Study on the temporal and spatial distribution of Culex mosquitoes in Hanoi, Vietnam.

Arboviruses transmitted by mosquitoes, including Japanese encephalitis virus (JEV), present a substantial global health threat. JEV is transmitted by mosquitoes in the genus Culex, which are common in both urban and rural areas in Vietnam. In 2020, we conducted a 1-year survey of Culex mosquito abundance in urban, suburban, and peri-urban areas of Hanoi using CDC-light traps. Mosquitoes were identified to species and sorted into pools based on species, sex, and trap location. The mosquito pools were also investigated by RT-qPCR for detection of JEV. In total, 4829 mosquitoes were collected over a total of 455 trap-nights, across 13 months. Collected mosquitoes included Culex, Aedes, Anopheles, and Mansonia species. Culex mosquitoes, primarily Cx. quinquefasciatus, predominated, especially in peri-urban areas. Most Culex mosquitoes were caught in the early months of the year. The distribution and abundance of mosquitoes exhibited variations across urban, suburban, and peri-urban sites, emphasizing the influence of environmental factors such as degree of urbanization, temperature and humidity on Culex abundance. No JEV was detected in the mosquito pools. This study establishes baseline knowledge of Culex abundance and temporal variation, which is crucial for understanding the potential for JEV transmission in Hanoi.

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.

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/ß 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-ß, and IFN-γ, was observed in the brain, while upregulation of IL-6, IFN-ß, 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.

Short-chain fatty acids abrogate Japanese encephalitis virus-induced inflammation in microglial cells via miR-200a-3p/ZBTB20/IKßα axis.

Japanese encephalitis virus (JEV), a member of the Flaviviridae family, is a leading cause of viral encephalitis in humans. Survivors of this infection often develop lifelong neurological sequelae. Short-chain fatty acids (SCFAs) produced in the gut are vital mediators of the gut-brain axis. We aimed to study microRNA-based mechanisms of SCFAs in an in vitro model of JEV infection. N9 microglial cells were pretreated with SCFA cocktail before JEV infection. Cytokine bead analysis, immunoblotting, and PCR were performed to analyze relevant inflammatory markers. microRNA sequencing was performed using Illumina Hiseq, and bioinformatics tools were used for differentially expressed (DE) miRNAs and weighted gene co-expression network analysis (WGCNA). microRNA mimic/inhibitor experiments and luciferase assay were performed to study miRNA-target interaction. A significant reduction in monocyte chemoattractant protein (MCP1) and tumor necrosis factor alpha (TNFα) along with reduced expression of phospho-nuclear factor kappa B (phospho-NF-κB) was observed in SCFA conditions. Significant attenuation of histone deacetylase activity and protein expression was recorded. miRNA sequencing revealed 160 DE miRNAs in SCFA + JEV-treated cells at 6 h post-infection. WGCNA revealed miR-200a-3p, a hub miRNA significantly upregulated in SCFA conditions. Transcription factor ZBTB20 was bioinformatically predicted and validated as a gene target for miR-200a-3p. Further miRNA mimic/inhibitor assay demonstrated that miR-200-3p regulated ZBTB20 along with Iκßα that possibly dampened NF-κB signal activation downstream. IMPORTANCE: The gut-brain axis plays a pivotal role in the physiological state of an organism. Gut microbiota-derived metabolites are known to play a role in brain disorders including neuroviral infections. Short-chain fatty acids (SCFAs) appear to quench inflammatory markers in Japanese encephalitis virus-infected microglial cells in vitro. Mechanistically, we demonstrate the interaction between miR-200a-3p and ZBTB20 in regulating the canonical nuclear factor kappa B (NF-κB) signaling pathway via transcriptional regulation of Iκßα. Findings of this study pave the way to a better understanding of SCFA mechanisms that can be used to develop strategies against viral neuroinflammation.

In vivo rescue of arboviruses directly from subgenomic DNA fragments.

