Japanese Encephalitis Virus Infection Research Articles

Japanese encephalitis virus inhibits superinfection of Zika virus in cells by the NS2B protein.

Superinfection exclusion (SIE) is a phenomenon in which a preexisting infection prevents a secondary infection. SIE has been described for several flaviviruses, such as West Nile virus vs Nhumirim virus and Dengue virus vs yellow fever virus. Zika virus (ZIKV) is an emerging flavivirus posing threats to human health. The SIE between ZIKV and Japanese encephalitis virus (JEV) is investigated in this study. Our results demonstrate for the first time that JEV inhibits ZIKV infection in both mammalian and mosquito cells, whether co-infects or subsequently infects after ZIKV. The exclusion effect happens at the stage of ZIKV RNA replication. Further studies show that the expression of JEV NS2B protein is sufficient to inhibit the replication of ZIKV, and the outer membrane region of NS2B (46-103 aa) is responsible for this SIE. JEV infection and NS2B expression also inhibit the infection of the vesicular stomatitis virus. In summary, our study characterized a SIE caused by JEV NS2B. This may have potential applications in the prevention and treatment of ZIKV or other RNA viruses.IMPORTANCEThe reemerged Zika virus (ZIKV) has caused severe symptoms in humans and poses a continuous threat to public health. New vaccines or antiviral agents need to be developed to cope with possible future pandemics. In this study, we found that infection of Japanese encephalitis virus (JEV) or expression of NS2B protein well inhibited the replication of ZIKV. It is worth noting that both the P3 strain and vaccine strain SA14-14-2 of JEV exhibited significant inhibitory effects on ZIKV. Additionally, the JEV NS2B protein also had an inhibitory effect on vesicular stomatitis virus infection, suggesting that it may be a broad-spectrum antiviral factor. These findings provide a new way of thinking about the prevention and treatment of ZIKV.

Short Communication: Mosquito Histone 2A Protein Facilitate Japanese Encephalitis Virus Infection in the Mosquito.

Japanese encephalitis virus is mainly prevalent in the tropical and subtropical regions of Asia and Oceania. Through immunoprecipitation-mass spectrometry analysis using monoclonal antibodies targeting JEV E protein, we found that mosquito Histone 2A protein could bind to JEV particles. The binding of H2A and JEV was detected in the salivary gland and supernatant of mosquito cells. Furthermore, RNA interference experiments in vitro and in vivo confirmed that H2A protein promotes JEV infection in mosquitoes. In summary, we found that mosquito H2A is a factor that supports JEV infection and can potentially facilitate cross-species transmission of JEV.

Marine Brown Algae-Derived Compounds as Potential Inhibitors of Japanese Encephalitis Virus RNA-Dependent RNA Polymerase.

The Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus that primarily affects people in Asia and seriously threatens public health. Considering the rising occurrence rates and lack of targeted antiviral treatments, it is essential to comprehend and tackle obstacles related to JEV in order to lessen its influence on world health. This investigation explores compounds derived from marine brown algae (Phaeophyceae) as potential inhibitors of JEV RNA-dependent RNA polymerase (RdRp), a critical enzyme in the virus's replication cycle. Employing the computational virtual screen approach, four compounds, i.e., CMNPD16749, CMNPD2606, CMNPD27817, and CMNPD23662, with favorable binding energies ranging from -15.7 Kcal/mol to -13.9 kcal/mol were identified. Subsequently, through molecular docking analysis, the interactions responsible for the binding stability between the target protein and hit molecules compared to the reference molecule Galidesvir were studied. Further, through extensive molecular dynamic (MD) simulation studies at 200 ns, it was confirmed that each docked complex showed acceptable dynamic stability compared to the reference molecule. These findings were further validated using MM/PBSA free binding energy calculations, PCA analysis and free energy landscape construction. These computational findings suggested that the brown algae-derived compounds may act as an antiviral drug against JEV infection and lay a crucial foundation for future experimental studies against JEV.

Regulation of Onecut2 by miR-9-5p in Japanese encephalitis virus infected neural stem/progenitor cells.

