

Molecular characterization of the full-length genome of the Japanese encephalitis viral strain K87P39

We have determined the complete nucleotide and deduced amino acid sequences of the Japanese encephalitis virus (JEV) strain K87P39, isolated from a pool of circulating Culex tritaeniorhynchus mosquitoes in Korea. In comparison with 27 fully sequenced JEV genomes currently available, we found that the 10968-nucleotide RNA genome of K87P39 has a nine-nucleotide deletion in the 3′ nontranslated variable region and that its single open reading frame has a total of eight amino acid substitutions. The K87P39 isolate is highly similar to other JEV isolates, and homology ranges from 97.9 to 89.0% at the nucleotide level, and 99.1 to 96.7% at the deduced amino acid level. Phylogenetic analyses using the full-length sequence of the 27 available JEV genomes showed that the K87P39 strain is most closely related to six Chinese SA14 derivatives and that it is distantly related to the Australian FU, Korean K94P05 and Japanese Ishikawa strains. In addition, we also found that phylogenetic relationships based on the full-length genome are highly similar to those based on the E gene, indicating that phylogenetic analysis of the E gene will be useful for studying the genetic relationships among JEV isolates. We therefore performed a more extensive E gene-based phylogenetic analysis on a selection of 70 JEV isolates available from GenBank, which represent a temporally and geographically wide variety of JEV strains.

SHORT REPORT: A MAJOR GENOTYPE OF JAPANESE ENCEPHALITIS VIRUS CURRENTLY CIRCULATING IN JAPAN

A 240-nucleotide sequence of the capsid/premembrane gene region of 23 Japanese encephalitis virus (JEV) strains isolated in Tokyo and Oita, Japan was determined and phylogenetic analyses were performed. All the strains clustered into two distinct genotypes (III and I). All strains isolated before 1991 belonged to genotype III, while those isolated after 1994 belonged to genotype I. In addition, the strains of the genotype I isolated in Japan showed a close genetic relationship with those from Korea and Malaysia.

Induction of cross-protection against two wild-type Taiwanese isolates of Japanese encephalitis virus using Beijing-1 strain DNA vaccine

Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, is an important human pathogen. Mouse brain-derived, inactivated JEV vaccines have contributed greatly to the reduction in numbers of JE patients in several countries, including Taiwan. However, mice immunized with the Nakayama strain inactivated vaccine show lower protection against a lethal strain of Beijing-1 JEV than those immunized with the homologous vaccine. DNA vaccine encoding the envelope (E) protein gene appears to provide protection against JEV in the mouse model, but it is not known whether such vaccines would confer cross-protection for mice against different strains of JEV. In this study, we evaluated the ability of pE, a plasmid DNA vaccine encoding the Beijing-1 envelope protein to elicit cross-protective immunity against infection with the homologous Beijing-1 strain and two lethal Taiwanese isolates of JEV, CH2195 and CEN. Our results showed that mice immunized with pE were protected against lethal challenge with Beijing-1 JEV as well as two Taiwanese isolates. In addition, naı̈ve mice were cross-protected by passive transfer of sera from immunized animals, indicating the crucial role of humoral immunity in protection. These results demonstrate that JEV DNA vaccine can provide effective protection against infection not only with homologous virus, but also with heterologous virus.

Origin and evolution of Japanese encephalitis virus in southeast Asia.

Since it emerged in Japan in the 1870s, Japanese encephalitis has spread across Asia and has become the most important cause of epidemic encephalitis worldwide. Four genotypes of Japanese encephalitis virus (JEV) are presently recognized (representatives of genotypes I to III have been fully sequenced), but its origin is not known. We have determined the complete nucleotide and amino acid sequence of a genotype IV Indonesian isolate (JKT6468) which represents the oldest lineage, compared it with other fully sequenced genomes, and examined the geographical distribution of all known isolates. JKT6468 was the least similar, with nucleotide divergence ranging from 17.4 to 19.6% and amino acid divergence ranging from 4.7 to 6.5%. It included an unusual series of amino acids at the carboxy terminus of the core protein unlike that seen in other JEV strains. Three signature amino acids in the envelope protein (including E327 Leu-->Thr/Ser on the exposed lateral surface of the putative receptor binding domain) distinguished genotype IV strains from more recent genotypes. Analysis of all 290 JEV isolates for which sequence data are available showed that the Indonesia-Malaysia region has all genotypes of JEV circulating, whereas only more recent genotypes circulate in other areas (P < 0.0001). These results suggest that JEV originated from its ancestral virus in the Indonesia-Malaysia region and evolved there into the different genotypes which then spread across Asia. Our data, together with recent evidence on the origins of other emerging viruses, including dengue virus and Nipah virus, imply that tropical southeast Asia may be an important zone for emerging pathogens.