Reverse genetic systems are mainly used to rescue recombinant viral strains in cell culture. These tools have also been used to generate, by inoculating infectious clones, viral strains directly in living animals. We previously developed the "Infectious Subgenomic Amplicons" (ISA) method, which enables the rescue of single-stranded positive sense RNA viruses in vitro by transfecting overlapping subgenomic DNA fragments. Here, we provide proof-of-concept for direct in vivo generation of infectious particles following the inoculation of subgenomic amplicons. First, we rescued a strain of tick-borne encephalitis virus in mice to transpose the ISA method in vivo. Subgenomic DNA fragments were amplified using a 3-fragment reverse genetics system and inoculated intramuscularly. Almost all animals were infected when quantities of DNA inoculated were at least 20 µg. We then optimized our procedure in order to increase the animal infection rate. This was achieved by adding an electroporation step and/or using a simplified 2- fragment reverse genetics system. Under optimal conditions, a large majority of animals were infected with doses of 20 ng of DNA. Finally, we demonstrated the versatility of this method by applying it to Japanese encephalitis and Chikungunya viruses. This method provides an efficient strategy for in vivo rescue of arboviruses. Furthermore, in the context of the development of DNA-launched live attenuated vaccines, this new approach may facilitate the generation of attenuated strains in vivo. It also enables to deliver a substance free of any vector DNA, which seems to be an important criterion for the development of human vaccines.

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.

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.

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

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.

Dissemination of the Flavivirus Subgenomic Replicon Genome and Viral Proteins by Extracellular Vesicles.

Extracellular vesicles (EVs) such as exosomes have been shown to play physiological roles in cell-to-cell communication by delivering various proteins and nucleic acids. In addition, several studies revealed that the EVs derived from the cells that are infected with certain viruses could transfer the full-length viral genomes, resulting in EVs-mediated virus propagation. However, the possibility cannot be excluded that the prepared EVs were contaminated with infectious viral particles. In this study, the cells that harbor subgenomic replicon derived from the Japanese encephalitis virus and dengue virus without producing any replication-competent viruses were employed as the EV donor. It was demonstrated that the EVs in the culture supernatants of those cells were able to transfer the replicon genome to other cells of various types. It was also shown that the EVs were incorporated by the recipient cells primarily through macropinocytosis after interaction with CD33 and Tim-1/Tim-4 on HeLa and K562 cells, respectively. Since the methods used in this study are free from contamination with infectious viral particles, it is unequivocally indicated that the flavivirus genome can be transferred by EVs from cell to cell, suggesting that this pathway, in addition to the classical receptor-mediated infection, may play some roles in the viral propagation and pathogenesis.

Identification critical host factors for Japanese encephalitis virus replication via CRISPR screening of human sgRNA library.

Japanese encephalitis virus (JEV) is a pathogen with a substantial impact on both livestock and human health. However, the critical host factors in the virus life cycle remain poorly understood. Using a library comprising 123411 small guide RNAs (sgRNAs) targeting 19050 human genes, we conducted a genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-based screen to identify essential genes for JEV replication. By employing knockout or knockdown techniques on genes, we identified eleven human genes crucial for JEV replication, such as prolactin releasing hormone receptor (PRLHR), activating signal cointegrator 1 complex subunit 3 (ASCC3), acyl-CoA synthetase long chain family member 3 (ACSL3), and others. Notably, we found that PRLHR knockdown blocked the autophagic flux, thereby inhibiting JEV infection. Taken together, these findings provide effective data for studying important host factors of JEV replication and scientific data for selecting antiviral drug targets.

Efficacy of genotype-matched vaccine against re-emerging genotype V Japanese encephalitis virus.

