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.
Japanese encephalitis virus (JEV) is the most important cause of epidemic encephalitis worldwide but its origin is unknown. Epidemics of encephalitis suggestive of Japanese encephalitis (JE) were described in Japan from the 1870s onwards. Four genotypes of JEV have been characterised and representatives of each genotype have been fully sequenced. Based on limited information, a single isolate from Malaysia is thought to represent a putative fifth genotype. We have determined the complete nucleotide and amino acid sequence of Muar strain and compared it with other fully sequenced JEV genomes. Muar was the least similar, with nucleotide divergence ranging from 20.2 to 21.2% and amino acid divergence ranging from 8.5 to 9.9%. Phylogenetic analysis of Muar strain revealed that it does represent a distinct fifth genotype of JEV. We elucidated Muar signature amino acids in the envelope (E) protein, including E327 Glu on the exposed lateral surface of the putative receptor binding domain which distinguishes Muar strain from the other four genotypes. Evolutionary analysis of full-length JEV genomes revealed that the mean evolutionary rate is 4.35 × 10(-4) (3.4906 × 10(-4) to 5.303 × 10(-4)) nucleotides substitutions per site per year and suggests JEV originated from its ancestral virus in the mid 1500s in the Indonesia-Malaysia region and evolved there into different genotypes, which then spread across Asia. No strong evidence for positive selection was found between JEV strains of the five genotypes and the E gene has generally been subjected to strong purifying selection.
Forty-six strains of Japanese encephalitis (JE) virus from a variety of geographic areas in Asia were examined by primer-extension sequencing of the RNA template. A 240 nucleotide sequence from the pre-M gene region was selected for study because it provided sufficient information for determining genetic relationships among the virus isolates. Using 12% divergence as a cutoff point for virus relationships, the 46 isolates fell into three distinct genotypic groups. One genotypic group consisted of JE virus isolates from northern Thailand and Cambodia. A second group was composed of isolates from southern Thailand, Malaysia, Sarawak and Indonesia. The remainder of the isolates, from Japan, China, Taiwan, the Philippines, Sri Lanka, India and Nepal, made up a third group. The implications of these findings in relation to the epidemiology of JE are discussed. Results of this study demonstrate that the comparison of short nucleotide sequences can provide insight into JE virus evolution, transmission and, possibly, pathogenesis.
Mosquito-borne flaviviruses are emerging pathogens of an increasing global public health concern because of their rapid increase in geographical range and the impact of climate change. Japanese encephalitis virus (JEV) and West Nile virus (WNV) are of concern because of the risk of reemergence and introduction by migratory birds. In Singapore, human WNV infection has never been reported and human JEV infection is rare. Four sentinel vector surveillance sites were established in Singapore to understand the potential risk posed by these viruses. Surveillance was carried out from August 2011 to December 2012 at Pulau Ubin, from March 2011 to March 2013 at an Avian Sanctuary (AS), from December 2010 from October 2012 at Murai Farmway, and from December 2010 to December 2013 at a nature reserve. The present study revealed active JEV transmission in Singapore through the detection of JEV genotype II in Culex tritaeniorhynchus collected from an Avian Sanctuary. Culex flavivirus (CxFV), similar to the Quang Binh virus isolated from Cx. tritaeniorhynchus in Vietnam and CxFV-LSFlaviV-A20-09 virus isolated in China, was also detected in Culex spp. (vishnui subgroup). No WNV was detected. This study demonstrates the important role that surveillance plays in public health and strongly suggests the circulation of JEV among wildlife in Singapore, despite the absence of reported human cases. A One Health approach involving surveillance, the collaboration between public health and wildlife managers, and control of mosquito populations remains the key measures in risk mitigation of JEV transmission in the enzootic cycle between birds and mosquitoes.
