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

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

TLR9 promotes monocytic myeloid-derived suppressor cell induction during JEV infection.

Myeloid-derived suppressor cells (MDSCs) are a group of heterologous populations of immature bone marrow cells consisting of progenitor cells of macrophages, dendritic cells and granulocytes. Recent studies have revealed that the accumulation of MDSCs in the mouse spleen plays a pivotal role in suppressing the immune response following JEV infection. However, the mechanisms by which JEV induces MDSCs are poorly understood. Here, it was found that JEV infection induces mitochondrial damage and the release of mitochondrial DNA (mtDNA), which further leads to the activation of TLR9. TLR9 deficiency decreases the M-MDSCs population and their suppressive function both in vitro and in vivo. Moreover, the increase of MHCⅡ expression on antigen-presenting cells and CD28 expression on T cells in TLR9-/- mice was positively correlated with M-MDSCs reduction. Accordingly, the survival rate of TLR9-/- mice dramatically increased after JEV infection. These findings reveal the connections of mitochondrial damage and TLR9 activation to the induction of M-MDSCs during JEV infection.

A fusion protein of vimentin with Fc fragment inhibits Japanese encephalitis virus replication.

Japanese encephalitis virus (JEV), a member of the Flaviviridae family and a flavivirus, is known to induce acute encephalitis. Vimentin protein has been identified as a potential receptor for JEV, engaging in interactions with the viral membrane protein. The Fc fragment, an integral constituent of immunoglobulins, plays a crucial role in antigen recognition by dendritic cells (DCs) or phagocytes, leading to subsequent antigen presentation, cytotoxicity, or phagocytosis. In this study, we fused the receptor of JEV vimentin with the Fc fragment of IgG and expressed the resulting vimentin-Fc fusion protein in Escherichia coli. Pull-down experiments demonstrated the binding ability of the vimentin-Fc fusion protein to JEV virion in vitro. Additionally, we conducted inhibition assays at the cellular level, revealing the ability of vimentin-Fc protein suppressing JEV replication, it may be a promising passive immunotherapy agent for JEV. These findings pave the way for potential therapeutic strategies against JEV.

Single-cell RNA sequencing reveals the immune features and viral tropism in the central nervous system of mice infected with Japanese encephalitis virus.

Japanese encephalitis virus (JEV) is a neurotropic pathogen that causes lethal encephalitis. The high susceptibility and massive proliferation of JEV in neurons lead to extensive neuronal damage and inflammation within the central nervous system. Despite extensive research on JEV pathogenesis, the effect of JEV on the cellular composition and viral tropism towards distinct neuronal subtypes in the brain is still not well comprehended. To address these issues, we performed single-cell RNA sequencing (scRNA-seq) on cells isolated from the JEV-highly infected regions of mouse brain. We obtained 88,000 single cells and identified 34 clusters representing 10 major cell types. The scRNA-seq results revealed an increasing amount of activated microglia cells and infiltrating immune cells, including monocytes & macrophages, T cells, and natural killer cells, which were associated with the severity of symptoms. Additionally, we observed enhanced communication between individual cells and significant ligand-receptor pairs related to tight junctions, chemokines and antigen-presenting molecules upon JEV infection, suggesting an upregulation of endothelial permeability, inflammation and antiviral response. Moreover, we identified that Baiap2-positive neurons were highly susceptible to JEV. Our findings provide valuable clues for understanding the mechanism of JEV induced neuro-damage and inflammation as well as developing therapies for Japanese encephalitis.

Japanese encephlu emergence in Australia: the potential population at risk.

