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.

Increased IL-6 Levels and the Upregulation of Iron Regulatory Biomarkers Contribute to the Progression of Japanese Encephalitis Virus Infection's Pathogenesis.

Integrated analysis of iron regulatory biomarkers and inflammatory response could be an important strategy for Japanese encephalitis viral (JEV) infection disease management. In the present study, the inflammatory response was assessed by measuring serum Interleukin-6 (IL-6) levels using ELISA, and the transcription levels of iron homeostasis regulators were analyzed via RT-PCR. Furthermore, inter-individual variation in the transferrin gene was analyzed by PCR-RFLP and their association with clinical symptoms, susceptibility, severity, and outcomes was assessed through binary logistic regression and classification and regression tree (CART) analysis. Our findings revealed elevated levels of IL-6 in serum as well as increased expression of hepcidin (HAMP), transferrin (TF), and transferrin receptor-1 (TFR1) mRNA in JEV infection cases. Moreover, we found a genetic variation in TF (rs4481157) associated with clinical symptoms of meningoencephalitis. CART analysis indicates that individuals with the wild-type TF genotype are more susceptible to moderate JEV infection, while those with the homozygous type are in the high-risk group to develop a severe JEV condition. In summary, the study highlights that JEV infection induces alteration in both IL-6 levels and iron regulatory processes, which play pivotal roles in the development of JEV disease pathologies.

CD4 is an important host factor for Japanese encephalitis virus entry and replication in PK-15 cells.

Japanese encephalitis virus (JEV) is a flavivirus that is spread through mosquito bites and is the leading cause of viral encephalitis in Asia. JEV can infect a variety of cell types; however, crucial receptor molecules remain unclear. The purpose of this study was to determine whether porcine CD4 protein is a receptor protein that impacts JEV entry into PK15 cells and subsequent viral replication. We confirmed the interaction between the JEV E protein and the CD4 protein through Co-IP, virus binding and internalization, antibody blocking, and overexpression and created a PK-15 cell line with CD4 gene knockdown by CRISPR/Cas9. The results show that CD4 interacts with JEV E and that CD4 knockdown cells altered virus adsorption and internalization, drastically reducing virus attachment. The level of viral transcription in CD4 antibody-blocked cells, vs. control cells, was decreased by 49.1%. Based on these results, we believe that CD4 is a receptor protein for JEVs. Furthermore, most viral receptors appear to be associated with lipid rafts, and colocalization studies demonstrate the presence of CD4 protein on lipid rafts. RT‒qPCR and WB results show that virus replication was suppressed in PK-15-CD4KD cells. The difference in viral titer between KD and WT PK-15 cells peaked at 24 h, and the viral titer in WT PK-15 cells was 5.6 × 106, whereas in PK-15-CD4KD cells, it was only 1.8 × 106, a 64% drop, demonstrating that CD4 deficiency has an effect on the process of viral replication. These findings suggest that JEV enters porcine kidney cells via lipid raft-colocalized CD4, and the proliferation process is positively correlated with CD4.

CXCR3 antagonist rescues ER stress and reduces inflammation and JEV infection in mice brain.

The endoplasmic reticulum (ER) is crucial for maintaining cellular homeostasis, and synthesis and folding of proteins and lipids. The ER is sensitive to stresses including viral infection that perturb the intracellular energy level and redox state, and accumulating unfolded/misfolded proteins. Viruses including Japanese encephalitis virus (JEV) activates unfolded protein response (UPR) causing ER stress in host immune cells and promotes inflammation and apoptotic cell death. The chemokine receptor CXCR3 has been reported to play important role in the accumulation of inflammatory immune cells and neuronal cell death in several disease conditions. Recently we described the involvement of CXCR3 in regulating inflammation and JEV infection in mice brain. Supplementation with a CXCR3 antagonist AMG487 significantly reduced JEV infection in the mice brain in conjunction with the downregulation of UPR pathway via PERK:eIF2α:CHOP, and decreased mitochondrial ROS generation, inflammation and apoptotic cell death. Alongside, AMG487 treatment improved interferon (IFN)-α/ß synthesis in JEV-infected mice brain. Thus, suggesting a potential therapeutic role of CXCR3 antagonist against JEV infection.

