Japanese encephalitis virus in India: An update on virus genotypes

Japanese encephalitis (JE) is a leading cause of viral encephalitis in Southeast Asia. It is a serious public health issue in India, and cases have been emerging in newer areas of the country. Although vaccination efforts have already been initiated in the country since 2006 and later through the Universal Immunization Programme in 2011, still a significant reduction in the number of cases has to be achieved since an escalating trend of JE incidence has been reported in certain States such as Assam, Uttar Pradesh and West Bengal. Moreover, fresh cases of JE have been reported from certain pockets in Odisha as well. Despite the mass JE vaccination programme implemented in prioritized endemic zones in the country in 2011, a shift in the age group of JE virus (JEV) infection was noticed affecting the adult population in West Bengal. The recent detection of the circulation of genotype I (GI) in Gorakhpur, Uttar Pradesh and the co-circulation of GI and genotype III (GIII) in West Bengal are probably a warning signal for the public health personnel to strengthen the surveillance system in all endemic hotspots in the country. The abrupt emergence of JEV genotype V (GV) in China and Korea in 2009, after its first detection in Malaya in 1952, endemic countries have been cautioned to strengthen their surveillance, because GV has been suspected of getting dispersed efficiently in other parts of Asia. Moreover, the reduced protection efficiency of the JEV GIII-based vaccine against the JEV genotype V further warrants careful evaluation of the ongoing vaccination strategies in the endemic countries, anticipating the possible incursion of GV and its impact on future control strategies. In view of the above facts, the present communication reviews the current knowledge on the molecular epidemiology of JEV in India vis-a-vis the global scenario and discusses the future priorities in JEV research in India for effectively designing control strategies.

First report on the detection of Japanese encephalitis virus in fruit bats from India

Japanese encephalitis (JE) is a mosquito borne viral zoonotic disease and JE virus (JEV) is responsible for causing several children deaths every year in India. Since 1978, cases of JE have been reported from Gorakhpur district of Uttar Pradesh state annually. The knowledge on the role played by wildlife reservoirs in the sylvatic transmission and maintenance of JE virus remains limited. Bats are reservoir hosts for several emerging and re-emerging viral pathogens but their role in zoonotic cycle of JEV has not been elucidated yet. In Gorakhpur district of Uttar Pradesh, 52 fruit bats were found dead on 26 May 2020. The post-mortem report of the bat samples conducted at the Indian Veterinary Research Institute stated that the bats died due to brain hemorrhage, caused by excessive heat. The brain tissue samples of the bats were subjected to investigation using molecular techniques to determine the presence of JEV. The present work reports for the first time the detection of JEV in brain samples of bats from India. The viral load ranging from 8 to 18 copies/reaction was detected in brain samples by TaqMan real Time RT-PCR. The low viral load might be the reason for the absence of apparent clinical signs in bats and suggests the probable role of fruit bats in maintaining the JEV in nature.

Alleviating Japanese encephalitis virus infection with multiple receptor tyrosine kinase inhibitor sunitinib / 中国热带医学

@#Abstract: Objective　To evaluate the effects of sunitinib on Japanese encephalitis virus (JEV) infection in vitro and vivo. Methods　The 4-week-old BALB/c mice infected with JEV by intraperitoneal injection. The infected mice were treated with sunitinib for 5 days and 10 days respectively. After that, the change of weight and survival rate were evaluated continuously. The viral load variation in mice brain were detected by qRT-PCR. Indirect immunohistochemical staining assay (IFA) was used to detect the number and distribution of CD4+/CD8+T cells in mouse brain. IFA was also used to observe the expression of virus E protein in the brain of mice. Vero cells were infected with JEV in vitro and given a certain concentration of sunitinib to observe the cell survival status. The expression of virus E protein in cells was detected by IFA. Results　Continuous administration of sunitinib significantly improved the survival rate of infected mice. Survival rate and body weight changes showed that the 5-day's administration strategy was significantly better than the 10-day's administration strategy. The treatment of sunitinib decreased the infiltration of CD4+/CD8+T cells in the brain and reduced the changes of vascular sleeve. However, the variation of viral load and E protein expression in brain were not obvious. The cytopathic effect (CPE) of infected Vero cells were slightly relieved after giving sunitinib, and the expression of E protein was also slightly changed. Conclusion　Sunitinib can significantly reduce the mortality of infected mice, and the 5-day's administration strategy is significantly better than the 10-day's administration strategy. Sunitinib decrease T lymphocyte infiltration in brain of mice, relieve the encephalitis symptoms ,and prolonged the life of mice.

