Japanese Encephalitis Virus Research Articles

Inflammatory & Apoptotic Factor Fluctuations Associated with Japanese Encephalitis Virus Infection in Transgenic IFNAR1-/- Mice.

Japanese encephalitis virus (JEV) is an orthoflavivirus that causes Japanese encephalitis, a mosquito-borne viral infection that primarily affects humans and animals. JEV is a major cause of encephalitis in many parts of Asia, particularly in rural and agricultural areas. In this study, we used the IFNAR1-/- mice model to investigate alterations in cytokine and apoptotic factor levels in IFNAR1-/- mice upon JEV infection. A 5-week-adult female C57BL/6 IFN-α/β receptor knockout (IFNAR1-/-) transgenic mice were intramuscularly inoculated with several viral titers and monitored within 10 dpi. The weight changes and survival rates were evaluated during the study period. Gene expression analysis was performed using RT-qPCR, targeting genes related to specific cytokines and apoptotic factors, to identify the inflammatory factors fluctuations associated with JEV strain KBPV-VR-27 infection in IFNAR1-/- mice. The expression of cytokine genes was enhanced in IFNAR1-/- mice infected with JEV KBPV-VR-27. Notably, a significant induction of cytokines, such as IL-13, IL-17α, IFN-β, and IFN-γ, was observed in the brain, while upregulation of IL-6, IFN-β, and IFN-γ was exhibited in the lung. In addition, among the targeted apoptotic factors, only significant induction of Bak was observed in the brain. We also found that the spleen exhibited a higher viral load compared to the brain and lungs. In conclusion, the findings of this study shed light on the varying viral loads across targeted organs, with the brain exhibiting a lower viral load but pronounced expression of targeted pro-inflammatory cytokines in IFNAR1-/- mice.

Interferon-stimulated gene 56 positively regulates Toll-like receptor 3-mediated CXCL10 expression in human renal proximal tubular epithelial cells.

Viral infections in tubular epithelial cells lead to the production of inflammatory cytokines by innate immunity, causing tubulointerstitial nephritis. TLR3 recognizes viral infections and acts via the activation of interferon (IFN)/IFN-stimulated genes (ISGs). This study investigates the role of ISG56, a representative ISG, in TLR3 signaling in cultured human renal proximal tubular epithelial cells (hRPTECs). To this end, hRPTECs were stimulated by a synthetic TLR3 ligand, polyinosinic-polycytidylic acid (poly IC), recombinant human interferon-β [r(h)IFN-β] or Japanese encephalitis virus (JEV) infection and assayed for inflammatory cytokine mRNA expression by RT-qPCR, and protein expression via western blotting or ELISA. ISG56 was expressed by poly IC or r(h)IFN-β and IFN-β knockdown reduced poly IC-induced expression of ISG56 and CXCL10. Moreover, ISG56 knockdown reduced poly IC- or r(h)IFN-β-induced expression of CXCL10 at the same time as increasing JEV growth and reducing CXCL10 expression induced by JEV infection. Overall, TLR3 signaling induced IFN-β-dependent expression of ISG56 and CXCL10. We show that ISG56 possibly plays a critical role in antiviral immunity of hRPTECs by positive regulation of IFN-β-mediated CXCL10 expression downstream of TLR3.

Characterization of a Small Plaque Variant Derived from Genotype V Japanese Encephalitis Virus Clinical Isolate K15P38.

Genotype V (GV) Japanese encephalitis virus (JEV) has been predominantly reported in the Republic of Korea (ROK) since 2010. GV JEV exhibits higher virulence and distinct antigenicity compared to other genotypes, which results in reduced efficacy of existing vaccines. Research on GV JEV is essential to minimize its clinical impact, but the only available clinical strain in the ROK is K15P38, isolated from the cerebrospinal fluid of a patient in 2015. We obtained this virus from National Culture Collection for Pathogens (NCCP) and isolated a variant forming small plaques during our research. We identified that this variant has one amino acid substitution each in the PrM and NS5 proteins compared to the reported K15P38. Additionally, we confirmed that this virus exhibits delayed propagation in vitro and an attenuated phenotype in mice. The isolation of this variant is a critical reference for researchers intending to study K15P38 obtained from NCCP, and the mutations in the small plaque-forming virus are expected to be useful for studying the pathology of GV JEV.

