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

OBJECTIVES: 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. STUDY DESIGN: Cross-sectional serosurvey study of the seroprevalence of JEV-specific antibodies in NSW. SETTING, PARTICIPANTS: 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. MAIN OUTCOME MEASURES: 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. RESULTS: 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. CONCLUSION: 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.

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

OBJECTIVES: 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). STUDY DESIGN: Cross-sectional serosurvey (part of a national JEV serosurveillance program). SETTING: Three northern Victorian local public health units (Ovens Murray, Goulburn Valley, Loddon Mallee), 8 August - 1 December 2022. PARTICIPANTS: 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. MAIN OUTCOME MEASURES: 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). RESULTS: 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). CONCLUSIONS: 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.

The RNA binding protein Quaking represses splicing of the Fibronectin EDA exon and downregulates the interferon response.

Quaking (QKI) controls RNA metabolism in many biological processes including innate immunity, where its roles remain incompletely understood. To illuminate these roles, we performed genome scale transcriptome profiling in QKI knockout cells with or without poly(I:C) transfection, a double-stranded RNA analog that mimics viral infection. Analysis of RNA-sequencing data shows that QKI knockout upregulates genes induced by interferons, suggesting that QKI is an immune suppressor. Furthermore, differential splicing analysis shows that QKI primarily controls cassette exons, and among these events, we noted that QKI silences splicing of the extra domain A (EDA) exon in fibronectin (FN1) transcripts. QKI knockout results in elevated production and secretion of FN1-EDA protein, which is a known activator of interferons. Consistent with an upregulation of the interferon response in QKI knockout cells, our results show reduced production of dengue virus-2 and Japanese encephalitis virus in these cells. In conclusion, we demonstrate that QKI downregulates the interferon system and attenuates the antiviral state.

CD8+ T cells are crucial for humoral immunity establishment by SA14-14-2 live attenuated Japanese encephalitis vaccine in mice.

The live attenuated SA14-14-2 Japanese encephalitis (JE) vaccine is a historical vaccine that protects against JE. Despite its extensive use, the mechanism of protective immunity conferred by the SA14-14-2 vaccine is not well established. Here, we used mouse models to understand the mechanism of the development of humoral immunity against the vaccine. The vaccine induces robust GC responses within a week postimmunization. In lethal virus challenge, we show that CD4+ T cells alone, but not CD8+ T cells, are sufficient to confer vaccine-mediated protection. However, the CD4-mediated protection was potentiated in the presence of vaccine-primed CD8+ T cells. Employing CD8-deficient mice, we show that both the protective traits of CD4+ T cells and the quality of antibody response to the vaccine are impaired in absence of CD8+ T cells. We further demonstrate that the poor protective immune response induced by the vaccine in absence of CD8+ T cells is mainly due to the impaired differentiation and function of follicular Th cells, leading to suboptimal GC reaction. Our study highlights an unprecedented role of CD8+ T cells in the establishment of humoral responses to the vaccine. By elucidating underlying cellular determinants of vaccine-induced protective immunity, our work has implications for rational design of vaccines against JE virus and related flaviviruses.

Lack of Interferon (IFN) Regulatory Factor 8 Associated with Restricted IFN-γ Response Augmented Japanese Encephalitis Virus Replication in the Mouse Brain.