MicroRNAs have emerged as key disease pathogenic markers and potential therapeutic targets. In this study, we solidify this concept by studying a key miRNA, miR-9-5p, in Japanese encephalitis virus infection of neural stem/progenitor cells. miRNA target Onecut2 has a possible role in stem cell pool biology. Here, we show a possible mechanistic axis worth investing in neurotropic viral biology.

Development of Colloidal Gold-Based Immunochromatographic Strips for Rapid Detection and Surveillance of Japanese Encephalitis Virus in Dogs across Shanghai, China.

Japanese encephalitis virus (JEV) causes acute encephalitis in humans and is of major public health concern in most Asian regions. Dogs are suitable sentinels for assessing the risk of JEV infection in humans. A neutralization test (NT) or an enzyme-linked immunosorbent assay (ELISA) is used for the serological detection of JEV in dogs; however, these tests have several limitations, and, thus, a more convenient and reliable alternative test is needed. In this study, a colloidal gold immunochromatographic strip (ICS), using a purified recombinant EDIII protein, was established for the serological survey of JEV infection in dogs. The results show that the ICSs could specifically detect JEV antibodies within 10 min without cross-reactions with antibodies against other canine viruses. The test strips could detect anti-JEV in serum with dilution up to 640 times, showing high sensitivity. The coincidence rate with the NT test was higher than 96.6%. Among 586 serum samples from dogs in Shanghai examined using the ICS test, 179 (29.98%) were found to be positive for JEV antibodies, and the high seropositivity of JEV in dogs in China was significantly correlated with the season and living environment. In summary, we developed an accurate and economical ICS for the rapid detection of anti-JEV in dog serum samples with great potential for the surveillance of JEV in dogs.

CRISPR/Cas9-Mediated Knockout of the HuR Gene in U251 Cell Inhibits Japanese Encephalitis Virus Replication.

Human antigen R (HuR) is an RNA-binding protein that regulates the post-transcriptional reaction of its target mRNAs. HuR is a critical factor in cancer development and has been identified as a potential target in many cancer models. It participates in the viral life cycle by binding to viral RNAs. In prior work, we used CRISPR/Cas9 screening to identify HuR as a prospective host factor facilitating Japanese encephalitis virus (JEV) infection. The HuR gene was successfully knocked out in U251 cell lines using the CRISPR/Cas9 gene-editing system, with no significant difference in cell growth between U251-WT and U251-HuR-KO2 cells. Here, we experimentally demonstrate for the first time that the knockout of the HuR gene inhibits the replication ability of JEV in U251 cell lines. These results play an essential role in regulating the replication level of JEV and providing new insights into virus-host interactions and potential antiviral strategies. It also offers a platform for investigating the function of HuR in the life cycle of flaviviruses.

Incongruence between confirmed and suspected clinical cases of Japanese encephalitis virus infection.

Japanese encephalitis (JE) is a notifiable infectious disease in China. Information on every case of JE is reported to the superior health administration department. However, reported cases include both laboratory-confirmed and clinically diagnosed cases. This study aimed to differentiate between clinical and laboratory-confirmed cases of Japanese encephalitis virus (JEV) infection, and improve the accuracy of reported JE cases by analyzing the acute-phase serum and cerebrospinal fluid of all reported JE cases in the Sichuan province from 2012 to 2022. All acute-phase serum and/or cerebrospinal fluid samples of the reported JE cases were screened for IgM(ImmunoglobulinM)to JEV using the enzyme-linked immunosorbent assay (ELISA), and the detection of the viral genes of JEV and 9 other pathogens including enterovirus (EV), using reverse transcription PCR was attempted. Epidemiological analyses of JE and non-JE cases based on sex, age, onset time, and geographical distribution were also performed. From 2012 to 2022, 1558 JE cases were reported in the Sichuan province. The results of serological (JEV-specific IgM) and genetic testing for JEV showed that 81% (1262/1558) of the reported cases were confirmed as JEV infection cases (laboratory-confirmed cases). Among the 296 cases of non-JEV infection, 6 viruses were detected in the cerebrospinal fluid in 62 cases, including EV and the Epstein-Barr virus (EBV), constituting 21% (62/296) of all non-JE cases. Among the 62 non-JEV infection cases with confirmed pathogens, infections with EV and EBV included 17 cases each, herpes simplex virus (HSV-1/2) included 14 cases, varicella- zoster virus included 6 cases, mumps virus included 2 cases, and human herpes viruses-6 included 1 case. Additionally, there were five cases involving mixed infections (two cases of EV/EBV, one case of HSV-1/HSV-2, one case of EBV/HSV-1, and one case of EV/herpes viruses-6). The remaining 234 cases were classified as unknown viral encephalitis cases. Our analysis indicated that those aged 0-15 y were the majority of the patients among the 1558 reported JE cases. However, the incidence of laboratory-confirmed JE cases in the >40 y age group has increased in recent years. The temporal distribution of laboratory-confirmed cases of JE revealed that the majority of cases occurred from May to September each year, with the highest incidence in August. The results of this study indicate that there is a certain discrepancy between clinically diagnosed and laboratory-confirmed cases of JE. Each reported case should be based on laboratory detection results, which is of great importance in improving the accuracy of case diagnosis and reducing misreporting. Our results are not only important for addressing JE endemic to the Sichuan province, but also provide a valuable reference for the laboratory detection of various notifiable infectious diseases in China and other regions outside China.