Analyzing the factors affecting virus invasion by quantitative single-particle analysis

ABSTRACT Viral diseases are among the main threats to public health. Understanding the factors affecting viral invasion is important for antiviral research. Until now, it was known that most viruses have very low plaque-forming unit (PFU)-to-particle ratios. However, further investigation is required to determine the underlying factors. Here, using quantitative single-particle analysis methods, the invasion of Semliki Forest virus (SFV), Japanese encephalitis virus (JEV), and influenza A virus (IAV) containing attachment to the cell surface, entry into the cell, transport towards the cell interior, and fusion with endosomes to release nucleocapsids were quantitatively analysed in parallel. It was found that for SFV with an PFU-to-particle ratio of approximately 1:2, an entry efficiency of approximately 31% limited infection. For JEV, whose PFU-to-particle ratio was approximately 1:310, an attachment efficiency of approximately 27% and an entry efficiency of 10% were the main factors limiting its infection. Meanwhile, for IAV with PFU-to-particle ratios of 1:8100, 5% attachment efficiency, 9% entry efficiency, and 53% fusion efficiency significantly limited its infection. These results suggest that viruses with different infectivities have different limited steps in the invasion process. Moreover, there are significant differences in attachment efficiencies among viruses, emphasizing the pivotal role of attachment in viral invasion. The influence of the virus purification method on virus invasion was also investigated. This study, for the first time, reports the efficiencies of different stages of virus invasion, leading to a better understanding of virus invasion and providing a protocol to quantitatively analyse the virus invasion efficiency.

Generation of rescued Japanese encephalitis virus genotype 1 from infectious full-size clone using reverse genetics

Japanese encephalitis virus (JEV) is a pathogen responsible for high mortality and morbidity rates among children with encephalitis. Since JEV genotype 1 (GI) is the most prevalent strain in South Korea these days, corresponding research and vaccine development is urgently required. Molecular genetic studies on JEV vaccines can be boosted by obtaining genetically stable full-length infectious JEV complementary DNA (cDNA) clones. Furthermore, the significance of the reverse genetics system in facilitating molecular biological analyses of JEV properties has been demonstrated. This study constructed a recombinant JEV-GI strain using a reverse genetics system based on a Korean wild-type GI isolate (K05GS). RNA extracted from JEV-GI was used to synthesize cDNA, a recombinant full-length JEV clone, pTRE-JEVGI, was generated from the DNA fragment, and the virus was rescued. We performed in vitro and in vivo experiments to analyze the rescued JEV-GI virus. The rescued JEV-GI exhibited similar characteristics to wild-type JEV. These results suggest that our reverse genetics system can generate full-length infectious clones that can be used to analyze molecular biological factors that influence viral properties and immunogenicity. Additionally, it may be useful as a heterologous gene expression vector and help develop new strains for JEV vaccines.

JEV infection leads to dysfunction of lysosome by downregulating the expression of LAMP1 and LAMP2

Japanese Encephalitis Virus (JEV), the predominant cause of viral encephalitis in many Asian countries, affects approximately 68,000 people annually. Lysosomes are dynamic structures that regulate cellular metabolism by mediating lysosomal biogenesis and autophagy. Here, we showed that lysosome-associated membrane protein 1 (LAMP1) and LAMP2 were downregulated in cells after JEV infection, resulting in a decrease in the quantity of acidified lysosomes and impaired lysosomal catabolism. What’s more, JEV nonstructural protein 4B plays key roles in the reduction of LAMP1/2 via the autophagy-lysosome pathway. JEV NS4B also promoted abnormal aggregation of SLA-DR, an important component of the swine MHC-II molecule family involved in antigen presentation and CD4+ cell activation initiation. Mechanistically, NS4B localized to the ER during JEV infection and interacted with GRP78, leading to the activation of ER stress-mediated autophagy. The 131–204 amino acid (aa) region of NS4B is essential for autophagy induction and LAMP1/2 reduction. In summary, our findings reveal a novel pathway by which JEV induces autophagy and disrupts lysosomal function.