Sodium Butyrate Induced Neural Stem/Progenitor Cell Death in an Experimental Model of Japanese Encephalitis.

The anti-inflammatory and neuroprotective effects of short chain fatty acid (SCFA) butyrate have been explored in a wide array of neurological pathologies. It is a 4-carbon SCFA produced from the fermentation of dietary fibers by the gut-microbiota. As evident from previous literature, butyrate plays a wide array of functions in CNS and interestingly enhances the differentiation potential of Neural stem/Progenitor Cells (NSPCs). Japanese encephalitis virus (JEV) is a well-known member of the Flaviviridae family and has been shown to alter neural stem cell pool of the brain, causing devastating consequences. In this study, we administered sodium butyrate (NaB) post JEV infection in BALB/c mouse model to examine any possible amelioration of the viral infection in NSPCs. In addition, ex vivo neurospheres and in vitro model of NSPCs were also used to study the effect of sodium butyrate in JEV infection. As an unprecedented finding, butyrate treated infected animals presented early onset of symptoms, as compared to their respective JEV infected groups. Alongside, we observed an increased viral load in NSPCs isolated from these animals as well as in cell culture models upon sodium butyrate treatment. Cytometric bead array analysis also revealed an increase in inflammatory cytokines, particularly, MCP-1 and IL-6. Further, increased expression of the key members of the canonical NF-κB pathway, viz-a-viz p-NF-κB, p-Iκ-Bα and p-IKK was observed. Overall, the increased inflammation and cell death caused early symptom progression in NaB-treated JEV infected animal model, which is contradictory to the well documented protective nature of NaB and therefore a better understanding of SCFA-based modulation of the gut-brain axis in viral infections is required.

VEGFR-3 signaling restrains the neuron-macrophage crosstalk during neurotropic viral infection.

Upon recognizing danger signals produced by virally infected neurons, macrophages in the central nervous system (CNS) secrete multiple inflammatory cytokines to accelerate neuron apoptosis. The understanding is limited about which key effectors regulate macrophage-neuron crosstalk upon infection. We have used neurotropic-virus-infected murine models to identify that vascular endothelial growth factor receptor 3 (VEGFR-3) is upregulated in the CNS macrophages and that virally infected neurons secrete the ligand VEGF-C. When cultured with VEGF-C-containing supernatants from virally infected neurons, VEGFR-3+ macrophages suppress tumor necrosis factor α (TNF-α) secretion to reduce neuron apoptosis. Vegfr-3ΔLBD/ΔLBD (deletion of ligand-binding domain in myeloid cells) mice or mice treated with the VEGFR-3 kinase inhibitor exacerbate the severity of encephalitis, TNF-α production, and neuron apoptosis post Japanese encephalitis virus (JEV) infection. Activating VEGFR-3 or blocking TNF-α can reduce encephalitis and neuronal damage upon JEV infection. Altogether, we show that the inducible VEGF-C/VEGFR-3 module generates protective crosstalk between neurons and macrophages to alleviate CNS viral infection.

Swine IFI6 confers antiviral effects against Japanese encephalitis virus in vitro and in vivo.

Swine are considered to be an important intermediate host in the cycle of Japanese encephalitis virus (JEV) infection. Most existing antiviral studies of JEV mainly focus on the host factor of the dead-end hosts. However, little research has addressed this in swine. Here, we found that swine interferon alpha-inducible protein 6 (sIFI6) possessed antiviral activity against JEV. In vitro studies showed that overexpression of sIFI6 inhibited the infection of JEV, while sIFI6 knockdown enhanced the infection of JEV in PK-15 cells. In addition, we also found that the structural integrity of sIFI6 was required by anti-JEV activity and that sIFI6 interacted with JEV nonstructural protein 4A (NS4A), an integral membrane protein with a pivotal function in replication complex during JEV replication. The interaction domain was mapped to the fourth transmembrane domain (TMD), also known as the 2K peptide of NS4A. The antiviral activity of sIFI6 was regulated by endoplasmic reticulum (ER) stress-related protein, Bip. In vivo studies revealed that sIFI6 alleviated symptoms of JEV infection in C57BL/6 mice. In addition, the antiviral spectrum of sIFI6 showed that sIFI6 specifically inhibited JEV infection. In conclusion, this study identified sIFI6 as a host factor against JEV infection for the first time. Our findings provide a potential drug target against JEV infection.

