Fitness adaptations of Japanese encephalitis virus in pigs following vector-free serial passaging.

Japanese encephalitis virus (JEV) is a zoonotic mosquito-transmitted Flavivirus circulating in birds and pigs. In humans, JEV can cause severe viral encephalitis with high mortality. Considering that vector-free direct virus transmission was observed in experimentally infected pigs, JEV introduction into an immunologically naïve pig population could result in a series of direct transmissions disrupting the alternating host cycling between vertebrates and mosquitoes. To assess the potential consequences of such a realistic scenario, we passaged JEV ten times in pigs. This resulted in higher in vivo viral replication, increased shedding, and stronger innate immune responses in pigs. Nevertheless, the viral tissue tropism remained similar, and frequency of direct transmission was not enhanced. Next generation sequencing showed single nucleotide deviations in 10% of the genome during passaging. In total, 25 point mutations were selected to reach a frequency of at least 35% in one of the passages. From these, six mutations resulted in amino acid changes located in the precursor of membrane, the envelope, the non-structural 3 and the non-structural 5 proteins. In a competition experiment with two lines of passaging, the mutation M374L in the envelope protein and N275D in the non-structural protein 5 showed a fitness advantage in pigs. Altogether, the interruption of the alternating host cycle of JEV caused a prominent selection of viral quasispecies as well as selection of de novo mutations associated with fitness gains in pigs, albeit without enhancing direct transmission frequency.

Epitope(s) involving amino acids of the fusion loop of Japanese encephalitis virus envelope protein is(are) important to elicit protective immunity.

Dengue vaccine candidates have been shown to improve vaccine safety and efficacy by altering the residues or accessibility of the fusion loop on the virus envelope protein domain II (DIIFL) in an ex vivo animal study. The current study aimed to comprehensively investigate the impact of DIIFL mutations on the antigenicity, immunogenicity, and protective efficacy of Japanese encephalitis virus (JEV) virus-like particles (VLPs) in mice. We found the DIIFL G106K/L107D (KD) and W101G/G106K/L107D (GKD) mutations altered the binding activity of JEV VLP to cross-reactive monoclonal antibodies but had no effect on their ability to elicit total IgG antibodies in mice. However, JEV VLPs with KD or GKD mutations induced significantly less neutralizing antibodies against JEV. Only 46% and 31% of the KD and GKD VLPs-immunized mice survived compared to 100% of the wild-type (WT) VLP-immunized mice after a lethal JEV challenge. In passive protection experiments, naïve mice that received sera from WT VLP-immunized mice exhibited a significantly higher survival rate of 46.7% compared to those receiving sera from KD VLP- and GKD VLP-immunized mice (6.7% and 0%, respectively). This study demonstrated that JEV DIIFL is crucial for eliciting potently neutralizing antibodies and protective immunity against JEV. IMPORTANCE: Introduction of mutations into the fusion loop is one potential strategy for generating safe dengue and Zika vaccines by reducing the risk of severe dengue following subsequent infections, and for constructing live-attenuated vaccine candidates against newly emerging Japanese encephalitis virus (JEV) or Japanese encephalitis (JE) serocomplex virus. The monoclonal antibody studies indicated the fusion loop of JE serocomplex viruses primarily comprised non-neutralizing epitopes. However, the present study demonstrates that the JEV fusion loop plays a critical role in eliciting protective immunity in mice. Modifications to the fusion loop of JE serocomplex viruses might negatively affect vaccine efficacy compared to dengue and zika serocomplex viruses. Further studies are required to assess the impact of mutant fusion loop encoded by commonly used JEV vaccine strains on vaccine efficacy or safety after subsequent dengue virus infection.

Japanese encephalitis virus NS5 protein interacts with nucleolin to enhance the virus replication.