Role of inflammatory cytokine burst in neuro-invasion of Japanese Encephalitis virus infection: an immunotherapeutic approaches.

Japanese Encephalitis remains a significant global health concern, contributing to millions of deaths annually worldwide. Microglial cells, as key innate immune cells within the central nervous system (CNS), exhibit intricate cellular structures and possess molecular phenotypic plasticity, playing pivotal roles in immune responses during CNS viral infections. Particularly under viral inflammatory conditions, microglial cells orchestrate innate and adaptive immune responses to mitigate viral invasion and dampen inflammatory reactions. This review article comprehensively summarizes the pathophysiology of viral invasion into the CNS and the cellular interactions involved, elucidating the roles of various immune mediators, including pro-inflammatory cytokines, in neuroinflammation. Leveraging this knowledge, strategies for modulating inflammatory responses and attenuating hyperactivation of glial cells to mitigate viral replication within the brain are discussed. Furthermore, current chemotherapeutic and antiviral drugs are examined, elucidating their mechanisms of action against viral replication. This review aims to provide insights into therapeutic interventions for Japanese Encephalitis and related viral infections, ultimately contributing to improved outcomes for affected individuals.

The mutation of Japanese encephalitis virus envelope protein residue 389 attenuates viral neuroinvasiveness

The envelope (E) protein of the Japanese encephalitis virus (JEV) is a key protein for virus infection and adsorption of host cells, which determines the virulence of the virus and regulates the intensity of inflammatory response. The mutation of multiple aa residues in the E protein plays a critical role in the attenuated strain of JEV. This study demonstrated that the Asp to Gly, Ser, and His mutation of the E389 site, respectively, the replication ability of the viruses in cells was significantly reduced, and the viral neuroinvasiveness was attenuated to different degrees. Among them, the mutation at E389 site enhanced the E protein flexibility contributed to the attenuation of neuroinvasiveness. In contrast, less flexibility of E protein enhanced the neuroinvasiveness of the strain. Our results indicate that the mechanism of attenuation of E389 aa mutation attenuates neuroinvasiveness is related to increased flexibility of the E protein. In addition, the increased flexibility of E protein enhanced the viral sensitivity to heparin inhibition in vitro, which may lead to a decrease in the viral load entering brain. These results suggest that E389 residue is a potential site affecting JEV virulence, and the flexibility of the E protein of aa at this site plays an important role in the determination of neuroinvasiveness.

Aetiological profile of acute encephalitis syndrome in Assam, India, during a 4-year period from 2019 to 2022.

Acute encephalitis syndrome (AES) is a major public health concern in India as the aetiology remains unknown in the majority of cases with the current testing algorithm. We aimed to study the incidence of Japanese encephalitis (JE) and determine the aetiology of non-JE AES cases to develop an evidence-based testing algorithm. Cerebrospinal fluid (CSF) samples were tested for Japanese encephalitis virus by ELISA and polymerase chain reaction (PCR). Multiplex real-time PCR was done for Dengue, Chikungunya, West Nile, Zika, Enterovirus, Epstein Barr Virus, Herpes Simplex Virus, Adenovirus, Cytomegalovirus, Herpesvirus 6, Parechovirus, Parvovirus B19, Varicella Zoster Virus, Scrub typhus, Rickettsia species, Leptospira, Salmonella species, Streptococcus pneumoniae, Haemophilus influenzae, Neisseria meningitidis, Plasmodium species and by ELISA for Mumps and Measles virus. Of the 3173 CSF samples, 461 (14.5%) were positive for JE. Of the 334 non-JE AES cases, 66.2% viz. Scrub typhus (25.7%), Mumps (19.5%), Measles (4.2%), Parvovirus B19 (3.9%) Plasmodium (2.7%), HSV 1 and 2 (2.4%), EBV and Streptococcus pneumoniae (2.1% each), Salmonella and HHV 6 (1.2% each) were predominant. Hence, an improved surveillance system and our suggested expanded testing algorithm can improve the diagnosis of potentially treatable infectious agents of AES in India.