QKI deficiency in macrophages protects mice against JEV infection by regulating cell migration and antiviral response.

Japanese encephalitis (JE) is a major reason to cause viral encephalitis, with 50% patients suffering from severe neuro-inflammation and permanent neural injury. Effective anti-viral treatment is urgently needed. Here, we found RNA binding protein quaking (QKI) was involved in the progression of JE by regulating migration and anti-viral response of macrophages. After JE virus (JEV) infection, QKI-deficient mice had lower viral loads in the brain and fewer neurological symptoms. In comparison with control mice, proinflammatory cytokines in the brain of QKI-deficient animals revealed distinct patterns, with lower levels of IL-6 (interleukin-6) and IFN-ß (interferon-ß) at the early stage but higher levels at the end of JE. Then we found infiltration of CCR2 positive ((C-C motif) receptor 2) peripheral macrophages and CCR2 expression on macrophages were inhibited in QKI-deficient mice, while the expression of CCR2 ligands was not changed. Bioinformatical analysis showed that a QRE (quaking response element) located on 3'UTR (untranslated region) of Ccr2. We further verified that QKI was able to interact with Ccr2 mRNA and regulate its degradation in vitro. Additionally, since the IFN-ß production was increased in QKI-ablation mice after JEV infection, the anti-viral response was analyzed. Results in QKI-silenced N9 cells showed that the expression of RIG-I (retinoic acid-inducible gene-I) and TBK1 (TANK binding kinase 1) was increased, thus further inducing IRF3 (interferon regulatory factor 3) phosphorylation and interferon activation. Overall, these results revealed QKI mediated the anti-viral process via interfering migration of macrophages to CNS (central nervous system) and enhancing RIG-I/IRF3/IFN-ß pathway to restrict virus dissemination.

JEV Infection Induces M-MDSC Differentiation Into CD3+ Macrophages in the Brain.

Japanese encephalitis virus (JEV) is one of the most important members of the flavivirus family. It is a typical zoonotic pathogen that has caused substantial social and economic losses worldwide. The relation between JEV-induced immunosuppression and inflammatory responses has not been thoroughly investigated. In this study, cells infiltrating the brain tissue of JEV-infected mice were mainly identified as monocytic myeloid-derived suppressor cells (M-MDSCs), which subsequently differentiated into CD3+ macrophages. Co-culture with T cells showed that both splenic M-MDSCs and brain infiltrated M-MDSCs isolated from JEV-infected mice inhibited T cell proliferation through ARG1 and iNOS. The splenectomy model revealed that JEV-induced M-MDSCs were mainly derived from bone marrow and migrated to the spleen and central nervous system (CNS). The results of the transcriptome analysis and IRF7-deficient mice indicated that the ZBP1-IRF7 signaling pathway stimulated by JEV RNA played a central role in the induction of M-MDSCs. M-MDSCs migrated into the CNS through the chemokine CCL2/N-CCL2 derived from astrocytes and brain infiltrated M-MDSCs differentiated into CD3+ macrophages through a mechanism mediated by M-CSF, IL-6 and IFN-γ in the brain microenvironment. These findings provide evidence for the mechanism that JEV regulates the differentiation of M-MDSCs and thereby exacerbates pathogenicity, which represents a potential therapeutic target for Japanese encephalitis (JE).

Japanese Encephalitis Virus NS4A Protein Interacts with PTEN-Induced Kinase 1 (PINK1) and Promotes Mitophagy in Infected Cells.

