• Journal of Microbiology and Biotechnology
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• v.32 no.9
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• pp.1120-1125
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• 2022

Japanese encephalitis virus (JEV), the causative agent of Japanese encephalitis (JE), is an importantly zoonotic, vector-borne virus widely prevalent in Asia. Although JE has been well controlled in China, its prevalence remains a huge threat to the pig industry as well as human health. Herein, we report on our molecular and serological investigations of JEV among pigs from different regions in Hunan Province of China from 2019 to 2021. Collectively, 19.27% (583/3026, 95% Confidential Interval (CI) 17.86-20.68) of sampled pigs were positive for JEV IgG antibody as revealed by indirect enzyme-linked immunosorbent assay, and the seroprevalence of JEV among pigs was significantly associated with the development stage and breeding scale (p < 0.01). Meanwhile, 10.99% (42/382, 95% CI 7.86-14.13) of tissue samples of pigs with suspected clinical symptoms of JE and 23.44% (15/64, 95% CI 13.06-33.82) of mosquito batches were JEV-positive via reverse polymerase chain reaction. In addition, the complete E gene sequences of 14 JEV strains identified in this study were amplified and sequenced. Phylogenetic analysis showed that all 14 JEV strains belonged to genotype I-b and displayed a distinct genetic relationship to the present JEV vaccine strain (SA14-14-2). In conclusion, our results revealed not only the severe prevalence of JEV in Hunan Province, but also that JEV I-b might be the predominant genotype in Hunan Province, suggesting therefore that effective measures for JE control are urgently needed.

• Proceedings of the Microbiological Society of Korea Conference
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• 2000.05a
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• pp.23-36
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• 2000

Japanese encephalitis virus (JEV) is the causative agent of a mosquito-borne encephalitis and is transmitted to human via persistently infected mosquito vectors. Although the virus is known to cause only acute infection, there were reports that showed neurological sequelae, latent infection in peripheral mononuclear cells, and recurrence of the disease after acute encephalitis. Innate resistance of certain cell lines, abnormal SN1 expression of the virus, and anti-apoptotic effect of cullular bcl-2 have been suggested as probable causes of JEV persistence even in the absence of defective interfering (DI) particles. Although possible involvement of DI particles in JEV persistence was suggested, neither has a direct evidence for DI presence nor its molecular characterization been made. Two questions asked in this study are whether the DI virus plays any role in JEV persistent infection if it is associated with and what type of change(s) can be made in persistently infected cells to avoid apoptosis even with the continuous virus replication, DI-free standard stock of JEV was infected in BHK-21, Vero, and SW13 cells and serial high multiplicity passages were performed in order to generate DI particles. There different-sized DI RNA species which were defective in both structural and nonstructural protein coding genes. Rescued ORFs of the DI genome maintained in-frame and the presence of replicative intermediate or replicative form RNA of the DI particles confirmed their replication competence. On the other hand, several clones with JEV persistent infection were established from the cells survived acute infections during the passages. Timing of the DI virus generation during the passages seemed coincide to the appearance of persistently infected cells. The DI RNAs were identified in most of persistently infected cells and were observed throughout the cell maintenance. One of the cloned cell line maintained the viral persistence without DI RNA coreplication. The cells with viral persistence released the reduced but continuous infectious JEV particle for up to 9 months and were refractory to homologous virus superinfection but not to heterologous challenges. Unlike the cells with acute infection these cells were devoid of characteristic DNA fragmentation and JEV-induced apoptosis with or without homologous superinfection. Therefore, the DI RNA generated during JEV undiluted serial passage on mammalian cells was shown to be biologically active and it seemed to be responsible, at least in part, for the establishment and maintenance of the JEV persistence in mammalian cells. Viral persistence without DI RNA coreplication, as in one of the cell clones, supports that JEV persistent infection could be maintained with or without the presence of DI particles. In addition, the fact that the cells with JEV persistence were resistant against homologous virus superinfection, but not against heterologous one, suggests that different viruses have their own and independent pathway for cytopathogenesis even if viral cytopathic effect could be converged to an apoptosis after all.

• Korean Journal of Veterinary Service
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• v.35 no.3
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• pp.169-174
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• 2012

Japanese encephalitis virus (JEV) is one of the main causes of viral encephalitis in human and animals. For over 30 years, a live attenuated JEV vaccine strain has been used in the veterinary field, and it is required to conduct quality evaluation studies on the commercial vaccines. For the quality control of live attenuated JEV vaccine, we investigated the nucleotide sequence similarity of prME gene derived from five JEV vaccines commercially available in pigs in Korea. The Vero cells infected with JEV vaccines showed specific cytopathic effect, which was characterized by rounding and detached cells. In the phylogenetic analysis, all of the vaccine strains showed a close relationship with the original vaccine seed strain (Anyang 300) and clustered into the genotype 3. In comparison of the nucleotide and deduced amino acid sequences of prME genes with the original strain, all JEV vaccine strains showed high amino acid similarity ranging from 98.9% to 99.5%, but had several point mutations, probably due to high mutation rates of viral RNA polymerase by several virus passages. Even though the current JEV vaccine strains have been maintained and produced for a long period of time, the genetic characterization of them have been rarely changed. However, since the mid 1990's, molecular epidemiology of JEV has been changed sharply from genotype 3 to genotype 1 in Korea, further studies on new vaccine strains to genotype 1 is required for more effective prevention in the field.