Detection and isolation of Japanese encephalitis (JE) virus were attempted from female mosquitoes collected in Kampong Pasir Panjang, Sabak Bernam, Selangor, from May to November 1992. A total of 7,400 mosquitoes consisting of 12 species in 148 pools were processed and inoculated into Aedes albopictus clone C6/36 cell cultures. Of these, 26 pools showed the presence of viral antigens in the infected C6/36 cells by specific immunoperoxidase staining using an anti-JE virus polyclonal antibody. Presence of JE virus genome was confirmed in the infected culture fluid for 16 pools by using reverse transcriptase-polymerase chain reaction and JE virus-specific primers. Of these, 3 pools were from Culex tritaeniorhynchus, 4 from Culex vishnui, 3 from Culex bitaeniorhynchus, 2 from Culex sitiens, one from Aedes species, and 3 from Culex species. Isolation of JE virus from Cx. sitiens, Cx. bitaeniorhynchus, and Aedes sp. (Aedes butleri and Ae. albopictus) is reported for the first time in Malaysia.
Isolation of Japanese encephalitis virus (JEV) from mosquitoes in Sabak Bernam, Selangor, Malaysia, was attempted. An aliquot of homogenate from each pool of mosquitoes, 50 per tube, was inoculated into Aedes albopictus clone C6/36 cells for virus isolation. Each cell culture was tested for the presence of viral antigen by immunoperoxidase staining using an anti-JEV polyclonal antibody. Out of 4 Culex sitiens mosquito pools, 2 pools were positive for JEV by cell culture. Presence of JEV genome in the cell cultures for Cx. sitiens was confirmed by using reverse transcriptase-polymerase chain reaction and JEV-specific primers. This is the first report on the isolation of JEV from Cx. sitiens.
In mid-January 2000, the reappearance of Japanese encephalitis (JE) virus activity in the Australasian region was first demonstrated by the isolation of JE virus from 3 sentinel pigs on Badu Island in the Torres Strait. Further evidence of JE virus activity was revealed through the isolation of JE virus from Culex gelidus mosquitoes collected on Badu Island and the detection of specific JE virus neutralizing antibodies in 3 pigs from Saint Pauls community on Moa Island. Nucleotide sequencing and phylogenetic analyses of the premembrane and envelope genes were performed which showed that both the pig and mosquito JE virus isolates (TS00 and TS4152, respectively) clustered in genotype I, along with northern Thai, Cambodian, and Korean isolates. All previous Australasian JE virus isolates belong to genotype II, along with Malaysian and Indonesian isolates. Therefore, for the first time, the appearance and transmission of a second genotype of JE virus in the Australasian region has been demonstrated.
The role of the cytoskeleton, actin, and microtubules were examined during the process of Japanese encephalitis (JEV) infection in a human neuroblastoma cell line, IMR32. Cytochalasin D and nocodazole were used to depolymerise the cellular actin and microtubules, respectively, in order to study the effect of JEV infection in the cell. This study shows that depolymerisation of the actin cytoskeleton at early process of infection inhibits JEV infection in the cell; however infection was not inhibited when depolymerisation occurred at the later stage of infection. The microtubules, on the other hand, are required at 2 points in infection. The antigen production in the cells was inhibited when the infected cells were treated at time up to 2 hours after inoculation and there was no significant effect at later times, while the viable virus released continued to be affected until 10 hours after inoculation. In conclusion, infection of JEV in IMR32 cells required actin to facilitate early process in infection and the microtubular network is utilised as the transport system to the virus replication site and the release of mature virus.
Although a previous study predicted that Japanese encephalitis virus (JEV) originated in the Malaysia/Indonesia region, the virus is known to circulate mainly on the Asian continent. However, there are no reported systematic studies that adequately define how JEV then dispersed throughout Asia.