The nonstructural protein 4A (NS4A) of flaviviruses has been implicated as a "central organizer" of the membrane-bound replication complex during virus replication. However, its role in the host responses to virus infection is not understood. Using the yeast-two-hybrid library screen, we identified a multitude of host proteins interacting with the Japanese encephalitis virus (JEV) NS4A protein. Several of these interacting proteins are known to localize to the mitochondria. One of these proteins was PTEN-induced kinase 1 (PINK1), a serine/threonine-protein kinase known for its role in mitophagy. Here, we demonstrate the JEV-NS4A localization to the mitochondria and its interaction with PINK1 in Huh7 cells during JEV infection. The JEV-infected cells showed an enhanced mitophagy flux with a concomitant decline in the mitochondrial mass. We present data showing that JEV-NS4A alone was sufficient to induce mitophagy. Interference with mitochondrial fragmentation and mitophagy resulted in reduced virus propagation. Overall, our study provides the first evidence of mitochondrial quality control dysregulation during JEV infection, largely mediated by its NS4A protein. IMPORTANCE The JEV-infected mammalian cells show an enhanced mitophagy flux with a concomitant decline in the mitochondrial mass. We show that the NS4A protein of JEV localized to the mitochondria and interacted with PINK1 in Huh7 cells during infection with the virus and demonstrate that JEV-NS4A alone is sufficient to induce mitophagy. The study provides the first evidence of mitochondrial quality control dysregulation during JEV infection, largely mediated by its NS4A protein.

Japanese encephalitis viral infection modulates proinflammatory cyto/chemokine profile in primary astrocyte and cell line of astrocytic origin.

Japanese Encephalitis Virus (JEV) is a neurotropic virus that invades Central Nervous System (CNS) and causes severe neuroinflammation. Given the abundance and the position of astrocytes in the CNS, we speculate that they might play a critical role in the process of neuroinflammation. Unfortunately, the role of astrocytes in JEV-mediated neuroinflammation has long been understated. In this study, we have attempted to assess the role of astrocyte-mediated neuroinflammation upon JEV infection. Mouse model of JEV infection, generated by intraperitoneal injection, showed severe reactive astrogliosis. To further address our hypothesis, we employed immortalized astrocytic cell line (in vitro) and primary astrocyte-enriched culture (ex vivo) as experimental models. JEV infection in the astrocytes induces proinflammatory cytokines like MCP1/CCL2 and IL6 in both ex vivo and in vitro cultures as observed from the cytometric bead array analysis. A significantly altered cytokine profile was observed using PCR analysis in in vitro and ex vivo models upon infection, with respect to control, validating our previous results. We also show that there exists a major inconsistency in the viral replication kinetics, wherein the cell line showed a robust rate of replication whereas the primary astrocyte-enriched culture showed negligibly low number of plaques, underlining the importance of the selection of appropriate experimental model system. In conclusion, we claim that astrocytes significantly contribute to JEV-mediated neuroinflammation, despite not being a CNS immune cell.

Proteomic landscape of Japanese encephalitis virus-infected fibroblasts.

Advances in proteomics have enabled a comprehensive understanding of host-pathogen interactions. Here we have characterized Japanese encephalitis virus (JEV) infection-driven changes in the mouse embryonic fibroblast (MEF) proteome. Through tandem mass tagging (TMT)-based mass spectrometry, we describe changes in 7.85 % of the identified proteome due to JEV infection. Pathway enrichment analysis showed that proteins involved in innate immune sensing, interferon responses and inflammation were the major upregulated group, along with the immunoproteasome and poly ADP-ribosylation proteins. Functional validation of several upregulated anti-viral innate immune proteins, including an active cGAS-STING axis, was performed. Through siRNA depletion, we describe a crucial role of the DNA sensor cGAS in restricting JEV replication. Further, many interferon-stimulated genes (ISGs) were observed to be induced in infected cells. We also observed activation of TLR2 and inhibition of TLR2 signalling using TLR1/2 inhibitor CU-CPT22-blocked production of inflammatory cytokines IL6 and TNF-α from virus-infected N9 microglial cells. The major proteins that were downregulated by infection were involved in cell adhesion (collagens), transport (solute carrier and ATP-binding cassette transporters), sterol and lipid biosynthesis. Several collagens were found to be transcriptionally downregulated in infected MEFs and mouse brain. Collectively, our data provide a bird's-eye view into how fibroblast protein composition is rewired following JEV infection.