Atorvastatin ameliorates viral burden and neural stem/ progenitor cell (NSPC) death in an experimental model of Japanese encephalitis

Japanese encephalitis virus, a neurotropic flavivirus, causes sporadic encephalitis with nearly 25% fatal casereports. JEV infects neural stem/progenitor cells (NSPCs) and decreases their proliferation. Statin, a commonlyused class of cholesterol lowering drug, has been shown to possess potent anti-inflammatory and neuroprotective effects in acute brain injury and chronic neurodegenerative conditions. Here, we aimed to check theefficacy of atorvastatin in alleviating the symptoms of Japanese encephalitis (JE). Using BALB/c mouse modelof JEV infection, we observed that atorvastatin effectively reduces viral load in the subventricular zone (SVZ)of infected pups and decreases the resultant cell death. Furthermore, atorvastatin abrogates microglial activation and production of proinflammatory cyto/chemokine production post JEV infection in vivo. It alsoreduced interferon-b response in the neurogenic environs. The neuroprotective role of atorvastatin is againevident from the rescued neurosphere size and decreased cell death in vitro. It has also been observed that uponatorvastatin administration, cell cycle regulatory proteins and cell survival proteins are also restored to theirrespective expression level as observed in uninfected animals. Thus the antiviral, immunomodulatory andneuroprotective roles of atorvastatin reflect in our experimental observations. Therefore, this drug broadens apath for future therapeutic measures against JEV infection.

Japanese encephalitis virus: Biological clones from a clinical isolate quasispecies show differing neurovirulence in vitro and in a mouse model

@#The Japanese encephalitis virus (JEV), a leading cause of encephalitis, exists as quasispecies in clinical isolates. Using a limiting dilution method combined with immunohistochemistry to detect viral antigens, 10 biological clones were isolated and purified from a clinical JEV isolate (CNS138/9) derived from an autopsy brain. These biological clones were tested for neurovirulence in SK-N-MC and NIE-115 neuronal cells, and a 2-week-old, footpad-infected, JE mouse model. Nine clones were found to be neurovirulent; one clone neuroattenuated. Although further studies are needed to determine genotypic differences, if any, in these clones, the limiting dilution purification and neurovirulence testing methods described herein should be useful for phenotypic studies of quasispecies of neurotropic viruses in general, and JEV and other flaviviruses in particular.

The progress and challenge of Japanese encephalitis control and prevention in China / 中华预防医学杂志

Japanese encephalitis (JE) is the important viral encephalitis in China. In the 1940s, JE was confirmed to be epidemic in China. In 1971, the annual incidence rate was 20.92/100 000. Since 2008, JE vaccine was included in the national Expanded Program of Immunization (EPI). In 2013, the incidence of Japanese encephalitis decreased to 0.16/100 000. JE virus is divided into five genotypes, and genotype 1, 3 and 5 JE virus was isolated in China. Genotype 1 JE virus was the mainly genotype currently circulated in China. In recent years, the characteristics of the population of JE have been changed to adult, especially in the northern provinces of China. JE prevention and control faces new challenges.

Co-infection of Dirofilaria immitis and Japanese encephalitis virus in a spotted seal (Phoca largha) in the Republic of Korea

A 10-year-old male spotted seal presented with loss of appetite and decreased activity. Grossly, the internal organs revealed several filarial nematodes in the right ventricle of the heart and the pulmonary vessels. Histopathological examination of the brain revealed moderate nonsuppurative meningoencephalitis with glial nodules and neuronophagia. Japanese encephalitis virus (JEV) of genotype I was isolated from the brain. All nematodes were identified as Dirofilaria immitis. This is the first clinical case of co-infection with D. immitis and JEV in a seal, suggesting that the seal, may be a dead-end host, like the human and horse, for JEV.