Japanese encephalitis virus (JEV), an RNA virus transmitted by Culex mosquitoes, primarily cycles between aquatic birds and mosquitoes with pigs as amplifying hosts, posing a significant global encephalitis threat. The emergence and spread of the JEV in new epidemiological regions, such as recent cases in Australia and nonendemic areas like Pune, India, raise significant concerns. With an estimated 68 000 clinical cases and 13 600 to 20 400 deaths annually, JEV poses a substantial global health threat. The virus primarily affects children, with a case-fatality ratio of 20-30% and long-term neurological sequelae in survivors. The changing epidemiology, influenced by factors like bird migration, climate change, and increased urbanization, contributes to the geographic expansion of JEV. The recent outbreaks underscore the potential for the virus to establish itself in nonendemic regions, posing a threat to populations previously considered at low-risk. With limited treatment options and high rates of neurological complications, continued surveillance, traveler vaccination, and research into treatments are crucial to mitigate the impact of JEV on human health. The evolving scenario necessitates proactive measures to prevent and control the spread of the virus in both endemic and newly affected areas.

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

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

Current Advances in Japanese Encephalitis Virus Drug Development.

Japanese encephalitis virus (JEV) belongs to the Flaviviridae family and is a representative mosquito-borne flavivirus responsible for acute encephalitis and meningitis in humans. Despite the availability of vaccines, JEV remains a major public health threat with the potential to spread globally. According to the World Health Organization (WHO), there are an estimated 69,000 cases of JE each year, and this figure is probably an underestimate. The majority of JE victims are children in endemic areas, and almost half of the surviving patients have motor or cognitive sequelae. Thus, the absence of a clinically approved drug for the treatment of JE defines an urgent medical need. Recently, several promising and potential drug candidates were reported through drug repurposing studies, high-throughput drug library screening, and de novo design. This review focuses on the historical aspects of JEV, the biology of JEV replication, targets for therapeutic strategies, a target product profile, and drug development initiatives.

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.

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.

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

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

Ferroptosis contributes to JEV-induced neuronal damage and neuroinflammation.

Ferroptosis is a newly discovered prototype of programmed cell death (PCD) driven by iron-dependent phospholipid peroxidation â€‹accumulation, and it has been linked to numerous organ injuries and degenerative pathologies. Although studies have shown that a variety of cell death processes contribute to JEV-induced neuroinflammation and neuronal injury, there is currently limited research on the specific involvement of ferroptosis. In this study, we explored the neuronal ferroptosis induced by JEV infection in vitro and in vivo. Our results indicated that JEV infection induces neuronal ferroptosis through inhibiting the function of the antioxidant system mediated by glutathione (GSH)/glutathione peroxidase 4 (GPX4), as well as by promoting lipid peroxidation mediated by yes-associated protein 1 (YAP1)/long-chain acyl-CoA synthetase 4 (ACSL4). Further analyses revealed that JEV E and prM proteins function as agonists, inducing ferroptosis. Moreover, we found that treatment with a ferroptosis inhibitor in JEV-infected mice reduces the viral titers and inflammation in the mouse brains, ultimately improving the survival rate of infected mice. In conclusion, our study unveils a critical role of ferroptosis in the pathogenesis of JEV, providing new ideas for the prevention and treatment of viral encephalitis.

Docking and MM study of non-structural protein (NS5) of Japanese Encephalitis Virus (JEV) with some derivatives of adenosyl.

Introduction: The flavivirus NS5, a non-structural protein of Japanese Encephalitis Virus (JEV), a serious deadly human pathogen responsible for epidemics in South East Asia, consists of N-terminal methyl transferase (MTase) domain and RNA-dependent RNA polymerase (RdRp) is known for unique viral genome replication and cap formation activity. S-adenosyl executes a crucial function in these viral activities. S-adenosyl derivatives are chosen as potential binders with the MTase domain of NS5 based on MM and docking studies. Methods: MM GBSA (Generalized Born Surface Area) simulation were performed to evaluate the binding energy, following the 100 nanosecond (ns) production MD simulation in the periodic boundary condition (PBC) for the selected docked ligands with NS5. Quasi-harmonic entropy of the ligands was also calculated with semi-empirical calculations at the PM3/PM6 level supporting docking and MM-GBSA results. Results and discussion: The residue-wise decomposition energy reveals that the key hydrophobic residues Gly 81, Phe 133, and Ile 147 in the RdRp-MTase interface, indicate the biological relevance. These residues act as the key residue stabilizer, binding vigorously with S-Adenosyl derivatives in the vicinity of the interface between the MTase domain and RdRp. This paves the way for the other potential drug as an inhibitor for the enzymatic activity of the NS5.