Immunogenicity of a Japanese encephalitis DNA vaccine candidate in cynomolgus monkeys

A Japanese encephalitis (JE) vaccine candidate encoding JE virus premembrane (prM) and envelope (E) genes, designated pNJEME, was evaluated for safety and immunogenicity in non-human primate, cynomolgus monkeys. pNJEME was constructed using a vector (pNGVL4a) designed to address some of the safety concerns of DNA vaccine. In two different experiments, two immunizations with 300 μg of pNJEME by intramuscular (i.m.) injection, and 3 μg of pNJEME using a gene gun, and three immunizations by i.m. injection with 500 μg of pNJEME were performed. All the three protocols induced low to high levels of neutralizing antibody, indicating an ability of pNJEME to induce neutralizing antibody in monkeys with a wide individual variation in response to pNJEME. In one experiment designed to compare the DNA vaccine with a commercial inactivated JE vaccine, three immunizations by i.m. inoculation with 300 μg of pNJEME or by gene gun administration with 3 μg of pNJEME induced similar levels of neutralizing antibody to those induced by three immunizations with a human dose of the inactivated vaccine in most monkeys. After intranasal challenge with the Beijing P3 or JaTH160 strain of JE virus, pNJEME-immunized monkeys showed anamnestic neutralizing antibody responses, indicating that pNJEME induced memory B cells which were responsive to infection with JE virus. No systemic and local reactions were observed in any monkeys after i.m. or gene gun inoculations with plasmid DNAs.

Cloning and sequencing of complete cDNA of Japanese encephalitis virus YL strain in Taiwan.

We determined the complete nucleotide sequence of the YL strain of Japanese encephalitis virus and its amino acid sequence was deduced. Our results displayed that the genome of YL strain contained a single open reading frame of 10,296 nucleotides (nts) which was flanked by untranslated region (UTR) containing 95 bases at the 5'-end and 586 bases at the 3'-end, respectively. Comparison of sequences showed that the overall amino acid sequence and 3' UTR of YL were similar to those of the virulent strain JaGAr0l. However, some significant amino acid differences of viral envelope (E) protein were observed between YL and JaGAr01; the amino acid sequence of E protein in YL strain possessed RGG(387-389) tripeptide instead of RGD(387-389) in JaGAr01 and in other strains; and another amino acid is K(138) in YL, not E(138) found in others. These differences suggested that the YL strain impairs in viral attachment to the cell surface and loses neuroinvasiveness, and therefore this strain was used as a live attenuated vaccine.

Vector competence of Australian mosquitoes (Diptera: Culicidae) for Japanese encephalitis virus.

Australian mosquitoes were evaluated for their ability to become infected with and transmit a Torres Strait strain of Japanese encephalitis virus. Mosquitoes, which were obtained from either laboratory colonies and collected using Centers for Disease Control and Prevention light traps baited with CO2 and octenol or reared from larvae, were infected by feeding on a blood/sucrose solution containing 10(4.5 +/- 0.1) porcine stable-equine kidney (PS-EK) tissue culture infectious dose50/mosquito of the TS3306 virus strain. After 14 d, infection and transmission rates of 100% and 81%, respectively, were obtained for a southeast Queensland strain of Culex annulirostris Skuse, and 93% and 61%, respectively, for a far north Queensland strain. After 13 or more days, infection and transmission rates of > 90% and > or = 50%, respectively, were obtained for southeast Queensland strains of Culex sitiens Wiedemann and Culex quinquefasciatus Say, and a far north Queensland strain of Culex gelidus Theobald. Although infection rates were > 55%, only 17% of Ochlerotatus vigilax (Skuse) and no Cx. quinquefasciatus, collected from far north Queensland, transmitted virus. North Queensland strains of Aedes aegypti L., Ochlerotatus kochi (Dönitz), and Verrallina funerea (Theobald) were relatively refractory to infection. Vertical transmission was not detected among 673 F1 progeny of Oc. vigilax. Results of the current vector competence study, coupled with high field isolation rates, host feeding patterns and widespread distribution, confirm the status of Cx. annulirostris as the major vector of Japanese encephalitis virus in northern Australia. The relative roles of other species in potential Japanese encephalitis virus transmission cycles in northern Australia are discussed.