• Korean Journal of Veterinary Research
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• v.62 no.3
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• pp.19.1-19.6
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• 2022

Japanese encephalitis virus (JEV) and Akabane virus (AKAV) are mosquito-borne viruses that cause encephalitis and reproductive disorders in horses and cattle, respectively. There is no treatment for JEV or AKAV infections in animals. Therefore, we evaluated the antiviral activity of 18-mer amphipathic peptides in the 1b-4/21-C series on JEV and AKAV using Vero cells in vitro and evaluated their effects on JEV in mice. Of 6 peptides, 1b-4/21-C12 had the lowest IC₅₀ of 0.313 against JEV and its use as an antiviral against JEV and AKAV was examined. The IC₅₀ of 1b-4/21-C12 against JEV and AKAV was 0.78 and 1.14 µM, respectively. Mice treated with 5 or 2 mg/kg of 1b-4/21-C12 had 32% and 16% survival rates, respectively, and the surviving mice treated with 1b-4/21-C12 began to gain weight beginning 8 days post challenge with the virulent Nakayama strain. Moreover, 20 µM 1b-4/21-C peptide had no cytotoxic effects on Vero cells. Our in vitro and in vivo results indicate that 1b-4/21-C12 has antiviral activity against enveloped JEV and AKAV and might be useful as a therapeutic substance.

• Proceedings of the Microbiological Society of Korea Conference
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• 2003.05a
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• pp.83-86
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• 2003

Major advances in positive-sense RNA virus research have been facilitated by the development of reverse genetics systems. These systems consist of an infectious cDNA clone that encompasses the genome of the virus in question. This clone is then used as a template for the subsequent synthesis of infectious RNA for the generation of synthetic viruses. However, the construction of infectious cDNA for the Japanese encephalitis virus (JEV) has been repeatedly thwarted by the instability of its cDNA. As JEV is an important human pathogen that causes permanent neuropsychiatric sequelae and even fatal disease, a reliable reverse genetics system for this virus is highly desirable. The availability of this tool would greatly and the development of effective vaccines as well as facilitate studies into the basic biology of the virus, including the molecular mechanisms of viral replication, neurovirulence, and pathogenesis. We have successfully constructed a genetically stable infectious JEV cDNA containing full-length viral RNA genome. Synthetic RNA transcripts generated in vitro from the cDNA were highly infectious upon transfection into susceptible cells, and the cDNA remained stable after it had been propagated in E. coli for 180 generations. Using this infectious JEV cDNA, we have successfully expressed a variety of reporter genes from the full-length genomic and various subgenomic RNAs in vitro transcribed from functional JEV cDNAS. In summary, we have developed a reverse genetics system for JEV that will greatly facilitate the research on this virus in a variety of different fields. It will also be useful as a heterologous gene expression vector and aid the development of a vaccine against JEV.

• Molecules and Cells
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• v.21 no.1
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• pp.112-120
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• 2006

It has been suggested that defective interfering (DI) RNA contributes to the persistence of Japanese encephalitis virus (JEV). In this study, we characterized molecular and biological aspects of the DI RNA and its relation to viral persistence. We identified a homologous DI virus intimately associated with JEV persistence in Vero cells. The production of DI RNA during undiluted serial passages of JEV coincided with the appearance of cells refractory to acute infection with JEV. We also established a Vero cell clone with a persistent JEV infection in which the DI RNA coreplicated efficiently at the expense of helper virus. The infectious virus yield of the clone fluctuated during its growth depending upon the amount of DI RNA accumulated in the previous replication cycle. Identification of the corresponding negative-sense RNA of the DI RNA indicated that the DI RNA functioned as a replication unit. Most of the DI RNA molecules retained their open reading frames despite a large deletion, encompassing most of the prM, the entire E, and the 5' half of the NS1 gene. Taken together, these observations suggest that the generation of homologous DI RNA during successive JEV acute infections in Vero cells probably participates actively in persistent JEV infection.