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.

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

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

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

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

Development of a quadruplex real-time quantitative RT-PCR for detection and differentiation of PHEV, PRV, CSFV, and JEV.

Porcine hemagglutinating encephalomyelitis virus (PHEV), porcine pseudorabies virus (PRV), classical swine fever virus (CSFV), and Japanese encephalitis virus (JEV) cause similar neurological symptoms in the infected pigs, and their differential diagnosis depends on laboratory testing. Four pairs of specific primers and probes were designed targeting the PHEV N gene, PRV gB gene, CSFV 5' untranslated region (5'UTR), and JEV NS1 gene, respectively, and a quadruplex real-time quantitative RT-PCR (qRT-PCR) was developed to detect and differentiate PHEV, PRV, CSFV, and JEV. The assay showed high sensitivity, with the limit of detection (LOD) of 1.5 × 101 copies/µL for each pathogen. The assay specifically detected only PHEV, PRV, CSFV, and JEV, without cross-reaction with other swine viruses. The coefficients of variation (CVs) of the intra-assay and the inter-assay were less than 1.84%, with great repeatability. A total of 1,977 clinical samples, including tissue samples, and whole blood samples collected from Guangxi province in China, were tested by the developed quadruplex qRT-PCR, and the positivity rates of PHEV, PRV, CSFV, and JEV were 1.57% (31/1,977), 0.35% (7/1,977), 1.06% (21/1,977), and 0.10% (2/1,977), respectively. These 1,977 samples were also tested by the previously reported qRT-PCR assays, and the coincidence rates of these methods were more than 99.90%. The developed assay is demonstrated to be rapid, sensitive, and accurate for detection and differentiation of PHEV, PRV, CSFV, and JEV.

Priming with Japanese encephalitis virus or yellow fever virus vaccination led to the recognition of multiple flaviviruses without boosting antibody responses induced by an inactivated Zika virus vaccine.

BACKGROUND: Complex patterns of cross-reactivity exist between flaviviruses, yet there is no precise understanding of how sequential exposures due to flavivirus infections or vaccinations impact subsequent antibody responses. METHODS: We investigated whether B cell priming from Japanese encephalitis virus (JEV) or yellow fever virus (YFV) vaccination impacted binding and functional antibody responses to flaviviruses following vaccination with a Zika virus (ZIKV) purified inactivated virus (ZPIV) vaccine. Binding antibody responses and Fc gamma receptor engagement against 23 flavivirus antigens were characterized along with neutralization titres and Fc effector responses in 75 participants at six time points. FINDINGS: We found no evidence that priming with JEV or YFV vaccines improved the magnitude of ZPIV induced antibody responses to ZIKV. Binding antibodies and Fc gamma receptor engagement to ZIKV antigens did not differ significantly across groups, while antibody-dependent cellular phagocytosis (ADCP) and neutralizing responses were higher in the naïve group than in the JEV and YFV primed groups following the second ZPIV immunization (p ≤ 0.02). After a third dose of ZPIV, ADCP responses remained higher in the naïve group than in the primed groups. However, priming affected the quality of the response following ZPIV vaccination, as primed individuals recognized a broader array of flavivirus antigens than individuals in the naïve group. INTERPRETATION: While a priming vaccination to either JEV or YFV did not boost ZIKV-specific responses upon ZIKV vaccination, the qualitatively different responses elicited in the primed groups highlight the complexity in the cross-reactive antibody responses to flaviviruses. FUNDING: This work was supported by a cooperative agreement between The Henry M. Jackson Foundation for the Advancement of Military Medicine, Inc., and the U.S. Department of the Army [W81XWH-18-2-0040]. The work was also funded in part by the National Institute of Allergy and Infectious Diseases (NIAID) R01AI155983 to SJK and KM.