Genotype I of Japanese encephalitis virus as the dominant genotype among mosquitoes in Shandong / 中华实验和临床病毒学杂志

Objective@#To analysis the genotype of Japanese encephalitis virus (JEV) in mosquitoes from Shandong province.@*Methods@#Mosquitoes were collected between August and September in Weishan county, Junan county, and Kenli county of Shandong province in 2016. Viruses were isolated by BHK-21 cell and identified by molecular method . Real-Time RT-PCR was conducted to detect the Japanese encephalitis virus carried by the mosquitoes.@*Results@#A total of 8418 mosquitoes divided into 81 pools including 3 species, Culex tritaeniorhynchus, Anopheles sinensis and Armigeres obturbans. Eight Japanese encephalitis viruses were isolated; 23 pools were positive by JEV specific real-time RT-PCR. Phylogenetic analysis on E sequence of JEV showed all JEV strains belonged to genotype Ⅰ JEV, and new strains that were homogenous with previous JEV strains isolated from Shandong.@*Conclusions@#Genotype Ⅰ JEV was the dominant genotype in Shandong province.

Pig IFITMs are restriction factors for Japanese encephalitis virus

Objective:Japanese encephalitis virus (JEV) is responsible for one of the most serious epidemics of encephalitis in the world. JEV uses pigs as its main hosts and spreads among vertebrates and humans mediated by Culex mosquitoes. The prevention and control of JEV spread in pigs is one of the most effective measures to protect global public health. Interferon-inducible transmembrane proteins (IFITMs) are small membrane-spanning proteins that were identified as innate antiviral factors against multiple pathogenic viruses, especially enveloped viral pathogens. This study aims to verify whether pig interferon-inducible transmembrane proteins (pIFITMs) inhibit JEV and investigate the related molecular mechanisms of anti-JEV.Methods:Transient expression and RNA interference technology were used to overexpress and silence IFITMs gene. Three different cell lines, PK15, HEK293 and Huh7, were transfected with recombinant pIFITMs-expressing plasmids. The lentiviral vectors harboring RNAi sequences targeting pIFITMs were introduced into PK15 cells. Quantitative real-time PCR was used to determine the antiviral activities of pIFITMs through measuring and analyzing the virus copy number of JEV (SA14-14-2 strain) in the supernant of pIFITMs overexpression or silencing cells 48 hours post transfection. The expression of the related proteins was examined by western blot. The fusion vectors inserted with pig IFITM1-EGFP were constructed and introduced into three different cell lines respectively. Then Laser Co-focus light microscopy was used to observe the subcellular localization. The key active amino acids of pIFITM1 were analyzed by investigating the anti-JEV effect of the cysteine mutants produced with PCR site directed mutagenesistechnology.Results:In three different cell lines, PK15, HEK293 and Huh7, all of three pig IFITM proteins, pIFITM1, pIFITM2, and pIFITM3 could inhibit the replication of JEV whether through transient gene over-expression methods or RNA interference silencing. And that, among three pig IFITMs, pIFITM1 showed the strongest anti-JEV effect. The anti-JEV activity of pIFITM1 manifested at the early entry stage. In PK15, BHK21, and HEK293 cells, before virus infection, pIFITM1 was located in the plasma membrane area, and after infection, transferred to the membranous structures outside the nucleus. The S-palmitoylated cysteines at position 50, 51 and 84 of pIFITM1 had significant effect on virus replication.Conclusions:Pig interferon-inducible transmembrane proteins are restriction factors for JEV infection and have potentials in the prevention of virus spread. Our results provide some new sights into understanding the antiviral activity of pig IFITMs.

Mosquitoes, midges and related arboviruses in southeast Sichuan province / 中华流行病学杂志

Objective To investigate the distribution patterns of mosquitoes,midges and related arboviruses in Sichuan province.Methods Blood-sucking insects were collected from houses and pens,using the ultraviolet lights.Mosquito samples were classified according to morphologic characteristics and then stored at liquid nitrogen.All samples were incubated with BHK-21 and C6/36 cells for virus isolation and then detected for their viral genes.Sequences of the virus were identified and analyzed by molecular biological software,such as BioEdit 7.0.5.3,MEGA 6.0.Results In total,17 019 mosquitoes from 3 genera and 4 species and 12 700 midges were collected from the southeast regions of Sichuan province in 2016 and 2017.Among them,79.4％ (13 519/17 019) belonged to Culex tritaeniorhynchus with 11.1％ (1 897/17 019) as Armigeres subalbatus,5.5％ (930/17 019) were Anopheles sinensis and 4.0％ (673/17 019) were Anopheles sinensis 3 virus strains that isolated from Culex tritaeniorhynchus were identified as type Ⅰ Japanese encephalitis virus.Seven pools of mosquitoes isolated from Hejiang county were identified Japanese encephalitis virus gene positive through PCR amplification.With 4 pool midges were detected positive for Akabane virus through PCR gene amplification while midges samples didn't have virus isolates.Conclusions Culex tritaeniorhynchus appeared the predominant species in the southeast regions of Sichuan.Japanese encephalitis virus transmitted by mosquitoes and Akabane virus by midges were prevalent in southeast Sichuan province.