Cellular nuclear-localized U2AF2 protein is hijacked by the flavivirus 3’UTR for viral replication complex formation and RNA synthesis

Japanese encephalitis virus (JEV) is a zoonotic pathogen belonging to the Flavivirus genus, causing viral encephalitis in humans and reproductive failure in swine. The 3’ untranslated region (3’UTR) of JEV contains highly conservative secondary structures required for viral translation, RNA synthesis, and pathogenicity. Identification of host factors interacting with JEV 3’UTR is crucial for elucidating the underlying mechanism of flavivirus replication and pathogenesis. In this study, U2 snRNP auxiliary factor 2 (U2AF2) was identified as a novel cellular protein that interacts with the JEV genomic 3’UTR (the SL-I, SL-II, SL-III, and DB region) via its 1 to 148 amino acids. JEV infection or JEV 3’ UTR on its own triggered the nuclear-localized U2AF2 redistributed to the cytoplasm and colocalized with viral replication complex. U2AF2 also interacts with JEV NS3 and NS5 protein, the downregulation of U2AF2 nearly abolished the formation of flavivirus replication vesicles. The production of JEV protein, RNA, and viral titers were all increased by U2AF2 overexpression and decreased by knockdown. U2AF2 also functioned as a pro-viral factor for Zika virus (ZIKV) and West Nile virus (WNV), but not for vesicular stomatitis virus (VSV). Mechanically, U2AF2 facilitated the synthesis of both positive- and negative-strand flavivirus RNA without affecting viral attachment, internalization or release process. Collectively, our work paves the way for developing U2AF2 as a potential flavivirus therapeutic target.

Methotrimeprazine is a neuroprotective antiviral in JEV infection via adaptive ER stress and autophagy

Japanese encephalitis virus (JEV) pathogenesis is driven by a combination of neuronal death and neuroinflammation. We tested 42 FDA-approved drugs that were shown to induce autophagy for antiviral effects. Four drugs were tested in the JE mouse model based on in vitro protective effects on neuronal cell death, inhibition of viral replication, and anti-inflammatory effects. The antipsychotic phenothiazines Methotrimeprazine (MTP) & Trifluoperazine showed a significant survival benefit with reduced virus titers in the brain, prevention of BBB breach, and inhibition of neuroinflammation. Both drugs were potent mTOR-independent autophagy flux inducers. MTP inhibited SERCA channel functioning, and induced an adaptive ER stress response in diverse cell types. Pharmacological rescue of ER stress blocked autophagy and antiviral effect. MTP did not alter translation of viral RNA, but exerted autophagy-dependent antiviral effect by inhibiting JEV replication complexes. Drug-induced autophagy resulted in reduced NLRP3 protein levels, and attenuation of inflammatory cytokine/chemokine release from infected microglial cells. Our study suggests that MTP exerts a combined antiviral and anti-inflammatory effect in JEV infection, and has therapeutic potential for JE treatment.