Interferon regulatory factor 8 (IRF8), a myeloid lineage transcription factor, emerges as an essential regulator for microglial activation. However, the precise role of IRF8 during Japanese encephalitis virus (JEV) infection in the brain remains elusive. Here, we report that JEV infection enhances IRF8 expression in the infected mouse brain. Comparative transcriptional profiling of whole-brain RNA analysis and validation by quantitative reverse transcription-PCR (qRT-PCR) reveals an impaired interferon gamma (IFN-γ) and related gene expression in Irf8 knockout (Irf8-/-)-infected mice. Further, Ifnγ knockout (Ifnγ-/-) mice exhibit a reduced level of Irf8. Both Ifnγ-/- and Irf8-/- mice exhibit significantly reduced levels of activated (CD11b+ CD45hi, CD11b+ CD45lo, Cd68, and CD86) and infiltrating immune cells (Ly6C+, CD4, and CD8) in the infected brain compared to those of wild-type (WT) mice. However, a higher level of granulocyte cell (Ly6G+) infiltration is evident in Irf8-/- mice as well as the increased concentration of tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), monocyte chemoattractant protein 1 (MCP1) levels in the brain. Interestingly, neither the Irf8-/- nor the Ifnγ-/- conferred protection against lethal JEV challenge to mice and exhibit augmentation in JEV replication in the brain. The gain of function of Irf8 by overexpressing functional IRF8 in an IRF8-deficient cell line attenuates viral replication and enhances IFN-γ production. Overall, we summarize that in the murine model of JEV encephalitis, IRF8 modulation affects JEV replication. We also show that lack of Irf8 affects immune cell abundance in circulation and the infected brain, leading to a reduction in IFN-γ level and increased viral load in the brain. IMPORTANCE Microglial cells, the resident macrophages in the brain, play a vital role in Japanese encephalitis virus (JEV) pathogenesis. The deregulated activity of microglia can be lethal for the brain. Therefore, it is crucial to understand the regulators that drive microglia phenotype changes and induce inflammation in the brain. Interferon regulatory factor 8 (IRF8) is a myeloid lineage transcription factor involved in microglial activation. However, the impact of IRF8 modulation on JEV replication remains elusive. Moreover, the pathways regulated by IRF8 to initiate and amplify pathological neuroinflammation are not well understood. Here, we demonstrated the effect of IRF8 modulation on JEV replication, microglial activation, and immune cells infiltration in the brain.

NS5-V372A and NS5-H386Y variations are responsible for differences in interferon α/ß induction and co-contribute to the replication advantage of Japanese encephalitis virus genotype I over genotype III in ducklings.

Japanese encephalitis virus (JEV) genotype I (GI) replicates more efficiently than genotype III (GIII) in birds, and this difference is considered to be one of the reasons for the JEV genotype shift. In this study, we utilized duck embryo fibroblasts and domestic ducklings as in vitro and in vivo models of a JEV amplifying avian host to identify the viral determinants of the differing replication efficiency between the GI and GIII strains in birds. GI strains induced significantly lower levels of interferon (IFN)-α and ß production than GIII strains, an effect orrelated with the enhanced replication efficiency of GI strains over GIII strains. By using a series of chimeric viruses with exchange of viral structural and non-structural (NS) proteins, we identified NS5 as the viral determinant of the differences in IFN-α and ß induction and replication efficiency between the GI and III strains. NS5 inhibited IFN-α and ß production induced by poly(I:C) stimulation and harbored 11 amino acid variations, of which the NS5-V372A and NS5-H386Y variations were identified to co-contribute to the differences in IFN-α and ß induction and replication efficiency between the strains. The NS5-V372A and NS5-H386Y variations resulted in alterations in the number of hydrogen bonds formed with neighboring residues, which were associated with the different ability of the GI and GIII strains to inhibit IFN-α and ß production. Our findings indicated that the NS5-V372A and NS5-H386Y variations enabled GI strains to inhibit IFN-α and ß production more efficiently than GIII strains for antagonism of the IFN-I mediated antiviral response, thereby leading to the replication and host adaption advantages of GI strains over GIII strains in birds. These findings provide new insight into the molecular basis of the JEV genotype shift.

Expression of Zinc-Finger Antiviral Protein in hCMEC/D3 Human Cerebral Microvascular Endothelial Cells: Effect of a Toll-Like Receptor 3 Agonist.