The future of dengue vaccines

Every year there are several hundred thousand acres of dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) resulting in thousands of deaths. The only method currently available to prevent dengue infections is the control of Aedes aegypti the mosquito vector. This approach has proved expensive and mostly unworkable. Serial infection occurs in most areas of the world where multiple dengue viruses circulate. A small percentage of second dengue infections result in DHF/DSS. Dengue vaccines must provide solid and long-lasting protection against all four dengue viruses or in dengue-endemic countries there is the risk of sensitizing recipients to severe disease. Many candidate dengue vaccines are moving towards clinical trials in human beings. These vaccines include several based on the formation of live-attenuated chimeric viruses. Yellow fever (YF) Japanese encephalitis (JE) and the four dengue viruses are all small single-strand positive-sense ribonucleic acid flaviviruses with similar genetic organization and replication strategies. Evidence that dengue vaccines should succeed comes from encouraging experience with attenuated vaccines against related viruses such as YF and JE and from a recent successful phase 1 trial of a chimera of these two viruses. This progress is timely because global dengue morbidity achieved an all time high during 2001-02. Thomas Monath and colleagues developed a live-attenuated chimeric vaccine against JE virus by replacing the premembrane (prM) and envelope (E) genes of the YF 17D vaccine with the corresponding genes from SA 14-14-2 an attenuated JE virus strain to produce YF-JE chimera. No unusual side-effects were seen in the phase 1 study and high titers of neutralizing antibodies to JE virus were found 3-4 weeks after inoculation of doses of 100000 and 1000. plaque-forming units. Similar methodology has been used to make chimera vaccines with YF virus for all four dengue viruses. Hence to ensure the introduction of safe and effective dengue vaccines for the worlds children ways must be found to accelerate phase 3 trials of tetravalent dengue vaccines in suitable people. Only testing in man can answer questions of safety and efficacy. A parallel effort is needed to understand protective mechanisms in dengue-infections especially those mediated by antibodies. The largest constraint to the solution of these problems is adequate funding which is now being addressed by a new international consortium. (authors)

The development and application of the latex agglutination test to detect serum antibodies against Japanese encephalitis virus.

The attenuated SA14-14-2 strain of Japanese encephalitis virus (JEV) was cultured in BHK-21 cells. The viral supernatant was purified and concentrated with PEG (MW 20,000). A suitable concentration of JEV antigen was used to sensitize latex to prepare the latex antigen. The specificity, sensitivity and stability of the antigen were assessed. A latex agglutination test (LAT) was developed for rapidly detecting antibody against JEV infection. The LAT and haemagglutination inhibition (HI) assay were compared by simultaneously testing 35 porcine serum samples from five farms. Ninety per cent (20/23) of the samples were seropositive by both assays. No significant difference was found between the two methods (p > 0.05). Furthermore, when 1,613 porcine sera from 120 farms were tested by LAT, the number of positive sera was 652, while that of negative sera was 961, ranging from 20% to 50% positive throughout the year. These results indicate that LAT is an appropriate candidate method for epidemiological surveys for and diagnosis of Japanese encephalitis.

Molecular epidemiology of Japanese encephalitis virus.

Japanese encephalitis (JE) virus causes an encephalitic disease in humans that is characterized by a wide range of symptoms from a nonspecific febrile illness to convulsions, seizure, and death (Solomon et al. 2000). Approximately 30% of patients presenting at hospital with JE will succumb to the disease and about 50% of those who survive will have severe neurological sequelae (Solomon et al. 2000). The JE virus is carried by mosquitoes, primarily Culex species, and is endemic from tropical to temperate regions of Asia and Australasia. JE virus is generally considered to circulate in an endemic cycle in southern regions of Asia whereas the virus tends to have a more epidemic epidemiological pattern in northern Asia with outbreaks occurring during the summer months (Vaughn and Hoke 1992). The virus infects a number of animal hosts, most importantly pigs, which act as amplifying hosts and are found in high numbers throughout rural Asia. The first isolate of JE virus, and the prototype strain, was isolated in 1935 in Tokyo from a human brain and is designated the Nakayama strain. Since that time, a large number of JE virus isolates have been identified from a wide range of geographical locales throughout Asia, Indonesia, and most recently, northern Australia. Many of these viruses have been analyzed serologically and/or genetically to examine the distribution of the virus strains over both temporal and geographical boundaries. To date, the complete genome of 20 strains of JE virus have been sequenced including the first isolate from Australia (Williams et al. 2000).