Japanese encephalitis virus (JEV), the prototype member of the JEV serocomplex, genus Flavivirus, family Flaviviridae, is the most significant arthropod-borne encephalitis worldwide in terms of morbidity and mortality. At least four genotypes (GI-GIV) of the virus have been identified; however, to date, the genomic nucleotide sequence of only one GII virus has been determined (FU strain, Australia, 1995). This study sequenced three additional GII strains of JEV isolated between 1951 and 1978 in Korea, Malaysia and Indonesia, respectively, and compared them with the FU strain, as well as with virus strains representing the other three genotypes. Based on nucleotide and amino acid composition, the genotype II strains were the most similar to GI strains; however, these two genotypes are epidemiologically distinct. Selection analyses revealed that the strains utilized in this study are under predominantly purifying selection, and evidence of positive selection was detected at aa 24 of the NS4B protein, a protein that functions as an alpha/beta interferon signalling inhibitor.
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.
Two hundred and forty nucleotides from the pre-M gene region of 10 Japanese encephalitis (JE) virus strains isolated in Malaysia in 1992 were sequenced and compared with the other JE virus strains from different geographic areas in Asia. Our JE virus strains belong to the largest genotypic group that includes strains isolated in temperate regions such as Japan, China, and Taiwan. Our Malaysian JE virus strains differed in 32 nucleotides (13.3%) from WTP/70/22 strain isolated from Malaysia in 1970, which belonged to another distinct genotypic group.
Two hundred forty nucleotides from the pre-membrane gene region of 12 Japanese encephalitis virus (JEV) strains isolated from three different regions of Malaysia from 1993 to 1994 were sequenced and compared with each other and with the JEV strains from different geographic areas in Asia. These 12 Malaysian isolates were classified into two genotypes. The four JEV strains isolated from Sarawak in 1994 and the four JEV strains isolated from Sepang, Selangor in 1993 were classified into one genotype that included earlier isolated strains from Malaysia (JE-827 from Sarawak in 1968 and WTP/70/22 from Kuala Lumpur in 1970). The four JEV strains from Ipoh, Perak in 1994 were classified into another genotype that included JEV strains isolated from northern Thailand and Cambodia. In an earlier report, 10 JEV strains from Sabak Bernam, Selangor in 1992 were classified into the largest genotype that included strains isolated in temperate regions such as Japan, China, and Taiwan. The data indicate that at least three genotypes of JEV have been circulating in Malaysia.
Japanese encephalitis virus (JEV) genotype V was originally isolated in Malaysia in 1952 and has long been restricted to the area. In 2009, sudden emergence of the genotype V in China and Korea was reported, suggesting expansion of its geographical distribution. Although studies on the genotype V are becoming more important, they have been limited partly due to lack of its infectious molecular clone. In this study, a plasmid carrying cDNA corresponding to the entire genome of JEV Muar strain, which belongs to genotype V, in the downstream of T7 promoter was constructed. Electroporation of viral RNA transcribed by T7 RNA polymerase (T7RNAP) in vitro from the plasmid led to production of progeny viruses both in mammalian and mosquito cells. Also, transfection of the infectious clone plasmid into mammalian cells expressing T7RNAP transiently or stably was demonstrated to generate infectious progenies. When the viral structural protein genes were partially deleted from the full-length cDNA, the subgenomic RNA transcribed in vitro from the modified plasmid was shown to replicate itself in mammalian cells as a replicon. The replicon carrying the firefly luciferase gene in place of the deleted structural protein genes was also shown to efficiently replicate itself and express luciferase in mammalian cells. Compared with the replicon derived from JEV genotype III (Nakayama strain), the genotype V-derived replicon appeared to be more tolerant to introduction of a foreign gene. The infectious clone and the replicons constructed in this study may serve as useful tools for characterizing JEV genotype V.