Regulation of microglia-mediated inflammation by host lncRNA Gm20559 upon flaviviral infection.

BACKGROUND: Japanese Encephalitis Virus (JEV) and West Nile Viruses (WNV) are neurotropic flaviviruses which cause neuronal death and exaggerated glial activation in the central nervous system. Role of host long non coding RNAs in shaping microglial inflammation upon flavivirus infections has been unexplored. This study attempted to decipher the role of lncRNA Gm20559 in regulating microglial inflammatory response in context of flaviviruses. METHODS: Antisense oligonucleotide LNA Gapmers designed against lncRNA Gm20559 and non-specific site (negative control) were used for Gm20559 knockdown in JEV and WNV-infected N9 microglial cells. Upon establishing successful Gm20559 knockdown, expression of various proinflammatory cytokines, chemokines, interferon-stimulated genes (ISGs) and RIG-I were checked by qRT-PCR and cytometric bead array. Western Blotting was done to analyse the phosphorylation level of various inflammatory markers and viral non-structural protein expression. Plaque Assays were employed to quantify viral titres in microglial supernatant upon knocking down Gm20559. Effect of microglial supernatant on HT22 neuronal cells was assessed by checking expression of apoptotic protein and viral non-structural protein by Western Blotting. RESULTS: Upregulation in Gm20559 expression was observed in BALB/c pup brains, primary microglia as well as N9 microglia cell line upon both JEV and WNV infection. Knockdown of Gm20559 in JEV and WNV-infected N9 cell led to the reduction of major proinflammatory cytokines - IL-1ß, IL-6, IP-10 and IFN-ß. Inhibition of Gm20559 upon JEV infection in N9 microglia also led to downregulation of RIG-I and OAS-2, which was not the case in WNV-infected N9 microglia. Phosphorylation level of P38 MAPK was reduced in case of JEV-infected N9 microglia and not WNV-infected N9 microglia. Whereas phosphorylation of NF-κB pathway was unchanged upon Gm20559 knockdown in both JEV and WNV-infected N9 microglia. However, treating HT22 cells with JEV and WNV-infected microglial supernatant with and without Gm20559 could not trigger cell death or influence viral replication. CONCLUSION: Knockdown studies on lncRNA Gm20559 suggests its pivotal role in maintaining the inflammatory milieu of microglia in flaviviral infection by modulating the expression of various pro-inflammatory cytokines. However, Gm20559-induced increased microglial proinflammatory response upon flavivirus infection fails to trigger neuronal death.

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.

PARP1 inhibition protects mice against Japanese encephalitis virus infection.

Japanese encephalitis (JE) is a vector-borne viral disease that causes acute encephalitis in children. Although vaccines have been developed against the JE virus (JEV), no effective antiviral therapy exists. Our study shows that inhibition of poly(ADP-ribose) polymerase 1 (PARP1), an NAD+-dependent (poly-ADP) ribosyl transferase, protects against JEV infection. Interestingly, PARP1 is critical for JEV pathogenesis in Neuro-2a cells and mice. Small molecular inhibitors of PARP1, olaparib, and 3-aminobenzamide (3-AB) significantly reduce clinical signs and viral load in the serum and brains of mice and improve survival. PARP1 inhibition confers protection against JEV infection by inhibiting autophagy. Mechanistically, upon JEV infection, PARP1 PARylates AKT and negatively affects its phosphorylation. In addition, PARP1 transcriptionally upregulates PTEN, the PIP3 phosphatase, negatively regulating AKT. PARP1-mediated AKT inactivation promotes autophagy and JEV pathogenesis by increasing the FoxO activity. Thus, our findings demonstrate PARP1 as a potential mediator of JEV pathogenesis that can be effectively targeted for treating JE.