Seroconversion, genotyping, and potential mosquito vector identification of Japanese encephalitis virus in pig sentinel settings in Bali, Indonesia.

Despite the endemicity of Japanese encephalitis virus (JEV) in humans and animals in the Province of Bali, Indonesia, there is little data on whether seroconversion to the virus occurs in pigs, JEV genotypes circulating, and it's potential mosquito vectors in the area. The aims of this study were to (i) Determine whether JEV infection in Balinese pigs occurs before reaching their sexual maturity, (ii) identify the genotypes of circulating JEV, and (iii) identify potential JEV mosquito vectors at the study sites in urban and peri-urban areas of Bali. Sixteen 1-week-old Landrace piglets from two different sows were housed in Denpasar. Similarly, 18 one-week-old mixed-breed piglets of two different sows were housed in Badung Regency. The piglets were bled every 1 to 4 weeks for up to 24 weeks. Serum samples from the 11 piglets were tested for antibodies against JEV, and seroconversion-suspected sera were titrated using an enzyme-linked immunosorbent assay. Blood of seroconverted sera from pigs were tested using polymerase chain reaction (PCR) to detect the genetic sequence of JEV. The mosquitoes in the sentinels were trapped throughout the study period to identify the potential mosquito vectors of JEV. Antibodies were detected in most of the selected piglets' sera from weeks 1 to 24 of their age. However, sera of pig B9 collected from the sentinel setting in Badung Regency showed a four-fold increase in antibody titer from week 4 to week 8, indicating seroconversion. PCR testing of blood from B9 (pooled blood sample collected from week 5 to week 8) identified JEV nucleic acids, which were phylogenetically classified as belonging to the JEV genotype III. Meanwhile, 1271 of two genera of mosquitoes, Anopheles spp. and Culex spp. were trapped in the pig sentinels. JEV seroconversion likely occurs before the pig reaches sexual maturity in Badung Regency. Sequence data indicate that JEV genotype III is circulating in the pig sentinel setting in the regency; however, circulating genotypes need to be clarified through increased surveillance. Meanwhile, Culex spp. and most likely Culex quinquefasciatus and Anopheles spp. were the dominant mosquitoes present in the study sites set in the urban area of Denpasar and peri-urban areas of Badung, Bali, indicating that these are likely vectors in spread of JEV in the region.

Novel antiviral discoveries for Japanese encephalitis virus infections through reporter virus-based high-throughput screening.

Japanese encephalitis (JE) caused by JEvirus (JEV), remains a global public health concern. Currently, there is no specific antiviral drug approved for the treatment of JE. While vaccines are available for prevention, they may not cover all at-risk populations. This underscores the urgent need for prophylaxis and potent anti-JEV drugs. In this context, a high-content JEV reporter system expressing Nanoluciferase (Nluc) was developed and utilized for a high-throughput screening (HTS) of a commercial antiviral library to identify potential JEV drug candidates. Remarkably, this screening process led to the discovery of five drugs with outstanding antiviral activity. Further mechanism of action analysis revealed that cepharanthine, an old clinically approved drug, directly inhibited virus replication by blocking GTP binding to the JEV RNA-dependent RNA polymerase. Additionally, treatment with cepharanthine in mice models alleviated JEV infection. These findings warrant further investigation into the potential anti-JEV activity of cepharanthine as a new therapeutic approach for the treatment of JEV infection. The HTS method employed here proves to be an accurate and convenient approach that facilitates the rapid development of antiviral drugs.

Serological Investigation of Japanese Encephalitis Virus Infection in Commercially Reared Pigs, Southwestern Nigeria.