INTRODUCTION: Invasion of viruses into the brain causes viral encephalitis, which can be fatal and causes permanent brain damage. The blood-brain barrier (BBB) protects the brain by excluding harmful substances and microbes. Brain microvascular endothelial cells are important components of the BBB; however, the mechanisms of antiviral reactions in these cells have not been fully elucidated. Zinc-finger antiviral protein (ZAP) is a molecule that restricts the infection of various viruses, and there are 2 major isoforms: ZAPL and ZAPS. Toll-like receptor 3 (TLR3), a pattern-recognition receptor against viral double-stranded RNA, is implicated in antiviral innate immune reactions. The aim of this study was to investigate the expression of ZAP in cultured hCMEC/D3 human brain microvascular endothelial cells treated with an authentic TLR3 agonist polyinosinic-polycytidylic acid (poly IC). METHODS: hCMEC/D3 cells were cultured and treated with poly IC. Expression of ZAPL and ZAPS mRNA was investigated using quantitative reverse transcription-polymerase chain reaction, and protein expression of these molecules was examined using western blotting. The role of nuclear factor-κB (NF-κB) was examined using the NF-κB inhibitor, SN50. The roles of interferon (IFN)-ß, IFN regulatory factor 3 (IRF3), tripartite motif protein 25 (TRIM25), and retinoic acid-inducible gene-I (RIG-I) in poly IC-induced ZAPS expression were examined using RNA interference. Propagation of Japanese encephalitis virus (JEV) was examined using a focus-forming assay. RESULTS: ZAPS mRNA and protein expression was upregulated by poly IC, whereas the change of ZAPL mRNA and protein levels was minimal. Knockdown of IRF3 or TRIM25 decreased the poly IC-induced upregulation of ZAPS, whereas knockdown of IFN-ß or RIG-I did not affect ZAPS upregulation. SN50 did not affect ZAPS expression. Knockdown of ZAP enhanced JEV propagation. CONCLUSION: ZAPL and ZAPS were expressed in hCMEC/D3 cells, and ZAPS expression was upregulated by poly IC. IRF3 and TRIM25 are involved in poly IC-induced upregulation of ZAPS. ZAP may contribute to antiviral reactions in brain microvascular endothelial cells and protect the brain from invading viruses such as JEV.

Antibody-dependent enhancement (ADE) of dengue virus: Identification of the key amino acid that is vital in DENV vaccine research.

BACKGROUND: The antibody-dependent enhancement (ADE) of dengue virus (DENV) has critically restricted vaccine development. Prior research suggested pr4 as the probable ADE epitope of DENV. METHODS: Chimeric DENV was constructed by replacing the DENV pr4 gene with the corresponding Japanese encephalitis virus (JEV) gene to determine whether it can reduce ADE activities. An alanine scanning method and bioinformatics analysis were utilized to identify the amino acid of pr4 that was crucial as an ADE epitope. RESULTS: Chimeric virus reduced ADE and virulence. The amino acids at the following locations on the mutant peptides showed significantly reduced binding ability to prM antibody: pr4.5 (position 5 - leucine), pr4.6 (position 6 - leucine), pr4.7 (position 7 - phenyalanine) and pr4.16 (position 16 - cysteine). The four amino acids had formed a pocket-like structure, which could increase the possibility of binding to an antibody. CONCLUSIONS: ADE activities could be reduced by replacing the DENV pr4 gene with the corresponding JEV gene. Leucine at position 5, leucine at position 6, phenyalanine at position 7 and cysteine at position 16 were the key amino acid sites in the ADE response of DENV. The occurrence of ADE can potentially be reduced by the replacement of key amino acids, hence highlighting its possible contribution to dengue vaccine design, paving a way for future vaccine research.

Type I IFN signaling limits hemorrhage-like disease after infection with Japanese encephalitis virus through modulating a prerequisite infection of CD11b+Ly-6C+ monocytes.

BACKGROUND: The crucial role of type I interferon (IFN-I, IFN-α/ß) is well known to control central nervous system (CNS) neuroinflammation caused by neurotrophic flaviviruses such as Japanese encephalitis virus (JEV) and West Nile virus. However, an in-depth analysis of IFN-I signal-dependent cellular factors that govern CNS-restricted tropism in JEV infection in vivo remains to be elucidated. METHODS: Viral dissemination, tissue tropism, and cytokine production were examined in IFN-I signal-competent and -incompetent mice after JEV inoculation in tissues distal from the CNS such as the footpad. Bone marrow (BM) chimeric models were used for defining hematopoietic and tissue-resident cells in viral dissemination and tissue tropism. RESULTS: The paradoxical and interesting finding was that IFN-I signaling was essentially required for CNS neuroinflammation following JEV inoculation in distal footpad tissue. IFN-I signal-competent mice died after a prolonged neurological illness, but IFN-I signal-incompetent mice all succumbed without neurological signs. Rather, IFN-I signal-incompetent mice developed hemorrhage-like disease as evidenced by thrombocytopenia, functional injury of the liver and kidney, increased vascular leakage, and excessive cytokine production. This hemorrhage-like disease was closely associated with quick viral dissemination and impaired IFN-I innate responses before invasion of JEV into the CNS. Using bone marrow (BM) chimeric models, we found that intrinsic IFN-I signaling in tissue-resident cells in peripheral organs played a major role in inducing the hemorrhage-like disease because IFN-I signal-incompetent recipients of BM cells from IFN-I signal-competent mice showed enhanced viral dissemination, uncontrolled cytokine production, and increased vascular leakage. IFN-I signal-deficient hepatocytes and enterocytes were permissive to JEV replication with impaired induction of antiviral IFN-stimulated genes, and neuron cells derived from both IFN-I signal-competent and -incompetent mice were vulnerable to JEV replication. Finally, circulating CD11b+Ly-6C+ monocytes infiltrated into the distal tissues inoculated by JEV participated in quick viral dissemination to peripheral organs of IFN-I signal-incompetent mice at an early stage. CONCLUSION: An IFN-I signal-dependent model is proposed to demonstrate how CD11b+Ly-6C+ monocytes are involved in restricting the tissue tropism of JEV to the CNS.