Japanese encephalitis virus (JEV) is a major cause of neurological disability in Asia and causes thousands of severe encephalitis cases and deaths each year. Although Japanese encephalitis (JE) is a WHO reportable disease, cases and deaths are significantly underreported and the true burden of the disease is not well understood in most endemic countries. Here, we first conducted a spatial analysis of the risk factors associated with JE to identify the areas suitable for sustained JEV transmission and the size of the population living in at-risk areas. We then estimated the force of infection (FOI) for JE-endemic countries from age-specific incidence data. Estimates of the susceptible population size and the current FOI were then used to estimate the JE burden from 2010 to 2019, as well as the impact of vaccination. Overall, 1,543.1 million (range: 1,292.6-2,019.9 million) people were estimated to live in areas suitable for endemic JEV transmission, which represents only 37.7% (range: 31.6-53.5%) of the over four billion people living in countries with endemic JEV transmission. Based on the baseline number of people at risk of infection, there were an estimated 56,847 (95% CI: 18,003-184,525) JE cases and 20,642 (95% CI: 2,252-77,204) deaths in 2019. Estimated incidence declined from 81,258 (95% CI: 25,437-273,640) cases and 29,520 (95% CI: 3,334-112,498) deaths in 2010, largely due to increases in vaccination coverage which have prevented an estimated 314,793 (95% CI: 94,566-1,049,645) cases and 114,946 (95% CI: 11,421-431,224) deaths over the past decade. India had the largest estimated JE burden in 2019, followed by Bangladesh and China. From 2010-2019, we estimate that vaccination had the largest absolute impact in China, with 204,734 (95% CI: 74,419-664,871) cases and 74,893 (95% CI: 8,989-286,239) deaths prevented, while Taiwan (91.2%) and Malaysia (80.1%) had the largest percent reductions in JE burden due to vaccination. Our estimates of the size of at-risk populations and current JE incidence highlight countries where increasing vaccination coverage could have the largest impact on reducing their JE burden.
Vaccination against JE ideally should be practiced in all areas of Asia where the virus is responsible for human disease. The WHO has placed a high priority on the development of a new vaccine for prevention of JE. Some countries in Asia (Japan, South Korea, North Korea, Taiwan, Vietnam, Thailand, and the PRC) manufacture JE vaccines and practice childhood immunization, while other countries suffering endemic or epidemic disease (India, Nepal, Laos, Cambodia, Bangladesh, Myanmar, Malaysia, Indonesia and the Philippines) have no JE vaccine manufacturing or policy for use. With the exception of the PRC, all countries practicing JE vaccination use formalin inactivated mouse brain vaccines, which are relatively expensive and are associated with rare but clinically significant allergic and neurological adverse events. New inactivated JE vaccines manufactured in Vero cells are in advanced preclinical or early clinical development in Japan, South Korea, Taiwan, and the PRC. An empirically derived, live attenuated vaccine (SA14-14-2) is widely used in the PRC. Trials in the PRC have shown SA14-14-2 to be safe and effective when administered in a two-dose regimen, but regulatory concerns over manufacturing and control have restricted international distribution. The genetic basis of attenuation of SA14-14-2 has been partially defined. A new live attenuated vaccine (ChimeriVax-JE) that uses a reliable flavivirus vaccine--yellow fever 17D--as a live vector for the envelope genes of SA14-14-2 virus is in early clinical trials and appears to be well tolerated and immunogenic after a single dose. Vaccinia and avipox vectored vaccines have also been tested clinically, but are no longer being pursued due to restricted effectiveness mediated by anti-vector immunity. Other approaches to JE vaccines--including naked DNA, oral vaccination, and recombinant subunit vaccines--have been reviewed.
The Sarawak strain of Japanese encephalitis virus (JE-Sar) is virulent in 3-week-old mice when inoculated intraperitoneally. The nucleotide sequence for the envelope glycoprotein (E) of this virus was determined and compared with the published sequences of four other strains. There were several silent nucleotide differences and five codon changes. Monoclonal antibodies (MAbs) against the E protein of JE-Sar virus were prepared and characterized. MAb-resistant mutants of JE-Sar were selected to determine if mutations in the E protein gene could affect its virulence for mice. Eight mutants were isolated using five different MAbs that identified virus-specific or group-reactive epitopes on the E protein. The mutants lost either complete or partial reactivity with selecting MAb. Several showed decreased virulence in 3-week-old mice after intraperitoneal inoculation. Two (r27 and r30) also showed reduced virulence in 2-week-old mice. JE-Sar and the derived mutants were comparable in their virulence for mice, when inoculated intracranially. Mutant r30 but not r27 induced protective immunity in adult mice against intracranial challenge with parent virus. However, r27-2 did induce protective immunity against itself. Nucleotide sequencing of the E coding region for the mutants revealed single base changes in both r30 and r27 resulting in a predicted change from isoleucine to serine at position 270 in r30 and from glycine to aspartic acid at position 333 in r27. The altered capacity of the mutants to induce protective immunity is consistent with the immunogenicity changes predicted by computer analysis using the Protean II program.