• Korean Journal of Veterinary Research
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• v.64 no.3
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• pp.26.1-26.9
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• 2024

Japanese encephalitis virus (JEV) is a mosquito-borne virus that can infect pigs, horses, and other mammals, including humans. Sero-epidemiological investigations of JEV have been performed using hemagglutination inhibition (HI), virus neutralization (VN) tests and enzyme-linked immunosorbent assay (ELISA). A need exists for a new ELISA that can detect JEV antibodies in the sera of several animal species. We aimed to develop a blocking ELISA (B-ELISA) for detecting JEV antibodies in pig and horse serum samples. JEV antibodies in 218 pig and 315 horse serum samples were measured using HI and VN tests. The purified KV1899-306 strain was used as an antigen for B-ELISA. The purified antibody (7A13) was conjugated with horseradish peroxidase and used as a detector antibody. The sera of pigs and horses to measure antibody against JEV were subjected to B-ELISA and analyzed. The B-ELISA had a diagnostic sensitivity of 94.6% to 100%, a specificity of 91.2 to 100%, and an accuracy of 94.9 to 98.6% compared with those of the HI and VN tests in pig and horse sera. The B-ELISA had a higher correlation with pig sera (r = 0.89 and 0.90 for VN and HI) than with horse sera (r = 0.75 and to 0.79). The new B-ELISA could be useful in the sero-surveillance of JEV in pig and horse sera and replace indirect ELISA.

• Korean Journal of Microbiology
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• v.38 no.3
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• pp.221-229
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• 2002

Japanese encephalitis virus (JEV) persistence was established and maintained in Vero cell culture for over 1 year. Eleven clones of plaque morphology mutant JEV, with large and small plaque sizes, were obtained from the cell culture supernatant. Genomic RNA replication efficiency of the mutants in naive Vero cell appeared to correspond to their different plaque sizes. No significant changes in envelop protein ORF or in non-coding regions at both ends of the RNA genome suggested that there could be an unidentified factor(s) playing role in JEV attenuation. Unlike to the replication of wild-type JEV, the mutants did not induce severe degree of cytopathic effect in Vero cells upon infection. While obvious decrease of Bcl-2 and its mRNA expression and sharp increase of p53 in naive Vero cells infected with either wild-type JEV or the large plaque-forming mutant, those changes were not observed with the small plaque-forming one. Together with these observation, internucleosomal DNA fragmentation and chromosomal DNA profile in the Vero cells infected with the mutants suggest that an overall changes in cytopathic effect in the plaque morphology mutants-infected cells should be primarily due to the reduced genomic RNA replication and the compromised degree of p53-independent apoptosis by the virus infection at least in part.

• KSBB Journal
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• v.19 no.1
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• pp.88-92
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• 2004

The immunogenicity of investigational combined vaccine, composed of the Japanese encephalis virus(JEV) and the polysaccharide capsule(Vi) of Salmonella typhi covalently bound to tetanus toxoid(TT) was evaluated in mice. The mice immunized with combined vaccine elicited higher anti-Vi Immunoglobulin G(IgG) as well as anti-JEV IgG levels than the mice immunized with Vi-TT or JEV alone. The combined vaccine produced four-fold increase in anti-Vi IgG level than Vi-TT alone. In JEV the combined vaccine was significantly more immunogenic than JEV alone and induced six-fold increase in IgG level. Adsorption of combined vaccine onto aluminium hydroxide gel also enhanced IgG level for both Vi and JEV.

• IMMUNE NETWORK
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• v.7 no.2
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• pp.66-74
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• 2007

Background: The genus Flavivirus consists of many emerging arboviruses, including Dengue virus (DV), Japanese encephalitis virus (JEV) and West Nile virus (WNV). Effective preventive vaccines remain elusive for these diseases. Mice are being increasingly used as the animal model for vaccine studies. However, the pathogenic mechanisms of these viruses are not clearly understood. Here, we investigated the interaction of DV and JEV with murine bone marrow-derived dendritic cells (bmDC). Methods: ELISA and FACS analysis were employed to investigate cytokine production and phenotypic changes of DCs obtained from bone marrow following flavivirus infection. Results: We observed that these viruses altered the cytokine profile and phenotypic markers. Although both viruses belong to the same family, JEV-infected bmDC produced anti-inflammatory cytokine (IL-10) along with pro-inflammatory cytokines, whereas DV infection induced production of large amounts of pro-inflammatory cytokines (IL-6 and TNF-${\alpha}$) and no IL-10 from murine bmDCs. Both flaviviruses also up-regulated the expression of co-stimulatory molecules such as CD40, CD80 and CD86. JEV infection led to down-regulation of MHC II expression on infected bmDCs. We also found that cytokine production induced by JEV and DV is MyD88-dependent. This dependence was complete for DV, as cytokine production was completely abolished in the absence of MyD88. With regard to JEV, the absence of MyD88 led to a partial reduction in cytokine levels. Conclusion: Here, we demonstrate that MyD88 plays an important role in the pathogenesis of flaviviruses. Our study provides insight into the pathogenesis of JEV and DV in the murine model.