C-type lectins and extracellular vesicles in virus-induced NETosis.

Dysregulated formation of neutrophil extracellular traps (NETs) is observed in acute viral infections. Moreover, NETs contribute to the pathogenesis of acute viral infections, including those caused by the dengue virus (DV) and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Furthermore, excessive NET formation (NETosis) is associated with disease severity in patients suffering from SARS-CoV-2-induced multiple organ injuries. Dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) and other members of C-type lectin family (L-SIGN, LSECtin, CLEC10A) have been reported to interact with viral glycans to facilitate virus spreading and exacerbates inflammatory reactions. Moreover, spleen tyrosine kinase (Syk)-coupled C-type lectin member 5A (CLEC5A) has been shown as the pattern recognition receptor for members of flaviviruses, and is responsible for DV-induced cytokine storm and Japanese encephalomyelitis virus (JEV)-induced neuronal inflammation. Moreover, DV activates platelets via CLEC2 to release extracellular vesicles (EVs), including microvesicles (MVs) and exosomes (EXOs). The DV-activated EXOs (DV-EXOs) and MVs (DV-MVs) stimulate CLEC5A and Toll-like receptor 2 (TLR2), respectively, to enhance NET formation and inflammatory reactions. Thus, EVs from virus-activated platelets (PLT-EVs) are potent endogenous danger signals, and blockade of C-type lectins is a promising strategy to attenuate virus-induced NETosis and intravascular coagulopathy.

miR-301a Regulates Inflammatory Response to Japanese Encephalitis Virus Infection via Suppression of NKRF Activity.

Microglia being the resident macrophage of brain provides neuroprotection following diverse microbial infections. Japanese encephalitis virus (JEV) invades the CNS, resulting in neuroinflammation, which turns the neuroprotective role of microglia detrimental as characterized by increased microglial activation and neuronal death. Several host factors, including microRNAs, play vital roles in regulating virus-induced inflammation. In the current study, we demonstrate that the expression of miR-301a is increased in JEV-infected microglial cells and human brain. Overexpression of miR-301a augments the JEV-induced inflammatory response, whereas inhibition of miR-301a completely reverses the effects. Mechanistically, NF-κB-repressing factor (NKRF) functioning as inhibitor of NF-κB activation is identified as a potential target of miR-301a in JEV infection. Consequently, miR-301a-mediated inhibition of NKRF enhances nuclear translocation of NF-κB, which, in turn, resulted in amplified inflammatory response. Conversely, NKRF overexpression in miR-301a-inhibited condition restores nuclear accumulation of NF-κB to a basal level. We also observed that JEV infection induces classical activation (M1) of microglia that drives the production of proinflammatory cytokines while suppressing alternative activation (M2) that could serve to dampen the inflammatory response. Furthermore, in vivo neutralization of miR-301a in mouse brain restores NKRF expression, thereby reducing inflammatory response, microglial activation, and neuronal apoptosis. Thus, our study suggests that the JEV-induced expression of miR-301a positively regulates inflammatory response by suppressing NKRF production, which might be targeted to manage viral-induced neuroinflammation.

Persistence of Immune Responses With an Inactivated Japanese Encephalitis Single-Dose Vaccine, JENVAC and Interchangeability With a Live-Attenuated Vaccine.