Deciphering the interaction surface between the West Nile virus NS3 and NS5 proteins.

West Nile virus (WNV) is the most prevalent mosquito-borne virus and the leading cause of viral encephalitis in the continental United States. It belongs to the family Flaviviridae which includes other important human pathogens such as dengue virus (DENV), Japanese encephalitis virus (JEV) and Zika viruses (ZIKV). Despite several decades of research, no specific antiviral drugs are available to treat flavivirus infections. The present study characterizes the interaction between the WNV NS3 and NS5 proteins for the purpose of identifying hotspots in the protein-protein interaction which could be targeted for the development of antiviral therapeutics. We previously developed an interaction model in silico based on data available in the literature. Here, potential interacting residues on NS3 and NS5 were mutated in a WNV replicon, and seven mutations in the NS3 protein were found to drastically reduce viral replication. In addition to being well conserved among mosquito-borne flaviviruses, these residues are located on the protein's surface in two clusters which might be interesting new targets for future drug development.

Novel envelope protein time-resolved fluoroimmunoassay as an alternative in vitro potency assay for quality control of inactivated Japanese encephalitis virus vaccine

Japanese encephalitis (JE) vaccination is the most effective way to prevent JE. Plaque reduction neutralization test (PRNT) as the standard method for potency testing for inactivated JE vaccine could not provide the exact potency value. Envelope (E) protein of JE virus induces the body to create neutralizing antibodies. There is a potential for using the determination of E protein to assess the immunogenicity and efficacy of JE vaccine. In this study, an automatic time-resolved fluoroimmunoassay for detection of E protein in JE vaccine was established as a simple and rapid in vitro potency assay to complement PRNT, including the expression and paired screening of monoclonal antibodies, the establishment of assay method and performance verification. A pair of anti-E protein neutralizing antibodies (L022 and L034) were screened to construct the sandwich detection pattern. After pre-treating the vaccine sample, the entire analysis was performed using a fully automated machine, which had a little detection time and eliminated manual error. The results of the validation experiment met the requirements for quality control. The linear range was from 0.78125 U/mL to 25 U/mL, the sensitivity was 0.01 U/mL, the intra-assay coefficient of variation was less than 5 %, and the inter-assay coefficient of variation was less than 10 %. The recovery from the dilution was between 90 % and 110 %. This present TRFIA shown good stability and effectiveness in quality control for samples related to JE vaccine production. The outcomes demonstrated that the present TRFIA could be an alternative in vitro potency assay in quality control for inactivated JE vaccine.

The seroprevalence of antibodies to Japanese encephalitis virus in five New South Wales towns at high risk of infection, 2022: a cross-sectional serosurvey.