Pre-treatment with Scopolamine Naturally Suppresses Japanese Encephalitis Viral Load in Embryonated Chick Through Regulation of Multiple Signaling Pathways.

Suitable recognition of invasive microorganisms is a crucial factor for evoking a strong immune response that can combat the pathogen. Toll-like receptors (TLRs) play a pivotal role in the induction of this innate immune response through stimulation of interferons (IFNs) that control viral replication in the host via distinct signaling pathways. Though the antiviral property of Atropa belladonna has been established, yet the role of one of its active components scopolamine in modulating various factors of the innate immune branch has not yet been investigated until date. Thus, the present study was conducted to assess the antiviral effects of scopolamine and its immunomodulatory role against Japanese encephalitis virus (JEV) infections in embryonated chick. Pre-treatment with scopolamine hydrobromide showed a significant decrease in the viral loads of chorioallantoic membrane (CAM) and brain tissues. Molecular docking analysis revealed that scopolamine hydrobromide binds to the active site of non-structural protein 5 (NS5) that has enzymatic activities required for replication of JEV, making it a highly promising chemical compound against the virus. The binding contributions of different amino acid residues at or near the active site suggest a potential binding of this compound. Pre-treatment with the scopolamine hydrobromide showed significant upregulation of different TLRs like TLR3, TLR7, and TLR8, interleukins like IL-4, and IL-10, as well as IFNs and their regulatory factors. However, virus-infected tissues (direct infection group) exhibited higher TLR4 expression as compared to scopolamine hydrobromide pre-treated, virus-infected tissues (medicine pre-treated group). These results indicate that scopolamine hydrobromide contributes much to launch antiviral effects by remoulding the TLR and IFN signaling pathways that are involved in sensing and initiating the much-needed anti-JEV responses.

p53 promotes ZDHHC1-mediated IFITM3 palmitoylation to inhibit Japanese encephalitis virus replication.

The tumor suppressor p53 as an innate antiviral regulator contributes to restricting Japanese encephalitis virus (JEV) replication, but the mechanism is still unclear. The interferon-induced transmembrane protein 3 (IFITM3) is an intrinsic barrier to a range of virus infection, whether IFITM3 is responsible for the p53-mediated anti-JEV response remains elusive. Here, we found that IFITM3 significantly inhibited JEV replication in a protein-palmitoylation-dependent manner and incorporated into JEV virions to diminish the infectivity of progeny viruses. Palmitoylation was also indispensible for keeping IFITM3 from lysosomal degradation to maintain its protein stability. p53 up-regulated IFITM3 expression at the protein level via enhancing IFITM3 palmitoylation. Screening of palmitoyltransferases revealed that zinc finger DHHC domain-containing protein 1 (ZDHHC1) was transcriptionally up-regulated by p53, and consequently ZDHHC1 interacted with IFITM3 to promote its palmitoylation and stability. Knockdown of IFITM3 significantly impaired the inhibitory role of ZDHHC1 on JEV replication. Meanwhile, knockdown of either ZDHHC1 or IFITM3 expression also compromised the p53-mediated anti-JEV effect. Interestingly, JEV reduced p53 expression to impair ZDHHC1 mediated IFITM3 palmitoylation for viral evasion. Our data suggest the existence of a previously unrecognized p53-ZDHHC1-IFITM3 regulatory pathway with an essential role in restricting JEV infection and provide a novel insight into JEV-host interaction.

Japanese Encephalitis Virus exploits microRNA-155 to suppress the non-canonical NF-κB pathway in human microglial cells.