Mutations in the NS3 gene and 3'-NCR of Japanese encephalitis virus isolated from an unconventional ecosystem and implications for natural attenuation of the virus.

The T1P1 strain of Japanese encephalitis (JE) virus was recently isolated from paddy-free Liu-Chiu Islet in which natural JE antibody has been prevalent. In mouse neuroblastoma-derived Neuro-2a cells, T1P1 appeared significantly lower in virus productivity than another local isolate, CH1392. It implied that this new isolate possesses a characteristic viral replication pattern other than that of CH1392. T1P1 has also shown lower neurovirulence, which was reflected by a significantly higher LD(50) (2.44 x 10(6) PFU) than CH1392 (2.87 x 10(2) PFU). In comparison of the full-length RNA sequences between T1P1 and CH1392, a total of 7 nucleotides, including 1 in preM/M and 2 each in NS3, NS5, and the 3'-end noncoding region (NCR), appeared different. Of them, only the changes in NS3 (position 325, T for CH1392, A for T1P1; and position 364, G for CH1392 and A for T1P1) resulted in substitutions of deduced amino acids. There were two additional nucleotide changes appearing in the 3'-NCR. The amino acids 109 Phe and 122 Glu in NS3 of CH1392 were substituted by Ile and Lys, respectively, in T1P1. The unique growth properties and low virulence of T1P1 presented in this report were likely related to abnormal enzymatic activity due to mutations of the NS3 gene (especially position 364) and possibly to the mutations in the 3'-NCR. The natural attenuation of T1P1 that has been circulating in paddy-free Liu-Chiu Islet may account for the absence of clinical JE cases in past years.

A purified inactivated Japanese encephalitis virus vaccine made in vero cells

A second generation, purified, inactivated vaccine (PIV) against Japanese encephalitis (JE) virus was produced and tested in mice where it was found to be highly immunogenic and protective. The JE-PIV was made from an attenuated strain of JE virus propagated in certified Vero cells, purified, and inactivated with formalin. Its manufacture followed current GMP guidelines for the production of biologicals. The manufacturing process was efficient in generating a high yield of virus, essentially free of contaminating host cell proteins and nucleic acids. The PIV was formulated with aluminum hydroxide and administered to mice by subcutaneous inoculation. Vaccinated animals developed high-titered JE virus neutralizing antibodies in a dose dependant fashion after two injections. The vaccine protected mice against morbidity and mortality after challenge with live, virulent, JE virus. Compared with the existing licensed mouse brain-derived vaccine, JE-Vax, the Vero cell-derived JE-PIV was more immunogenic and as effective as preventing encephalitis in mice. The JE-PIV is currently being tested for safety and immunogenicity in volunteers.

Monoclonal antibody raised against envelope glycoprotein peptide neutralizes Japanese encephalitis virus.

Epitopes on envelope glycoprotein of Indian strain of Japanese encephalitis virus were delineated by prediction methods. Monoclonal antibodies (MAb) raised against a putative B cell epitope peptide, reacted with the virion in ELISA and immunofluorescence assays. One MAb was also able to neutralize the virus. The reactivity of this MAb against a Sri Lankan strain was checked, since this strain had a substitution within the B cell epitope at position Egp 153 (G-->W). The MAb was able to bind to, but was not able to neutralize the Sri Lankan isolate. The data indicated that the predicted B cell epitope is a neutralizing epitope and may be included in a peptide-based vaccine against the virus.

Highly Sulfated Forms of Heparin Sulfate Are Involved in Japanese Encephalitis Virus Infection

Japanese encephalitis virus (JEV) infects a broad range of cell types in vitro, though little is known about the initial events of JEV infection. In the present study, we found that highly sulfated glycosaminoglycans (GAGs) are involved in infection of both neurovirulent (RP-9) and attenuated (RP-2ms) JEV strains. Competition experiments using highly sulfated GAGs, heparin and dextran sulfate, demonstrated an inhibition of JEV's attachment and subsequent infection of BHK-21 cells. Treatment of target cells by a potent sulfation inhibitor, sodium chlorate, greatly reduced viral binding ability as well as infection, suggesting a critical role of GAGs' sulfation status on the cellular surface in JEV infection. This phenomenon was confirmed by the manifestation of a distinct binding efficiency of JEV to the wild-type CHO cell line and its mutants with defects in GAG biosynthesis. We also demonstrated the binding of JEV particles and virus envelope glycoprotein to immobilized heparin beads. Furthermore, the addition of heparin suppressed the cytopathic effects induced by JEV infection in cultured cells. Our results establish that the highly sulfated form of GAGs on cell surfaces plays a determining role in the early stage of in vitro JEV infection.