To determine the proportion of people in New South Wales towns at high risk of Japanese encephalitis virus (JEV) infections during the 2022 outbreak; to identify risk factors for JEV infection. Cross-sectional serosurvey study of the seroprevalence of JEV-specific antibodies in NSW. Convenience sample of people (all ages) from five regional NSW towns deemed to be at high risk of JEV infections after first outbreak of Japanese encephalitis in southeastern Australia in early 2022 (Balranald, Corowa, Dubbo, Griffith, Temora), 21 June - 22 July 2022. Proportion of people seropositive for JEV total antibody, assayed by defined epitope-blocking enzyme-linked immunosorbent assay; prevalence odds ratios for exposure risk factors and protective behaviours. Eighty of 917 eligible participants (559 girls or women, 61%; 42 Aboriginal and Torres Strait Islander people, 4.6%; median age, 52 years [IQR, 37-62 years]) were seropositive for JEV-specific total antibody (8.7%); the median age of seropositive people was 61 years (IQR, 48-70 years). The seropositivity proportion was largest for people aged 65 years or more (30 of 192; weighted proportion, 13.7%) and larger for male than female participants (30 of 358, 10.6% v 50 of 559, 7.5%). Five of 42 samples from Aboriginal and Torres Strait Islander participants were seropositive (12%). We found mixed associations with a range of potential risk factors. We found evidence for a substantial number of JEV infections in five regional NSW towns during a single arbovirus season in 2022. Public health responses, including effective surveillance, vaccination against JEV, and mosquito management, are critical for controlling outbreaks. Promoting behaviours that reduce exposure to mosquitoes is a core component of prevention, particularly when the vaccine supply is limited.

Insights into the Pathogenesis and Development of Recombinant Japanese Encephalitis Virus Genotype 3 as a Vaccine.

Japanese encephalitis virus (JEV), a flavivirus transmitted by mosquitoes, has caused epidemics and severe neurological diseases in Asian countries. In this study, we developed a cDNA infectious clone, pBAC JYJEV3, of the JEV genotype 3 strain (EF571853.1) using a bacterial artificial chromosome (BAC) vector. The constructed infectious clone was transfected into Vero cells, where it exhibited infectivity and induced cytopathic effects akin to those of the parent virus. Confocal microscopy confirmed the expression of the JEV envelope protein. Comparative analysis of growth kinetics revealed similar replication dynamics between the parental and recombinant viruses, with peak titers observed 72 h post-infection (hpi). Furthermore, plaque assays demonstrated comparable plaque sizes and morphologies between the viruses. Cryo-electron microscopy confirmed the production of recombinant virus particles with a morphology identical to that of the parent virus. Immunization studies in mice using inactivated parental and recombinant viruses revealed robust IgG responses, with neutralizing antibody production increasing over time. These results showcase the successful generation and characterization of a recombinant JEV3 virus and provide a platform for further investigations into JEV pathogenesis and vaccine development.

Japanese encephalitis virus in Australia: an ecological and epidemiological enigma.

Japanese encephalitis virus in Australia: an ecological and epidemiological enigma.

Characterization of genotype V Japanese encephalitis virus isolates from Republic of Korea

ABSTRACT Japanese encephalitis (JE), caused by the Japanese encephalitis virus (JEV) infection, continues to pose significant public health challenges worldwide despite efficient vaccines. The virus is classified into five genotypes, among which genotype V (GV) was not detected for a long period after its initial isolation in 1952, until reports emerged from China and the Republic of Korea (ROK) since 2009. The characteristics of the virus are crucial in estimating its potential epidemiological impact. However, characterization of GV JEVs has so far been limited to two strains: Muar, the original isolate, and XZ0934, isolated in China. Two additional ROK GV JEV isolates, NCCP 43279 and NCCP 43413, are currently available, but their characteristics have not been explored. Our phylogenetic analysis revealed that GV virus sequences from the ROK segregate into two clades. NCCP 43279 and NCCP 43413 belong to different clades and exhibit distinct in vitro phenotypes. NCCP 43279 forms larger plaques but demonstrates inefficient propagation in cell culture compared to NCCP 43413. In vivo, NCCP 43279 induces higher morbidity and mortality in mice than NCCP 43413. Notably, NCCP 43279 shows more severe blood–brain barrier damage, suggesting superior brain invasion capabilities. Consistent with its higher virulence, NCCP 43279 displays more pronounced histopathological and immunopathological outcomes. In conclusion, our study confirms that the two ROK isolates are not only classified into different clades but also exhibit distinct in vitro and in vivo characteristics.