Japanese Encephalitis Virus (JEV) is a single positive strand RNA virus, belongs to the Flaviviridae family. JEV is neurotropic in nature which accounts for 30-50% neurological, psychiatric sequelae and movement disorder, with 20-30% case fatality rate among children or elder population. JEV causes neuronal loss and microglial activation which leads to neuroinflammation. The microRNAs are the molecular switches, which regulate the gene expression post-transcriptionally. The microRNA-155 has been reported to be associated with CNS-related pathologies like, experimental autoimmune encephalitis, multiple sclerosis and amyotrophic lateral sclerosis. In the present study, we infected microglial cells with JEV, which resulted in the up-regulation of microRNA-155; quantified by real-time polymerase chain reaction. The gene target prediction databases revealed pellino 1 as a putative gene target for microRNA-155. The over-expression based studies of microRNA-155 mimics, scrambles, inhibitors, and cy3 negative control demonstrated the role of PELI1 in the regulation of the non-canonical NF-κB pathway via TRAF3. The luciferase assay showed the regulation of NF-κB promoter via microRNA-155 in JEV infected microglial cells. The suppression of NF-κB in JEV infected microglial cells led to the reduced expression of IL-6 and TNF-α. JEV exploits cellular microRNA-155 to suppress the expression of PELI1 in human microglial cells as a part of their immune evasion strategy.

Ezrin is essential for the entry of Japanese encephalitis virus into the human brain microvascular endothelial cells.

Japanese encephalitis virus (JEV) remains the predominant cause of viral encephalitis worldwide. It reaches the central nervous system upon crossing the blood-brain barrier through pathogenic mechanisms that are not completely understood. Here, using a high-throughput siRNA screening assay combined with verification experiments, we found that JEV enters the primary human brain microvascular endothelial cells (HBMEC) through a caveolae-mediated endocytic pathway. The role of ezrin, an essential host factor for JEV entry based on our screening, in caveolae-mediated JEV internalization was investigated. We observed that JEV internalization in HBMEC is largely dependent on ezrin-mediated actin cytoskeleton polymerization. Moreover, Src, a protein predicted by a STRING database search, was found to be required in JEV entry. By a variety of pharmacological inhibition and immunoprecipitation assays, we found that Src, ezrin, and caveolin-1 were sequentially activated and formed a complex during JEV infection. A combination of in vitro kinase assay and subcellular analysis demonstrated that ezrin is essential for Src-caveolin-1 interactions. In vivo, both Src and ezrin inhibitors protected ICR suckling mice against JEV-induced mortality and diminished mouse brain viral load. Therefore, JEV entry into HBMEC requires the activation of the Src-ezrin-caveolin-1 signalling axis, which provides potential targets for restricting JEV infection.

Comparison of a human neuronal model proteome upon Japanese encephalitis or West Nile Virus infection and potential role of mosquito saliva in neuropathogenesis.

Neurotropic flavivirus Japanese encephalitis virus (JEV) and West Nile virus (WNV) are amongst the leading causes of encephalitis. Using label-free quantitative proteomics, we identified proteins differentially expressed upon JEV (gp-3, RP9) or WNV (IS98) infection of human neuroblastoma cells. Data are available via ProteomeXchange with identifier PXD016805. Both viruses were associated with the up-regulation of immune response (IFIT1/3/5, ISG15, OAS, STAT1, IRF9) and the down-regulation of SSBP2 and PAM, involved in gene expression and in neuropeptide amidation respectively. Proteins associated to membranes, involved in extracellular matrix organization and collagen metabolism represented major clusters down-regulated by JEV and WNV. Moreover, transcription regulation and mRNA processing clusters were also heavily regulated by both viruses. The proteome of neuroblastoma cells infected by JEV or WNV was significantly modulated in the presence of mosquito saliva, but distinct patterns were associated to each virus. Mosquito saliva favored modulation of proteins associated with gene regulation in JEV infected neuroblastoma cells while modulation of proteins associated with protein maturation, signal transduction and ion transporters was found in WNV infected neuroblastoma cells.

Transcriptomic Analysis Suggests the M1 Polarization and Launch of Diverse Programmed Cell Death Pathways in Japanese Encephalitis Virus-Infected Macrophages.