Japanese encephalitis virus (JEV) is an arthropod-borne, plus-strand flavivirus causing viral encephalitis in humans with a high case fatality rate. The JEV non-structural protein 5 (NS5) with the RNA-dependent RNA polymerase activity interacts with the viral and host proteins to constitute the replication complex. We have identified the multifunctional protein Nucleolin (NCL) as one of the several NS5-interacting host proteins. We demonstrate the interaction and colocalization of JEV NS5 with NCL in the virus-infected HeLa cells. The siRNA-mediated knockdown of NCL indicated that it was required for efficient viral replication. Importantly, JEV grew to higher titers in cells over-expressing exogenous NCL, demonstrating its pro-viral role. We demonstrated that NS5 interacted with the RRM and GAR domains of NCL. We show that the NCL-binding aptamer AS1411 containing the G-quadruplex (GQ) structure and the GQ ligand BRACO-19 caused significant inhibition of JEV replication. The antiviral effect of AS1411 and BRACO-19 could be overcome in HeLa cells by the overexpression of exogenous NCL. We demonstrated that the synthetic RNAs derived from the 3'-NCR of JEV genomic RNA containing the GQ sequence could bind NCL in vitro. The replication complex binding to the 3'-NCR is required for the viral RNA synthesis. It is likely that NCL present in the replication complex destabilizes the GQ structures in the genomic RNA, thus facilitating the movement of the replication complex resulting in efficient virus replication.IMPORTANCEJapanese encephalitis virus (JEV) is endemic in most parts of South-East Asia and the Western Pacific region, causing epidemics of encephalitis with a high case fatality rate. While a tissue culture-derived JEV vaccine is available, no antiviral therapy exists. The JEV NS5 protein has RNA-dependent RNA polymerase activity. Together with several host and viral proteins, it constitutes the replication complex necessary for virus replication. Understanding the interaction of NS5 with the host proteins could help design novel antivirals. We identified Nucleolin (NCL) as a crucial host protein interactor of JEV NS5 having a pro-viral role in virus replication. The NS5-interacting NCL binds to the G-quadruplex (GQ) structure sequence in the 3'-NCR of JEV RNA. This may smoothen the movement of the replication complex along the genomic RNA, thereby facilitating the virus replication. This study is the first report on how NCL, a host protein, helps in JEV replication through GQ-binding.

Japanese encephalitis virus NS1 and NS1' protein disrupts the blood-brain barrier through macrophage migration inhibitory factor-mediated autophagy.

Flaviviruses in the Japanese encephalitis virus (JEV) serogroup, such as JEV, West Nile virus, and St. Louis encephalitis virus, can cause severe neurological diseases. The nonstructural protein 1 (NS1) is a multifunctional protein of flavivirus that can be secreted by infected cells and circulate in the host bloodstream. NS1' is an additional form of NS1 protein with 52 amino acids extension at its carboxy-terminal and is produced exclusively by flaviviruses in the JEV serogroup. In this study, we demonstrated that the secreted form of both NS1 and NS1' can disrupt the blood-brain barrier (BBB) of mice, with NS1' exhibiting a stronger effect. Using the in vitro BBB model, we found that treatment of soluble recombinant JEV NS1 or NS1' protein increases the permeability of human brain microvascular endothelial cells (hBMECs) and leads to the degradation of tight junction proteins through the autophagy-lysosomal pathway. Consistently, NS1' protein exhibited a more pronounced effect compared to NS1 in these cellular processes. Further research revealed that the increased expression of macrophage migration inhibitory factor (MIF) is responsible for triggering autophagy after NS1 or NS1' treatment in hBMECs. In addition, TLR4 and NF-κB signaling was found to be involved in the activation of MIF transcription. Moreover, administering the MIF inhibitor has been shown to decrease viral loads and mitigate inflammation in the brains of mice infected with JEV. This research offers a novel perspective on the pathogenesis of JEV. In addition, the stronger effect of NS1' on disrupting the BBB compared to NS1 enhances our understanding of the mechanism by which flaviviruses in the JEV serogroup exhibit neurotropism.IMPORTANCEJapanese encephalitis (JE) is a significant viral encephalitis worldwide, caused by the JE virus (JEV). In some patients, the virus cannot be cleared in time, leading to the breach of the blood-brain barrier (BBB) and invasion of the central nervous system. This invasion may result in cognitive impairment, behavioral disturbances, and even death in both humans and animals. However, the mechanism by which JEV crosses the BBB remains unclear. Previous studies have shown that the flavivirus NS1 protein plays an important role in causing endothelial dysfunction. The NS1' protein is an elongated form of NS1 protein that is particularly produced by flaviviruses in the JEV serogroup. This study revealed that both the secreted NS1 and NS1' of JEV can disrupt the BBB by breaking down tight junction proteins through the autophagy-lysosomal pathway, and NS1' is found to have a stronger effect compared to NS1 in this process. In addition, JEV NS1 and NS1' can stimulate the expression of MIF, which triggers autophagy via the ERK signaling pathway, leading to damage to BBB. Our findings reveal a new function of JEV NS1 and NS1' in the disruption of BBB, thereby providing the potential therapeutic target for JE.

Japanese encephalitis virus NS1 and NS1' proteins induce vimentin rearrangement via the CDK1-PLK1 axis to promote viral replication.