Five genotypes (GI-V) of Japanese encephalitis virus (JEV) have been identified, all of which have distinct geographical distributions and epidemiologies. It is thought that JEV originated in the Indonesia-Malaysia region from an ancestral virus. From that ancestral virus GV diverged, followed by GIV, GIII, GII, and GI. Genotype IV appears to be confined to the Indonesia-Malaysia region, as GIV has been isolated in Indonesia from mosquitoes only, while GV has been isolated on three occasions only from a human in Malaysia and mosquitoes in China and South Korea. In contrast, GI-III viruses have been isolated throughout Asia and Australasia from a variety of hosts. Prior to this study only 13 JEV isolates collected from the Indonesian archipelago had been studied genetically. Therefore the sequences of the envelope (E) gene of 24 additional Indonesian JEV isolates, collected throughout the archipelago between 1974 and 1987, were determined and a series of molecular adaptation analyses were performed. Phylogenetic analysis indicated that over a 14-year time span three genotypes of JEV circulated throughout Indonesia, and a statistically significant association between the year of virus collection and genotype was revealed: isolates collected between 1974 and 1980 belonged to GII, isolates collected between 1980 and 1981 belonged to GIV, and isolates collected in 1987 belonged to GIII. Interestingly, three of the GII Indonesian isolates grouped with an isolate that was collected during the JE outbreak that occurred in Australia in 1995, two of the GIII Indonesian isolates were closely related to a Japanese isolate collected 40 years previously, and two Javanese GIV isolates possessed six amino acid substitutions within the E protein when compared to a previously sequenced GIV isolate collected in Flores. Several amino acids within the E protein of the Indonesian isolates were found to be under directional evolution and/or co-evolution. Conceivably, the tropical climate of the Indonesia/Malaysia region, together with its plethora of distinct fauna and flora, may have driven the emergence and evolution of JEV. This is consistent with the extensive genetic diversity seen among the JEV isolates observed in this study, and further substantiates the hypothesis that JEV originated in the Indonesia-Malaysia region.
Japanese encephalitis virus (JEV) genotype V reemerged in Asia (China) in 2009 after a 57-year hiatus from the continent, thereby emphasizing a need to increase regional surveillance efforts. Genotypic characterization was performed on 19 JEV-positive mosquito pools (18 pools of Culex tritaeniorhynchus and 1 pool of Cx. bitaeniorhynchus) from a total of 64 positive pools collected from geographically different locations throughout the Republic of Korea (ROK) during 2008 and 2010.
Duck Tembusu virus (DTMUV), a newly identified flavivirus, has rapidly spread to China, Malaysia and Thailand. The potential threats to public health have been well-highlighted; however its virulence and pathogenesis remain largely unknown. Here, by using reverse genetics, a recombinant chimeric DTMUV based on Japanese encephalitis live vaccine strain SA14-14-2 was obtained by substituting the corresponding prM and E genes (named ChinDTMUV). In vitro characterization demonstrated that ChinDTMUV replicated efficiently in mammalian cells with small-plaque phenotype in comparison with its parental viruses. Mouse tests showed ChinDTMUV exhibited avirulent phenotype in terms of neuroinvasiveness, while it retained neurovirulence from its parental virus DTMUV. Furthermore, immunization with ChinDTMUV was evidenced to elicit robust IgG and neutralizing antibody responses in mice. Overall, we successfully developed a viable chimeric DTMUV, and these results provide a useful platform for further investigation of the pathogenesis of DTMUV and development of a live attenuated DTMUV vaccine candidate.