Japanese encephalitis (JE), caused by the Japanese encephalitis virus (JEV), is a highly threatening disease with no specific treatment. Fortunately, the development of vaccines has enabled effective defense against JE. However, re-emerging genotype V (GV) JEV poses a challenge as current vaccines are genotype III (GIII)-based and provide suboptimal protection. Given the isolation of GV JEVs from Malaysia, China, and the Republic of Korea, there is a concern about the potential for a broader outbreak. Under the hypothesis that a GV-based vaccine is necessary for effective defense against GV JEV, we developed a pentameric recombinant antigen using cholera toxin B as a scaffold and mucosal adjuvant, which was conjugated with the E protein domain III of GV by genetic fusion. This GV-based vaccine antigen induced a more effective immune response in mice against GV JEV isolates compared to GIII-based antigen and efficiently protected animals from lethal challenges. Furthermore, a bivalent vaccine approach, inoculating simultaneously with GIII- and GV-based antigens, showed protective efficacy against both GIII and GV JEVs. This strategy presents a promising avenue for comprehensive protection in regions facing the threat of diverse JEV genotypes, including both prevalent GIII and GI as well as emerging GV strains.

First Isolation of Japanese Encephalitis Virus Genotype IV from Mosquitoes in Australia.

Introduction: Widespread transmission of Japanese encephalitis virus (JEV) genotype four (GIV) occurred across mainland Australia in 2022. This resulted in forty-five human cases, including seven deaths, and the identification of JEV infection in over 80 commercial piggeries. Materials and Methods: We collected mosquitoes which were trapped using CO2-baited light traps deployed near piggeries reporting disease or in regions linked to human cases in the Wide Bay region in the state of Queensland. Mosquitoes from four traps yielded JEV RNA by real-time RT-PCR. Pools containing RNA positive mosquitoes were inoculated onto mosquito cell monolayers. Discussion: A single isolate of JEV was obtained from a pool of mixed mosquito species. Near whole genome sequencing and phylogenetic analysis of the JEV isolate demonstrated its high genomic relatedness with JEV GIV pig sequences sampled from Queensland and the state of New South Wales in 2022. Conclusion: We report the first isolation of JEV GIV from mosquitoes collected in Australia. With only a few JEV GIV isolates available globally, the isolate we report will be essential for future research of JEV host interactions, evolution and disease markers, and development of effective therapies, vaccines, diagnostic assays, and mosquito control strategies.

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.

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

Japanese encephalitis virus (JEV) is one of the major neurotropic viral infections that is known to dysregulate the homeostasis of neural stem/progenitor cells (NSPCs) and depletes the stem cell pool. NSPCs are multipotent stem cell population of the central nervous system (CNS) which are known to play an important role in the repair of the CNS during insults/injury caused by several factors such as ischemia, neurological disorders, CNS infections, and so on. Viruses have evolved to utilize host factors for their own benefit and during JEV infection, host factors, including the non-coding RNAs such as miRNAs, are reported to be affected, thereby cellular processes regulated by the miRNAs exhibit perturbed functionality. Previous studies from our laboratory have demonstrated the role of JEV infection in dysregulating the function of neural stem cells (NSCs) by altering the cell fate and depleting the stem cell pool leading to a decline in stem cell function in CNS repair mechanism post-infection. JEV-induced alteration in miRNA expression in the NSCs is one of the major interest to us. In prior studies, we have observed an altered expression pattern of certain miRNAs following JEV infection. In this study, we have validated the role of JEV infection in NSCs in altering the expression of miR-9-5p, which is a known regulator of neurogenesis in NSCs. Furthermore, we have validated the interaction of this miRNA with its target, Onecut2 (OC2), in primary NSCs utilizing miRNA mimic and inhibitor transfection experiments. Our findings indicate a possible role of JEV mediated dysregulated interaction between miR-9-5p and its putative target OC2 in NSPCs. IMPORTANCE: 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.

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

Background: 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. Methods: 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. Results: 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. Conclusion: 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.

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.

Low-temperature culture enhances production of flavivirus virus-like particles in mammalian cells.