The Japanese encephalitis virus (JEV) is a Culex mosquito-borne flavivirus and is the pathogenic agent of Japanese encephalitis, which is the most important type of viral encephalitis in the world. Macrophages are a type of pivotal innate immunocyte that serve as sentinels and respond quickly to pathogen invasions. However, some viruses like JEV can hijack macrophages as a refuge for viral replication and immune escape. Despite their crucial involvement in early JEV infection, the transcriptomic landscapes of JEV-infected macrophages are void. Here, by using an in situ JEV infection model, we investigate the transcriptomic alteration of JEV-infected peritoneal macrophages. We found that, upon JEV infection, the macrophages underwent M1 polarization and showed the drastic activation of innate immune and inflammatory pathways. Interestingly, almost all the programmed cell death (PCD) pathways were activated, especially the apoptosis, pyroptosis, and necroptosis pathways, which were verified by the immunofluorescent staining of specific markers. Further transcriptomic analysis and TUNEL staining revealed that JEV infection caused apparent DNA damage. The transcriptomic analysis also revealed that JEV infection promoted ROS and RNS generation and caused oxidative stress, which activated multiple cell death pathways. Our work uncovers the pivotal pathogenic roles of oxidative stress and multiple PCD pathways in JEV infection, providing a novel perspective on JEV-host interactions.

Hsp40 Protein DNAJB6 Interacts with Viral NS3 and Inhibits the Replication of the Japanese Encephalitis Virus.

The Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus prevalent in east and southeast Asia, the Western Pacific, and northern Australia. Since viruses are obligatory intracellular pathogens, the dynamic processes of viral entry, replication, and assembly are dependent on numerous host-pathogen interactions. Efforts to identify JEV-interacting host factors are ongoing because their identification and characterization remain incomplete. Three enzymatic activities of flavivirus non-structural protein 3 (NS3), including serine protease, RNA helicase, and triphosphatase, play major roles in the flaviviruses lifecycle. To identify cellular factors that interact with NS3, we screened a human brain cDNA library using a yeast two-hybrid assay, and identified eight proteins that putatively interact with NS3: COPS5, FBLN5, PPP2CB, CRBN, DNAJB6, UBE2N, ZNF350, and GPR137B. We demonstrated that the DnaJ heat shock protein family (Hsp40) member B6 (DNAJB6) colocalizes and interacts with NS3, and has a negative regulatory function in JEV replication. We also show that loss of DNAJB6 function results in significantly increased viral replication, but does not affect viral binding or internalization. Moreover, the time-course of DNAJB6 disruption during JEV infection varies in a viral load-dependent manner, suggesting that JEV targets this host chaperone protein for viral benefit. Deciphering the modes of NS3-interacting host proteins functions in virion production will shed light on JEV pathogenic mechanisms and may also reveal new avenues for antiviral therapeutics.

Elevation of Cleaved p18 Bax Levels Associated with the Kinetics of Neuronal Cell Death during Japanese Encephalitis Virus Infection.

Japanese encephalitis virus (JEV) infection induces uncontrolled neuronal apoptosis, leading to irreversible brain damage. However, the mechanism of JEV-induced neuronal apoptosis has not been clearly elucidated. This study aimed to investigate both virus replication and neuronal cell apoptosis during JEV infection in human neuroblastoma SH-SY5Y cells. As a result, the kinetic productions of new viral progeny were time- and dose-dependent. The stimulation of SH-SY5Y cell apoptosis was dependent on the multiplicity of infections (MOIs) and infection periods, particularly during the late period of infection. Interestingly, we observed that of full-length Bax (p21 Bax) level started to decrease, which corresponded to the increased level of its cleaved form (p18 Bax). The formation of p18 Bax resulting in cytochrome c release into the cytosol appeared to correlate with JEV-induced apoptotic cell death together with the activation of caspase-3/7 activity, especially during the late stage of a robust viral infection. Therefore, our results suggest another possible mechanism of JEV-induced apoptotic cell death via the induction of the proteolysis of endogenous p21 Bax to generate p18 Bax. This finding could be a new avenue to facilitate novel drug discovery for the further development of therapeutic treatments that could relieve neuronal damage from JEV infection.