Demographic inference from the mt-DNA COI gene and wing geometry of Culex gelidus (Diptera: Culicidae), an important vector of Japanese encephalitis in Thailand

Culex gelidus (Diptera: Culicidae), an important vector of the Japanese encephalitis virus (JEV), contributes to human viral encephalitis in many Asian countries, including Thailand. This study represents the first investigation of the demographic patterns of Cx. gelidus populations in Thailand using cytochrome c oxidase subunit I (COI) gene analysis and wing geometric morphometrics (GM). Mosquitoes were collected from 10 provinces across six regions of Thailand in 2022. Analysis of the COI sequences (n = 182) indicated high haplotype diversity (0.882) and low nucleotide diversity (0.006), with 72 haplotypes identified. The haplotype network demonstrated no profound splits among the geographic populations. Neutral tests, including Tajima's D and Fu's Fs, displayed negative values, with a significant result observed for Fu's Fs (−33.048, p < 0.05). The mismatch distribution analysis indicated that the population does not statistically deviate from a model of sudden population expansion (SSD = 0.010, p > 0.05; Rg = 0.022, p > 0.05). The estimations suggest that the Cx. gelidus population in Thailand began its expansion approximately between 459,243 and 707,011 years ago. The Mantel test showed no significant relationship between genetic and geographic distances (r = 0.048, p > 0.05). Significant phenotypic differences (based on wing shape) were observed among most populations. Additionally, in this study, we found no significant relationships between phenotypic and genetic distances (r = 0.250, p > 0.05). Understanding the genetic and morphological dynamics of Cx. gelidus is vital for developing targeted surveillance and vector control measures. This knowledge will also help to predict how future environmental changes might affect these populations, thereby informing long-term vector management strategies.

Serosurvey for Japanese encephalitis virus antibodies following an outbreak in an immunologically naïve population, Victoria, 2022: a cross-sectional study.

To investigate the distribution and prevalence of Japanese encephalitis virus (JEV) antibody (as evidence of past infection) in northern Victoria following the 2022 Japanese encephalitis outbreak, seeking to identify groups of people at particular risk of infection; to investigate the distribution and prevalence of antibodies to two related flaviviruses, Murray Valley encephalitis virus (MVEV) and West Nile virus Kunjin subtype (KUNV). Cross-sectional serosurvey (part of a national JEV serosurveillance program). Three northern Victorian local public health units (Ovens Murray, Goulburn Valley, Loddon Mallee), 8 August - 1 December 2022. People opportunistically recruited at pathology collection centres and by targeted recruitment through community outreach and advertisements. People vaccinated against or who had been diagnosed with Japanese encephalitis were ineligible for participation, as were those born in countries where JEV is endemic. Seroprevalence of JEV IgG antibody, overall and by selected factors of interest (occupations, water body exposure, recreational activities and locations, exposure to animals, protective measures). 813 participants were recruited (median age, 59 years [interquartile range, 42-69 years]; 496 female [61%]); 27 were JEV IgG-seropositive (3.3%; 95% confidence interval [CI], 2.2-4.8%) (median age, 73 years [interquartile range, 63-78 years]; 13 female [48%]); none were IgM-seropositive. JEV IgG-seropositive participants were identified at all recruitment locations, including those without identified cases of Japanese encephalitis. The only risk factors associated with JEV IgG-seropositivity were age (per year: prevalence odds ratio [POR], 1.07; 95% CI, 1.03-1.10) and exposure to feral pigs (POR, 21; 95% CI, 1.7-190). The seroprevalence of antibody to MVEV was 3.0% (95% CI, 1.9-4.5%; 23 of 760 participants), and of KUNV antibody 3.3% (95% CI, 2.1-4.8%; 25 of 761). People living in northern Victoria are vulnerable to future JEV infection, but few risk factors are consistently associated with infection. Additional prevention strategies, including expanding vaccine eligibility, may be required to protect people in this region from Japanese encephalitis.