Flavivirus virus-like particles (VLPs) exhibit a striking structural resemblance to viral particles, making them highly adaptable for various applications, including vaccines and diagnostics. Consequently, increasing VLPs production is important and can be achieved by optimizing expression plasmids and cell culture conditions. While attempting to express genotype III (GIII) Japanese encephalitis virus (JEV) VLPs containing the G104H mutation in the envelope (E) protein, we failed to generate VLPs in COS-1 cells. However, VLPs production was restored by cultivating plasmid-transfected cells at a lower temperature, specifically 28 °C. Furthermore, we observed that the enhancement in JEV VLPs production was independent of amino acid mutations in the E protein. The optimal condition for JEV VLPs production in plasmid-transfected COS-1 cells consisted of an initial culture at 37 °C for 6 h, followed by a shift to 28 °C (37/28 °C) for cultivation. Under 37/28 °C cultivation conditions, flavivirus VLPs production significantly increased in various mammalian cell lines regardless of whether its expression was transiently transfected or clonally selected cells. Remarkably, clonally selected cell lines expressing flavivirus VLPs consistently achieved yields exceeding 1 µg/ml. Binding affinity analyses using monoclonal antibodies revealed similar binding patterns for VLPs of genotype I (GI) JEV, GIII JEV, West Nile virus (WNV), and dengue virus serotype 2 (DENV-2) produced under both 37 °C or 37/28 °C cultivation conditions. In summary, our study demonstrated that the production of flavivirus VLPs can be significantly improved under 37/28 °C cultivation conditions without affecting the conformational structure of the E protein. KEYPOINTS: • Low-temperature culture (37/28 °C) enhances production of flavivirus VLPs. • Flavivirus VLPs consistently achieved yields exceeding 1 µg/ml. • 37/28 °C cultivation did not alter the structure of flavivirus VLPs.

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.

Palmitoylation of hIFITM1 inhibits JEV infection and contributes to BBB stabilization.

Human brain microvascular endothelial cells (hBMECs) are the main component cells of the blood-brain barrier (BBB) and play a crucial role in responding to viral infections to prevent the central nervous system (CNS) from viral invasion. Interferon-inducible transmembrane protein 1 (IFITM1) is a multifunctional membrane protein downstream of type-I interferon. In this study, we discovered that hIFITM1 expression was highly upregulated in hBMECs during Japanese encephalitis virus (JEV) infection. Depletion of hIFITM1 with CRISPR/Cas9 in hBMECs enhanced JEV replication, while overexpression of hIFITM1 restricted the viruses. Additionally, overexpression of hIFITM1 promoted the monolayer formation of hBMECs with a better integrity and a higher transendothelial electrical resistance (TEER), and reduced the penetration of JEV across the BBB. However, the function of hIFITM1 is governed by palmitoylation. Mutations of palmitoylation residues in conserved CD225 domain of hIFITM1 impaired its antiviral capacity. Moreover, mutants retained hIFITM1 in the cytoplasm and lessened its interaction with tight junction protein Occludin. Taken together, palmitoylation of hIFITM1 is essential for its antiviral activity in hBMECs, and more notably, for the maintenance of BBB homeostasis.

Comparative genomics of Japanese encephalitis virus shows low rates of recombination and a small subset of codon positions under episodic diversifying selection.

Orthoflavivirus japonicum (JEV) is the dominant cause of viral encephalitis in the Asian region with 100,000 cases and 25,000 deaths reported annually. The genome is comprised of a single polyprotein that encodes three structural and seven non-structural proteins. We collated a dataset of 349 complete genomes from a number of public databases, and analysed the data for recombination, evolutionary selection and phylogenetic structure. There are low rates of recombination in JEV, subsequently recombination is not a major evolutionary force shaping JEV. We found a strong overall signal of purifying selection in the genome, which is the main force affecting the evolutionary dynamics in JEV. There are also a small number of genomic sites under episodic diversifying selection, especially in the envelope protein and non-structural proteins 3 and 5. Overall, these results support previous analyses of JEV evolutionary genomics and provide additional insight into the evolutionary processes shaping the distribution and adaptation of this important pathogenic arbovirus.