Japanese encephalitis virus (JEV) is a zoonotic arbovirus that causes abortion, stillbirth, and congenital defects in pigs, and epidemic encephalitis in humans. Currently, there is scarcity of information on JEV infection in pigs in Nigeria. Since the Culex tritaeniorhynchus vector of JEV is present in Nigeria and considering recent anecdotal reports of abortions and birth of weak piglets in some pig farms in southwestern Nigeria, there is a need for studies on the presence of the virus and its true burden among pig populations in the country. Serum samples (n=368) obtained from farm-reared pigs in four States of southwestern Nigeria were screened for JEV-specific IgG antibodies using a commercial ELISA kit. An overall JEV seropositivity of 35.1% (95% CI: 30.18 - 39.93%) was obtained, with detectable antibodies in pigs of all age groups, breeds, sex, and locations. Our results suggest natural exposure of these unvaccinated intensively reared pigs to JEV circulating silently in the swine population with significant association of the seropositivity with location (state/community in which the pig farms exist) and breed of the pigs studied. This first report of detection of anti-JEV antibodies in pigs in Nigeria indicates that JEV circulated among these pigs and underscores the need for active surveillance for JEV in humans, pigs, and mosquitoes to provide valuable epidemiological data for the design of effective control strategies against the virus, thus forestalling potential future outbreaks of the infection.

Attenuated Chimeric GI/GIII Vaccine Candidate against Japanese Encephalitis Virus.

Japanese encephalitis (JE) is a very severe disease characterized by high fatality rates and the development of permanent behavioral, psychiatric, and neurological sequelae among survivors. Japanese encephalitis virus (JEV), a flavivirus, is responsible for JE. In Asia, Genotype I (GI) has emerged as the dominant strain, replacing Genotype III (GIII). However, no clinically approved drug is available to treat JEV infection, and currently available commercial vaccines derived from JEV GIII strains provide only partial protection against GI. Utilizing a reverse genetics system, this study attempted to produce a novel chimeric JEV strain with high efficacy against JEV GI. Accordingly, a GI/GIII intertypic recombinant strain, namely SA14-GI env, was generated by substituting the E region of the GIII SA14-14-2 strain with that of the GI strain, K05GS. The neurovirulence of the mutant virus was significantly reduced in mice. Analysis of the immunogenicity of the chimeric virus revealed that it induced neutralizing antibodies against JEV GI in mice, and the protective efficacy of SA14-GI env was higher than that of SA14-14-2. These findings suggest that SA14-GI env may be a safe and effective live-attenuated vaccine candidate against JEV GI.

Porcine promyelocytic leukemia protein isoforms suppress Japanese encephalitis virus replication in PK15 cells

BackgroundPromyelocytic leukemia protein (PML) is a primary component of PML nuclear bodies (PML-NBs). PML and PML-NBs play critical roles in processes like the cell cycle, DNA damage repair, apoptosis, and the antiviral immune response. Previously, we identified five porcine PML alternative splicing variants and observed an increase in the expression of these PML isoforms following Japanese encephalitis virus (JEV) infection. In this study, we examined the functional roles of these PML isoforms in JEV infection.MethodsPML isoforms were either knocked down or overexpressed in PK15 cells, after which they were infected with JEV. Subsequently, we analyzed the gene expression of PML isoforms, JEV, and the interferon (IFN)-β signaling pathway using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blot. Viral titers were determined through 50% tissue culture infectious dose (TCID50) assays.ResultsOur results demonstrated that the knockdown of endogenous PML promoted JEV replication, while the overexpression of PML isoforms 1, 3, 4, and 5 (PML1, PML3, PML4, and PML5) inhibited JEV replication. Further investigation revealed that PML1, PML3, PML4, and PML5 negatively regulated the expression of genes involved in the interferon (IFN)-β signaling pathway by inhibiting IFN regulatory factor 3 (IRF3) post-JEV infection.ConclusionsThese findings demonstrate that porcine PML isoforms PML1, PML3, PML4, and PML5 negatively regulate IFN-β and suppress viral replication during JEV infection. The results of this study provide insight into the functional roles of porcine PML isoforms in JEV infection and the regulation of the innate immune response.