BACKGROUND: This study reports immunogenicity, safety, and interchangeability of a single-dose, inactivated, Vero-cell derived, JENVAC to the live-attenuated SA 14-14-2 vaccine in healthy children. METHODS: This phase 4, multicenter, open-label, randomized, control trial enrolled 360 children who were equally randomized to receive a single dose of either JENVAC or SA 14-14-2. Children were followed at various time points, until 2 years (day 720) postvaccination, upon which a subset from each group was divided and allocated to a receive a booster dose or the other vaccine. RESULTS: At all time points, immunological measures were statistically higher in the JENVAC group. In the interchangeability study, children receiving 2 doses of JENVAC reported significantly higher response compared with 2 doses of SA 14-14-2. No difference in adverse events was observed. These corroborate with excellent seroprotection after the first dose of an earlier JENVAC study. CONCLUSIONS: A single-dose vaccination with JENVAC induces protective titers that persist up to 1 year. We report appreciable interchangeability between both vaccines, with JENVAC/JENVAC combination exhibiting the highest immune response. JENVAC is now licensed as a single-dose Japanese encephalitis vaccine.

Role of pattern recognition receptors and interferon-beta in protecting bat cell lines from encephalomyocarditis virus and Japanese encephalitis virus infection.

Bats are potential natural hosts of Encephalomyocarditis virus (EMCV) and Japanese encephalitis virus (JEV). Bats appear to have some unique features in their innate immune system that inhibit viral replication causing limited clinical symptoms, and thus, contributing to the virus spill over to humans. Here, kidney epithelial cell lines derived from four bat species (Pteropus dasymallus, Rousettus leschenaultii, Rhinolophus ferrumequinum, and Miniopterus fuliginosus) and two non-bat species (Homo sapiens and Mesocricetus auratus) were infected with EMCV and JEV. The replication of EMCV and JEV was lower in the bat cell lines derived from R. leschenaultii, R. ferrumequinum, and M. fuliginosus with a higher expression level of pattern recognition receptors (PRRs) (TLR3, RIG-I, and MDA5) and interferon-beta (IFN-ß) than that in the non-bat cell lines and a bat cell line derived from P. dasymallus. The knockdown of TLR3, RIG-I, and MDA5 in Rhinolophus bat cell line using antisense RNA oligonucleotide led to decrease IFN-ß expression and increased viral replication. These results suggest that TLR3, RIG-I, and MDA5 are important for antiviral response against EMCV and JEV in Rhinolophus bats.

Efficient control of Japanese encephalitis virus in the central nervous system of infected pigs occurs in the absence of a pronounced inflammatory immune response.

BACKGROUND: Japanese encephalitis virus (JEV) is the leading cause of viral encephalitis in Asia. JEV infection of mice and humans can lead to an uncontrolled inflammatory response in the central nervous system (CNS), resulting in a detrimental outcome. Pigs act as important amplification and reservoir hosts, and JEV infection of pigs is mostly subclinical. Information on virus spread in the CNS and immune responses controlling JEV infection in the CNS of pigs, however remains scarce. METHODS: Nine-week-old pigs were inoculated intranasal or intradermal with a relevant dose of 105 TCID50 of JEV genotype 3 Nakayama strain. Clinical signs were assessed daily, and viral spread was followed by RT-qPCR. mRNA expression profiles were determined to study immune responses in the CNS. RESULTS: Besides a delay of 2 days to reach the peak viremia upon intranasal compared to intradermal inoculation, the overall virus spread via both inoculation routes was highly similar. JEV appearance in lymphoid and visceral organs was in line with a blood-borne JEV dissemination. JEV showed a particular tropism to the CNS but without the induction of neurological signs. JEV entry in the CNS probably occurred via different hematogenous and neuronal pathways, but replication in the brain was mostly efficiently suppressed and associated with a type I IFN-independent activation of OAS1 expression. In the olfactory bulb and thalamus, where JEV replication was not completely controlled by this mechanism, a short but strong induction of chemokine gene expression was detected. An increased IFNy expression was simultaneously observed, probably originating from infiltrating T cells, correlating with a fast suppression of JEV replication. The chemokine response was however not associated with the induction of a strong inflammatory response, nor was an induction of the NLRP3 inflammasome observed. CONCLUSIONS: These findings indicate that an adequate antiviral response and an attenuated inflammatory response contribute to a favorable outcome of JEV infection in pigs and help to explain the limited neurological disease compared to other hosts. We show that the NLRP3 inflammasome, a key mediator of neurologic disease in mice, is not upregulated in pigs, further supporting its important role in JEV infections.

Inhibition of Japanese encephalitis virus infection by the host zinc-finger antiviral protein.