TBE In Japan

In Japan the Japanese encephalitis virus (JEV), one of mosquito-borne flaviviruses, has been widely endemic on the main and on the southern islands with more than 1,000 Japanese encephalitis (JE) cases reported annually in the late 1960s.1 In contrast, until 1993, no TBE case had ever been reported and it was considered that there was no endemic focus of TBEV.

Development of immunochromatographic strip assay to detect recent infection of Japanese encephalitis virus in swine population

Japanese Encephalitis (JE) is a mosquito borne re-emerging viral zoonotic disease. Sero-conversion in swine occurs 2–3 weeks before human infection, thus swine act as a suitable sentinel for predicting JE outbreaks in humans. The present study was undertaken with the objective of developing immunochromatographic strip (ICS) assay to detect recent infection of Japanese Encephalitis virus (JEV) in swine population. The two formats of ICS assay were standardized. In the first format, gold nanoparticles (GNP) were conjugated with goat anti-pig IgM (50 μg/ml) followed by spotting of recombinant NS1 protein (1 mg/ml) of JEV on NCM as test line and protein G (1 mg/ml) as control line. In the format-II, GNP were conjugated with rNS1 protein (50 μg/ml) followed by spotting of Goat anti-pig IgM (1 mg/ml) as test line and IgG against rNS1 (1 mg/ml) as control line. To decrease the non- specific binding, blocking of serum and nitrocellulose membrane (NCM) was done using 5% SMP in PBS-T and 1% BSA, respectively. Best reaction conditions for the assay were observed when 10 μl of GNP conjugate and 50 μl of 1:10 SMP blocked sera was reacted on BSA blocked NCM followed by reaction time of 15 mins. Samples showing both test and control line were considered positive whereas samples showing only control line were considered negative. A total of 318 field swine sera samples were screened using indirect IgM ELISA and developed ICS assay. Relative diagnostic sensitivity and specificity of format-I was 81.25% and 93.0% whereas of format-II was 87.50% and 62.93%, respectively. Out of 318 samples tested, 32 were positive through IgM ELISA with sero-positivity of 10.06% while sero-positivity with format-I of ICS was 8.1%. Owing to optimal sensitivity and higher specificity of format-I, it was validated in three different labs and the kappa agreement ranged from 0.80 to 1, which signifies excellent repeatability of the developed assay to test field swine sera samples for detecting recent JEV infection.

Japanese encephalitis virus hijacks ER-associated degradation regulators for its replication.

Flaviviruses target their replication on membranous structures derived from the ER, where both viral and host proteins play crucial structural and functional roles. Here, we have characterized the involvement of the ER-associated degradation (ERAD) pathway core E3 ligase complex (SEL1L-HRD1) regulator proteins in the replication of Japanese encephalitis virus (JEV). Through high-resolution immunofluorescence imaging of JEV-infected HeLa cells, we observe that the virus replication complexes marked by NS1 strongly colocalize with the ERAD adapter SEL1L, lectin OS9, ER-membrane shuttle factor HERPUD1, E3 ubiquitin ligase HRD1 and rhomboid superfamily member DERLIN1. NS5 positive structures also show strong overlap with SEL1L. While these effectors show significant transcriptional upregulation, their protein levels remain largely stable in infected cells. siRNA mediated depletion of OS9, SEL1L, HERPUD1 and HRD1 significantly inhibit viral RNA replication and titres, with SEL1L depletion showing the maximum attenuation of replication. By performing protein translation arrest experiments, we show that SEL1L, and OS9 are stabilised upon JEV infection. Overall results from this study suggest that these ERAD effector proteins are crucial host-factors for JEV replication.