Mosquitoes, midges and related arboviruses in southeast Sichuan province / 中华流行病学杂志

Objective To investigate the distribution patterns of mosquitoes,midges and related arboviruses in Sichuan province.Methods Blood-sucking insects were collected from houses and pens,using the ultraviolet lights.Mosquito samples were classified according to morphologic characteristics and then stored at liquid nitrogen.All samples were incubated with BHK-21 and C6/36 cells for virus isolation and then detected for their viral genes.Sequences of the virus were identified and analyzed by molecular biological software,such as BioEdit 7.0.5.3,MEGA 6.0.Results In total,17 019 mosquitoes from 3 genera and 4 species and 12 700 midges were collected from the southeast regions of Sichuan province in 2016 and 2017.Among them,79.4％ (13 519/17 019) belonged to Culex tritaeniorhynchus with 11.1％ (1 897/17 019) as Armigeres subalbatus,5.5％ (930/17 019) were Anopheles sinensis and 4.0％ (673/17 019) were Anopheles sinensis 3 virus strains that isolated from Culex tritaeniorhynchus were identified as type Ⅰ Japanese encephalitis virus.Seven pools of mosquitoes isolated from Hejiang county were identified Japanese encephalitis virus gene positive through PCR amplification.With 4 pool midges were detected positive for Akabane virus through PCR gene amplification while midges samples didn't have virus isolates.Conclusions Culex tritaeniorhynchus appeared the predominant species in the southeast regions of Sichuan.Japanese encephalitis virus transmitted by mosquitoes and Akabane virus by midges were prevalent in southeast Sichuan province.

Serological and molecular epidemiology of Japanese encephalitis virus infections in swine herds in China, 2006–2012

Japanese encephalitis virus (JEV) is a mosquito-borne, zoonotic flavivirus causing viral encephalitis in humans and reproductive disorder in swine. JEV is prevalent throughout China in human; however, spatiotemporal analysis of JEV in Chinese swine herds has not been reported previously. Herein, we present serological and molecular epidemiological results and estimates of prevalence of JEV infections among swine herds in various regions of China. The results suggest that JEV infections are widespread and genotype I and III strains co-exist in the same regions. Therefore, there is an urgent need to monitor JEV infection status among swine herds in China.

TaqMan Real-time RT-PCR Assay for Detecting and Differentiating Japanese Encephalitis Virus / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To detect Japanese encephalitis virus (JEV) rapidly and distinguish its genotypes, a TaqMan-based reverse transcriptase quantitative polymerase chain reaction (RT-PCR) detection system was developed.</p><p><b>METHODS</b>By aligning the full-length sequences of JEV (G1-G5), six sets of highly specific TaqMan real-time RT-PCR primers and probes were designed based on the highly conserved NS1, NS2, and M genes of JEV, which included one set for non-specific JEV detection and five sets for the detection of specific JEV genotypes. Twenty batches of mosquito samples were used to evaluate our quantitative PCR assay.</p><p><b>RESULTS</b>With the specific assay, no other flavivirus were detected. The lower limits of detection of the system were 1 pfu/mL for JEV titers and 100 RNA copies/µL. The coefficients of variation of this real-time RT-PCR were all < 2.8%. The amplification efficiency of this method was between 90% and 103%.</p><p><b>CONCLUSION</b>A TaqMan real-time RT-PCR detection system was successfully established to detect and differentiate all five JEV genotypes.</p>

Secreted expression of Japanese encephalitis virus prME in Pichia pastoris and immunogenicity evaluation of the virus-like particles in mice / 生物工程学报