CCCH-type zinc-finger antiviral protein (ZAP) is a host factor that restricts the infection of many viruses mainly through RNA degradation, translation inhibition and innate immune responses. So far, only one flavivirus, yellow fever virus, has been reported to be ZAP-resistant. Here, we investigated the antiviral potential of human ZAP (isoform ZAP-L and ZAP-S) against three flaviviruses, Japanese encephalitis virus (JEV), dengue virus (DENV) and Zika virus (ZIKV). Infection of JEV but not DENV or ZIKV was blocked by ZAP overexpression, and depletion of endogenous ZAP enhanced JEV replication. ZAP hampered JEV translation and targeted viral RNA for 3'-5' RNA exosome-mediated degradation. The zinc-finger motifs of ZAP were essential for RNA targeting and anti-JEV activity. JEV 3'-UTR, especially in the region with dumbbell structures and high content of CG dinucleotide, was mapped to bind ZAP and confer sensitivity to ZAP. In summary, we identified JEV as the first ZAP-sensitive flavivirus. ZAP may act as an intrinsic antiviral factor through specific RNA binding to fight against JEV infection.

Serological evidence of DENV, JEV, and ZIKV among the indigenous people (Orang Asli) of Peninsular Malaysia.

Dengue virus (DENV), Japanese encephalitis virus (JEV), and Zika virus (ZIKV) are mosquito-borne flavivirus of medical importance in tropical countries such as Malaysia. However, much remains unknown regarding their prevalence among the underserved indigenous people (Orang Asli) living in communities in the forest fringe areas of Peninsular Malaysia. Information on the prevalence of diseases is necessary to elevate the effectiveness of disease control and preventive measures. This study aimed to determine the seroprevalence of the three major flaviviruses among the Orang Asli and investigate the association between demographic factors and seropositivities. Sampling activities were conducted in the Orang Asli villages to obtain serum samples and demographic data from consenting volunteers. The presence of DENV, JEV, and ZIKV immunoglobulin G (IgG) antibodies in the sera were examined using commercial enzyme-linked immunosorbent assay kits. A focus reduction neutralization assay was performed to measure virus-specific neutralizing antibodies. A total of 872 serum samples were obtained from the Orang Asli volunteers. Serological assay results revealed that DENV IgG, JEV IgG, and ZIKV IgG seropositivities among the Orang Asli were at 4.9%, 48.4%, and 13.2%, respectively. Neutralizing antibodies (FRNT50 ≥ 1:40) against JEV and ZIKV were found in 86.7% and 100.0%, respectively, out of the samples tested. Positive serology to all three viruses corresponded significantly to the age of the volunteers with increasing seropositivity in older volunteers. Findings from the study suggest that Orang Asli are at significant risk of contracting JEV and ZIKV infections despite the lack of active transmission of the viruses in the country.

A case series of inactivated Japanese encephalitis virus vaccination associated with positive West Nile virus blood donor screening nucleic acid tests.

BACKGROUND: West Nile Virus (WNV) is a member of the Japanese Encephalitis (JE) serocomplex within the Flaviviridae family. We report four whole blood donors and one plasma donor with WNV nucleic acid test (NAT)-reactive donations between September 2018 and November 2019, following recent Japanese Encephalitis virus (JEV) vaccination. CASE SERIES: Cases 1 and 4 had reactive WNV NAT donations 1 day after receiving the JEV vaccine. Case 2 had a reactive WNV donation 3 days after receiving the JEV vaccine. Case 3 had a reactive WNV NAT donation 3 days after returning from Arizona and 1 day after receiving the JEV vaccine. Case 5 had a reactive WNV donation the same day as receiving the JEV vaccine. STUDY DESIGN AND METHODS: WNV screening used the Roche cobas WNV nucleic acid test (NAT) (Roche Molecular Systems). Reference testing on WNV-reactive donations was carried out by the National Microbiology Laboratory (NML). JEV vaccine dilutions were also analyzed. RESULTS: Supplemental NAT was negative for WNV and JEV for Cases 1, 3, and 5. Case 2 had a weak amplification curve for one of two JEV NAT targets. Case 4 was JEV NAT-positive, WNV NAT-negative. Serologic testing on donation specimens for Cases 2, 4, and 5 did not support recent or remote WNV infection. JEV vaccine dilutions were detected by both cobas and supplemental NAT. CONCLUSIONS: We recommend implementing a temporary blood donor deferral following a JEV vaccination, if screening utilizes a WNV assay with the capability of detecting other members of the JE serocomplex.