Inhibition of cellular activation induced by platelet factor 4 via the CXCR3 pathway ameliorates Japanese encephalitis and dengue viral infections

BackgroundActivated platelets secrete platelet factor 4 (PF4), which contributes to viral pathogenesis. Recently, we reported the proviral role of PF4 in replication of closely related flaviviruses, Japanese encephalitis virus (JEV) and dengue virus (DENV). ObjectivesThis study aimed to investigate the detailed mechanism of PF4-mediated virus replication. MethodsPF4−/− or wild-type (WT) mice were infected with JEV, and host defense mechanisms, including autophagic/interferon (IFN) responses, were assessed. WT mice were pretreated with the CXCR3 antagonist AMG487 that inhibits PF4:CXCR3 pathway. This pathway was tested in PF4−/− monocytes infected with DENV or in monocytes isolated from patients with DENV infection. ResultsPF4−/− mice infected with JEV showed reduced viral load and improved brain inflammation and survival. PF4−/− mice synthesized more IFN-α/β with higher expression of phosphorylated IRF3 in the brain. PF4 treatment decreased IRF-3/7/9 and IFN-α/β expression and suppressed autophagic LC3-II flux and lysosomal degradation of viral proteins in JEV-infected cells. PF4 increased the expression of P-mTOR, P-p38, and P-ULK1Ser757 and decreased expression of LC3-II. Decreased autophagosome-lysosome fusion in turn promoted DENV2 replication. The above processes were reversed by AMG487. Uninfected PF4−/− monocytes showed elevated LC3-II and autophagosome-lysosome fusion. Microglia of JEV-infected PF4−/− mice exhibited elevated LC3-II inversely related to viral load. Similarly, monocytes from PF4−/− mice showed reduced infection by DENV2. In patients with DENV infection, higher plasma PF4 and viral load were inversely correlated with LC3-II, LAMP-1, and lysosomal degradation of DENV-NS1 in monocytes during the febrile phase. ConclusionThese studies suggest that PF4 deficiency or inhibition of the PF4:CXCR3 pathway prevents JEV and DENV infection. The studies also highlight the PF4:CXCR3 axis as a potential target to develop treatment regimens against flaviviruses.

An RBM10 and NF-κB interacting host lncRNA promotes JEV replication and neuronal cell death.

Central nervous system infection by flaviviruses such as Japanese encephalitis virus, Dengue virus, and West Nile virus results in neuroinflammation and neuronal damage. However, little is known about the role of long non-coding RNAs (lncRNAs) in flavivirus-induced neuroinflammation and neuronal cell death. Here, we characterized the role of a flavivirus-induced lncRNA named JINR1 during the infection of neuronal cells. Depletion of JINR1 during virus infection reduces viral replication and cell death. An increase in GRP78 expression by JINR1 is responsible for promoting virus replication. Flavivirus infection induces the expression of a cellular protein RBM10, which interacts with JINR1. RBM10 and JINR1 promote the proinflammatory transcription factor NF-κB activity, which is detrimental to cell survival.

The detection of Japanese encephalitis virus in municipal wastewater during an acute disease outbreak.

To demonstrate the capability of wastewater-based surveillance (WBS) as a tool for detecting potential cases of Japanese Encephalitis Virus (JEV) infection in the community. In this study, we explore the potential of WBS to detect cases of JEV infection by leveraging from an established SARS-CoV-2 wastewater surveillance program. We describe the use of two reverse transcriptase quantitative polymerase chain reaction (RTqPCR) assays targeting JEV to screen archived samples from two wastewater treatment plants (WWTPs). JEV was detected in wastewater samples collected during a timeframe coinciding with a cluster of acute human encephalitis cases, alongside concurrent evidence of JEV detection in mosquito surveillance and the sentinel chicken programs within South Australia's Riverland and Murraylands regions. Current surveillance measures for JEV encounter multiple constraints, which may miss the early stages of JEV circulation or fail to capture the full extent of transmission. The detection of JEV in wastewater during a disease outbreak highlights the potential WBS has as a complementary layer to existing monitoring efforts forming part of the One Health approach required for optimal disease response and control.

The critical role of interleukin-6 in protection against neurotropic flavivirus infection.