The study was to express prME protein of Japanese encephalitis virus (JEV) in Pichia pastoris and then to evaluate the immunological properties of the recombinant protein in mice, so as to explore a new way for subunit vaccine development of JEV. The JEV prME gene was amplified by RT-PCR with genome RNA of JEV vaccine strain SA14-14-2 and subcloned into pPICZa-A vector, designated as pPICZα-prME. pPICZα-SprME was constructed same as pPICZα-prME besides with the additional 19 Aa signal peptides coding gene of the JEV cap protein C terminal. The linearized expression vector was integrated into the genome of Pichia pastoris X33 under the control of the alcohol oxidase (AOX1) promoter and induced with methanol during fermentation expression. The expression of JEV prME protein was identified by SDS-PAGE and Western blotting, and then it was purified by S-400 High Resolution HiPrep 16/60 Sephacry. The expressed products of Pichia pastoris were visualized by electron microscopy. In the immunization test, four groups of four-week old female mice were immunized subcutaneously with different doses purified JEV prME protein with complete Freund's adjuvant at a volumetric ratio of 1:1 and a control group was injected with sterile PBS. 10 μg/dose purified JEV prME protein mixing different doses nucleic acid adjuvant (Naa) was vaccinated in mice as the same mode. SDS-PAGE and Western blotting indicate that JEV prME was not cleaved between prM and E during secreted expression in Pichia pastoris. The purified recombinant prME was eluted in the first eluting peak which indicated that its molecular weight about 1×10⁶ Da to 20×10⁶ Da and may form a multimeric. Both the culture supernatant and the purified protein, examined by electron microscopy, we found to contain JEV virus like particles (VLPs) with diameters of 30-50 nm. The anti-JEV VLPs antibody titration reached peak at 3 wpi and still maintained in mice at 7 wpi inoculated with 10 μg and 15 μg prME. The strong antibody response was observed when the mice immunized with prME mixing nucleic acid adjuvant, which elicited high neutralizing antibody titer among 1:80 to 1:160. In conclusion, although JEV prME protein expressed in Pichia pastoris was not cleaved, which formed VLPs and showed efficient immunological properties in mice experiments.

Isolation and identification of Japanese encephalitis virus in the experimental minipigs / 中国比较医学杂志

Objective To understand the characteristics of minipigs infected withJapanese encephalitis virus(JEV).Methods After the brain tissues were treated, the pig brain tissue treatment solution was inoculated with BHK21 cells.Then, virus culture,indirect immunofluorescence assay, neutralization test, electron microscopic observation, and reverse transcription-polymerase chain reaction (RT-PCR) amplification of the new isolate E segment and PrM segment nucleotide sequence were performed and the genotype was identified.Results BHK21 cells were inoculated into 25 pigbrain tissues.Among them, three tissue-treated fluid couldinduce shrinkage and aggregation of BHK21 cells, and immunofluorescence staining showed strong green fluorescence response.The results of neutralization test showed that the neutralization titer of these three new isolates was 1:64, and the size of the virus particles was about 40nm under the electron microscope.The homology of both RT-PCR product sequencing results and E-segment of vaccine strain were 95%.Three new isolates were type GIII JEV.Conclusion The results ofthisstudydemonstrate that there is G III type Japanese encephalitis virus infection in the minipig farm.

Progress in Zika virus and its vaccines / 生物工程学报

Like Yellow fever virus, Dengue virus, Japanese encephalitis virus and West Nile virus, Zika virus is also a mosquito-borne flavivirus. Since it was isolated in 1947, there has been little concern over Zika virus due to its limited distribution and mild symptoms. In recent years, especially since 2015, Zika virus has become a global concern because of its outbreak in Brazil and associated microcephaly. Vaccines against Zika virus, regarded as the effective measures to control Zika fever epidemic, are being developed in nearly thirty institutions worldwide. In this paper, biology, epidemiology and clinical features of Zika virus were reviewed along with current research and development of different types of Zika vaccines. In addition, several other flavivirus vaccines approved or in clinical trials were briefly introduced, to provide valuable reference for Zika vaccines researchers.