Complete protection for mice conferred by a DNA vaccine based on the Japanese encephalitis virus P3 strain used to prepare the inactivated vaccine in China.

BACKGROUND: The incidence of Japanese encephalitis (JE) has been dramatically reduced in China after sufficient vaccine coverage. The live-attenuated Japanese encephalitis virus (JEV) vaccine SA14-14-2 is believed to have strongly contribute to this decrease. Another vaccine that seems to have decreased in importance is an inactivated vaccine based on the JEV P3 strain, which is considered to be modifiable, such as being transformed into a DNA vaccine to improve its immunogenicity. METHODS: In this study, the protective efficacy induced by the Japanese encephalitis DNA vaccine candidate pV-JP3ME encoding the premembrane (prM) and envelope (E) proteins of the P3 strain was assessed in BALB/c mice. The prM/E genes of the JEV P3 strain were subcloned into the vector pVAX1 (pV) to construct pV-JP3ME. RESULTS: The plasmid DNA was immunized into BALB/c mice, and high titers of IgG antibody and neutralizing antibody (nAb) against JEV were detected. The key cytokines in splenocytes were secreted upon stimulation with JEV antigens. Finally, complete protective efficacy was generated after challenge with the JEV P3 strain in the mice. CONCLUSIONS: The DNA vaccine pV-JP3ME based on the JEV P3 strain in this study can induce specific humoral immune and cytokine responses and provide complete protection against JEV in mice.

Non-structural protein 1 from Japanese encephalitis virus expressed in E. coli retains its molecular weight and immunogenicity.

Japanese encephalitis virus (JEV) is a member of the Flavivirus genus and has recently attracted attention as a high-risk pathogen in the Asia-Pacific region, with up to 30% mortality in the afflicted patients. Recent outbreaks of flavivirus-associated infections around the world have put the focus on non-structural protein 1 (NS1) as a candidate for diagnostic and vaccine researches on flaviviruses. Although the JEV NS1 protein has been expressed in eukaryotic cells, attempts to express JEV NS1 in E. coli are on due to advantages such as rapid growth, easy manipulation, low cost, and high yield. However, the challenges of low yield and poor solubility of the proteins expressed in E. coli remain to be overcome. Herein, we reported successful expression of the JEV NS1 protein in E. coli Rosetta(DE3) strain. We standardized the temperature, induction time, as well as the concentration of the inducer for optimizing the expression of JEV NS1 in E. coli. Further, we successfully obtained soluble JEV NS1 from inclusion bodies by partial refolding during elution and gradual refolding during dialysis. Furthermore, the JEV NS1 protein was found to retain its molecular weight and was able to induce an immune response in the mouse. Western blot and indirect enzyme-linked immunosorbent assay were performed using the blood of the immunized mouse and purified JEV NS1 in this study. Hence, JEV NS1 expressed in and isolated from E. coli Rosetta(DE3) strain holds potential for application in vaccine development and diagnostic studies to combat Japanese encephalitis outbreaks in the future.

The effects of Japanese encephalitis virus antibodies on Zika virus infection.

Recently, Zika virus (ZIKV) has become more widespread, thus attracting global attention. The vaccine against Japanese encephalitis virus (JEV) is currently used in China, being included in planned immunisation regimes. Although ZIKV and JEV are closely related mosquito-borne Flaviviruses, and a complex cross-immune response within flaviviruses has been demonstrated, the effect of JEV vaccination on ZIKV infection has not been well described. Thus, this study aimed to explore the impact of different titres of anti-JEV antibodies (Abs) against ZIKV infection using sera from healthy human donors in Guangzhou and anti-JEV rabbit polyclonal antibodies (pAbs) in vitro and vivo. Human anti-JEV Ab titres were tested at decreasing concentrations as the age increased. A neutralising effect on ZIKV infection was observed when anti-JEV Ab titres in human sera or rabbit pAbs were high (the corresponding age was under 30 years). Even though a lower titre in human sera showed no apparent effect, whereas rabbit pAbs had an antibody-dependent enhancement(ADE)effect, we proved an ADE effect in vivo for the first time. This study suggests that individuals over 60 years of age are at high risk for JEV and ZIKV infection, and screening this age group for infection should strengthen. Furthermore, a deep exploration of the relationship between anti-JEV Abs and ZIKV infection is needed.