West Nile virus (WNV) and Japanese encephalitis virus (JEV) are emerging mosquito-borne flaviviruses causing encephalitis globally. No specific drug or therapy exists to treat flavivirus-induced neurological diseases. The lack of specific therapeutics underscores an urgent need to determine the function of important host factors involved in flavivirus replication and disease progression. Interleukin-6 (IL-6) upregulation has been observed during viral infections in both mice and humans, implying that it may influence the disease outcome significantly. Herein, we investigated the function of IL-6 in the pathogenesis of neurotropic flavivirus infections. First, we examined the role of IL-6 in flavivirus-infected human neuroblastoma cells, SK-N-SH, and found that IL-6 neutralization increased the WNV or JEV replication and inhibited the expression of key cytokines. We further evaluated the role of IL-6 by infecting primary mouse cells derived from IL-6 knockout (IL-6-/-) mice and wild-type (WT) mice with WNV or JEV. The results exhibited increased virus yields in the cells lacking the IL-6 gene. Next, our in vivo approach revealed that IL-6-/- mice had significantly higher morbidity and mortality after subcutaneous infection with the pathogenic WNV NY99 or JEV Nakayama strain compared to WT mice. The non-pathogenic WNV Eg101 strain did not cause mortality in WT mice but resulted in 60% mortality in IL-6-/- mice, indicating that IL-6 is required for the survival of mice after the peripheral inoculation of WNV or JEV. We also observed significantly higher viremia and brain viral load in IL-6-/- mice than in WT mice. Subsequently, we explored innate immune responses in WT and IL-6-/- mice after WNV NY99 infection. Our data demonstrated that the IL-6-/- mice had reduced levels of key cytokines in the serum during early infection but elevated levels of proinflammatory cytokines in the brain later, along with suppressed anti-inflammatory cytokines. In addition, mRNA expression of IFN-α and IFN-β was significantly lower in the infected IL-6-/- mice. In conclusion, these data suggest that the lack of IL-6 exacerbates WNV or JEV infection in vitro and in vivo by causing an increase in virus replication and dysregulating host immune response.

The reemerging and outbreak of genotypes 4 and 5 of Japanese encephalitis virus.

The Japanese encephalitis virus (JEV) is classified into five distinct genotypes, with genotypes 1 and 3 historically showing higher activity. These genotypes are the primary agents of viral encephalitis in the Asian continent. Genotypes 4 and 5 have remained silent in low-latitude tropical regions since their discovery. From 2009, the hidden genotype 5 suddenly emerged simultaneously in mosquitoes from the Tibetan region of China and those from South Korea in East Asia. The detection of genotype 5 of JEV in these mosquitoes was associated with cases of viral encephalitis in the local population. Similarly, in 2022, the long-silent genotype 4 of JEV emerged in Australia, resulting in a local outbreak of viral encephalitis that primarily affected adults and caused fatalities. The emergence and outbreaks of genotypes 4 and 5 of JEV present new challenges for the prevention and control of Japanese encephalitis (JE). This study not only analyzes the recent emergence of these new genotypes but also discusses their implications in the development of JE vaccines and laboratory tests for newly emerging JEV infections.

Laboratory evaluation of ELISA and indirect immunofluorescence assay in response to emergence of Japanese encephalitis virus genotype IV in Australia.

The unexpected recent emergence of Japanese encephalitis virus (JEV) genotype IV in multiple southern states of Australia necessitated an evaluation of JEV serological tests suitable for diagnosing acute infection and for seroprevalence studies. This study examined the analytical and clinical performance of two high-throughput JEV assays, Euroimmun immunofluorescence assay (IFA) and Euroimmun enzyme-linked immunosorbent assay (ELISA), across four cohorts; (1) surveillance of piggery workers in outbreak areas, (2) surveillance of residents in outbreak areas, (3) acute JEV infection and (4) post-JEV vaccination. ELISA and IFA IgM demonstrated minimal cross-reactivity (0-1.8%) with other endemic flaviviruses, with high sensitivity (100%) for acute JEV infection in this low endemicity setting. Differences in IgG serodynamics between the two assays suggest convalescent and paired testing with IgM are critical in diagnosing acute infection. High assay concordance was observed between ELISA and IFA when used in serosurveillance (97.4% agreement, Cohen' κ 0.74 [95% CI 0.614-0.860]) and vaccination cohorts (91.1% agreement, Cohen's κ 0.806 [95% CI 0.672-0.941]). In conclusion, this study highlights the clinical & epidemiological applications and limitations of these two commercial JEV assays.