Improvement of indirect enzyme-linked immunosorbent assay for detection of Japanese encephalitis virus antibodies in swine sera

Japanese encephalitis (JE) is an important zoonosis caused by the mosquito-transmitted JE virus (JEV), which is a causative agent of reproductive failure in pregnant sows. Detection of JEV antibodies in swine is performed by hemagglutination inhibition (HI), virus neutralization (VN), and the plaque reduction neutralization test (PRNT). The most stringent PRNT is the 90% endpoint PRNT (PRNT₉₀). These conventional assays are difficult to carry out in diagnostic laboratories with insufficient instruments or cell culture systems. An alternative assay that is easily conducted and time efficient is required. In this study, we improved the indirect enzyme-linked immunosorbent assay (I-ELISA) with clarified antigen for the detection of JEV antibodies. The I-ELISA results obtained from 175 swine serum samples were compared with HI, VN, and PRNT₉₀ results. The sensitivity of I-ELISA was 91.8%, 95.0%, and 94.7% compared with HI, VN, and PRNT₉₀ results, respectively. The specificity of I-ELISA was 92.2%, 94.7%, and 94.7% compared with HI, VN, and PRNT₉₀ results, respectively. Moreover, the I-ELISA results were significantly correlated with the HI (r = 0.93), VN (r = 0.95), and PRNT₉₀ (r = 0.92) results. These results suggest that the improved I-ELISA is useful for serosurveillance of JEV in swine.

Pharmacodynamics of aminoglycosides and tetracycline derivatives against Japanese encephalitis virus

Objective: To explore the antiviral activity of antibiotic compounds, mainly aminoglycosides and tetracyclines against Japanese encephalitis virus (JEV) induced infection in vitro. Methods: Antiviral activity were evaluated against JEV using cytopathic effect inhibition assay, virus yield reduction assay, caspase 3 level, extracellular viral detection by antigen capture ELISA and viral RNA levels. Results: JEV induced cytopathic effect along with reduction of viral progeny plaque formation indicated antiviral potential of the compounds suggesting that antibiotics had broad spectrum activity. Doxycycline and kanamycin administration in dose dependent manner declined viral RNA replication. Conclusions: The present study shows kanamycin and doxycycline can affect virion structure and alter replication causing inhibition of JEV induced pathogenesis in vitro.

Mechanism of Japanese encephalitis virus genotypes replacement based on human, porcine and mosquito-originated cell lines model

Objective: To examine the multiplication efficiency Japanese encephalitis virus (JEV) genotype I (GI) and genotype III (GIII) of different cell lines which originated from human, porcine, mosquitoes in order to prove mechanism of JEV GI replacement JEV GIII since it emerging in nature recent decades. Methods: The mixture of GI and GIII JEV isolates was inoculated on human rhabdomyosarcoma (RD), pig kidney epithelial (PS) and Aedes albopictus C6/36 clone (C6/36) which originated from human, porcine and mosquitoes, respectively. Plaque assays were performed to calculate virus titer and real-time RT-PCR with GI and GIII specific primer sets to quantify the number of GI and GIII RNA copies. Results: The highest virus titer reached at the 3rd day of post infection when GI and GIII mixture was inoculated on RD and PS and that of C6/36 was at the 4th day. JEVs were amplified and maintained by C6/36 cells after 10 passages whereas that by RD and PS only limited within 8 and 6 passages, respectively. GI strain amplified and maintained more efficiently on C6/36 and PS but not RD, whereas GIII strain amplified and maintained more efficiently on RD. Conclusions: There is a correlation between the multiplication efficiency of GI and GIII JEV strains when these two genotype strains co-infected on different cell lines with the predominance of GI strains in C6/36 and PS and the limited detection of GI strains in RD cells proving a possible mechanism of shift JEV genotypes in nature recent decades since GI emerging.

Establishment of a Multiplex RT-PCR for the Sensitive and Differential Detection of Japanese Encephalitis Virus Genotype 1 and 3

Japanese encephalitis (JE) is a zoonosis that affects the nervous system of humans and other animals. The genotype of JE virus (JEV) has shifted recently from genotype 3 (G3) to genotype 1 (G1) in Asia, including Korea. Thus, a rapid differential assay is required to make an accurate diagnosis of JEV genotype. In this study, we designed common and differential primer sets for JEV G1 and G3 to detect the JEV envelope (E) gene. The specific primer sets for JEV G1 and G3 specifically amplified the target gene. The detection limits of the three primer sets were 10(1.0), 10(2.0), and 10(2.0) TCID₅₀/reaction, respectively. No cross-reactivity was detected with non-JEV reference viruses. The multiplex reverse transcription-polymerase chain reaction (RT-PCR) assay specifically differentiated JEV G1 from G3. Thus, a one-step multiplex RT-PCR assay was established to rapidly and differentially detect JEV. This assay will be useful for confirming JEV infections in animals and checking the JEV genotype in veterinary biological products.