Complete nucleotide sequence and cell-line multiplication pattern of the attenuated variant CH2195LA of Japanese encephalitis virus

Strain-specific determinant of Japanese encephalitis virus (JEV) can be different among each virulence phenotype. We reported previously that the attenuated variant CH2195LA compared with the non-attenuated variant CH2195SA had four amino acid differences in E protein (E-85, E-306, E-331, and E-387) (Wu, S.-C., Lian, W.-C., Hsu, L.-C., Liau, M., 1997. Japanese encephalitis virus antigenic variants with characteristic differences in neutralization resistance and mouse virulence. Virus Res. 51, 173–181). Our present study determined the complete nucleotide sequences of these two variants and found another five amino acid changes in the nonstructural gene regions, including NS2A-215, NS3-350, NS4B-196, NS4B-197, and NS4B-198. The complete nucleotide sequences of the attenuated variant CH2195LA were compared with the non-attenuated variant CH2195SA and other 13 JEV strains. CH2195LA was mostly close to CH2195SA and JaOArS982, and phylogenetically distant to SA14-14-2, SA14-2-8, and RP-2ms of the attenuated JEV strains. The multiplication patterns for CH2195LA compared with CH2195SA in other seven cell lines were different from Vero cells. Relative fitness vector analysis based on a mixture of both variants during cell passage indicated the attenuated variant CH2195LA showed increased viral fitness in Vero cell adaptation. These results revealed that the attenuated variant CH2195LA was unique to culture in Vero cells.

Nucleotide sequence of envelope protein of Japanese encephalitis virus SA14-14-2 adapted to vero cells.

Live attenuated Japanese encephalitis (JE) virus SA(14)-14-2 produced in primary dog kidney cells (PDK) was adapted to Vero cells. In an effort to gain insight into the molecular basis of the biological characteristics of the SA14-14-2(Vero) strain, the 1500 nucleotide sequence encoding the envelope (E) gene which possesses major neutralizing epitopes was determined and compared with the sequences of two other attenuated JE virus strains, SA14-14-2(PHK) and SA14-14-2(PDK). The amino acid sequence of the C-terminal region (a.a. 280-500) was found to be identical for all three strains, while the N-terminal region (a.a. 1-279) shows sequence variation. The distribution of mutations in the N-terminal region was nearly the same among the three attenuated strains, suggesting that the N-terminal sequences might be related with virus-host cell specificity. However, it was found that Lys and Val (a.a. 138 and 176, respectively), known to be responsible for attenuation, are still conserved in SA(14)-14-2(Vero). Animal testing showed that SA(14)-14-2(Vero) has an attenuation phenotype similar to that of the parent SA(14)-14-2(PDK) strain in mice.

Molecular characterization of the first Australian isolate of Japanese encephalitis virus, the FU strain.

The complete genomic and predicted amino acid sequence of the Japanese encephalitis virus (JEV) FU strain, a human isolate recovered from the first outbreak of Japanese encephalitis in Australian territory, was determined. Comparison of the FU genome with 15 fully sequenced JEV genomes revealed high levels of sequence identity, ranging from 88.7% (GP78) to 89.7% (K94P05) for nucleotides and 96.8% (K94P05) to 98.0% (JaGAr01) for amino acid sequences. A total of 39 unique amino acid differences were found in the FU strain polyprotein. Phylogenetic analyses were performed on all available full-length JEV genomes and a selection of 64 E gene sequences from temporally and geographically diverse JEV strains. For comparison with the E gene phylogeny, phylogenetic analysis using cognate prM gene sequences was also carried out. The FU strain was found to be most closely related to Korean isolate K94P05 in the full-length analysis and to Southeast Asian strains in the E and prM gene analyses. The E gene analysis corresponded well with the prM gene analysis and with previous genotyping studies using the prM gene. The epidemiological implications of this investigation are discussed.