The host cytoskeleton plays crucial roles in various stages of virus infection, including viral entry, transport, replication, and release. However, the specific mechanisms by which intermediate filaments are involved in orthoflavivirus infection have not been well understood. In this study, we demonstrate that the Japanese encephalitis virus (JEV) remodels the vimentin network, resulting in the formation of cage-like structures that support viral replication. Mechanistically, JEV NS1 and NS1' proteins induce the translocation of CDK1 from the nucleus to the cytoplasm and interact with it, leading to the phosphorylation of vimentin at Ser56. This phosphorylation event recruits PLK1, which further phosphorylates vimentin at Ser83. Consequently, these phosphorylation modifications convert the typically filamentous vimentin into non-filamentous "particles" or "squiggles." These vimentin "particles" or "squiggles" are then transported retrogradely along microtubules to the endoplasmic reticulum, where they form cage-like structures. Notably, NS1' is more effective than NS1 in triggering the CDK1-PLK1 cascade response. Overall, our study provides new insights into how JEV NS1 and NS1' proteins manipulate the vimentin network to facilitate efficient viral replication. IMPORTANCE: Japanese encephalitis virus (JEV) is a mosquito-borne orthoflavivirus that causes severe encephalitis in humans, particularly in Asia. Despite the availability of a safe and effective vaccine, JEV infection remains a significant public health threat due to limited vaccination coverage. Understanding the interactions between JEV and host proteins is essential for developing more effective antiviral strategies. In this study, we investigated the role of vimentin, an intermediate filament protein, in JEV replication. Our findings reveal that JEV NS1 and NS1' proteins induce vimentin rearrangement, resulting in the formation of cage-like structures that envelop the viral replication factories (RFs), thus facilitating efficient viral replication. Our research highlights the importance of the interplay between the cytoskeleton and orthoflavivirus, suggesting that targeting vimentin could be a promising approach for the development of antiviral strategies to inhibit JEV propagation.

Sialic acids as attachment factors in mosquitoes mediating Japanese encephalitis virus infection.

The role of Culex mosquitoes in the transmission of Japanese encephalitis virus (JEV) is crucial, yet the mechanisms of JEV infection in these vectors remain unclear. Previous research has indicated that various host factors participate in JEV infection. Herein, we present evidence that mosquito sialic acids enhance JEV infection both in vivo and in vitro. By treating mosquitoes and C6/36 cells with neuraminidase or lectin, the function of sialic acids is effectively blocked, resulting in significant inhibition of JEV infection. Furthermore, knockdown of the sialic acid biosynthesis genes in Culex mosquitoes also leads to a reduction in JEV infection. Moreover, our research revealed that sialic acids play a role in the attachment of JEV to mosquito cells, but not in its internalization. To further explore the mechanisms underlying the promotion of JEV attachment by sialic acids, we conducted immunoprecipitation experiments to confirm the direct binding of sialic acids to the last α-helix in JEV envelope protein domain III. Overall, our study contributes to a molecular comprehension of the interaction between mosquitoes and JEV and offers potential strategies for preventing the dissemination of flavivirus in natural environments.IMPORTANCEIn this study, we aimed to investigate the impact of glycoconjugate sialic acids on mosquito infection with Japanese encephalitis virus (JEV). Our findings demonstrate that sialic acids play a crucial role in enhancing JEV infection by facilitating the attachment of the virus to the cell membrane. Furthermore, our investigation revealed that sialic acids directly bind to the final α-helix in the JEV envelope protein domain III, thereby accelerating virus adsorption. Collectively, our results highlight the significance of mosquito sialic acids in JEV infection within vectors, contributing to a better understanding of the interaction between mosquitoes and JEV.

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.

Peripheral Nerve Injury Induced by Japanese Encephalitis Virus in C57BL/6 Mouse.

Japanese encephalitis virus (JEV), with neurotoxic and neuroinvasive properties, is the major cause of human viral encephalitis in Asia. Although Guillain-Barré syndrome caused by JEV infections is not frequent, a few cases have been reported in recent years. To date, no existing animal model for JEV-induced peripheral nerve injury (PNI) has been established, and thus the pathogenic mechanism is not clarified. Therefore, an animal model is urgently required to clarify the correlation between JEV infection and PNI. In the present study, we used JEV GIb strain of NX1889 to establish a mouse model of JEV infection. The general neurological signs emerged on day 3 of modeling. The motor function continued to deteriorate, reaching a maximum at 8 to 13 days postinfection (dpi) and gradually recovered after 16 dpi. The injuries of 105 PFU and 106 PFU groups were the most severe. Transmission electron microscopy and immunofluorescence staining showed varying degrees of demyelination and axonal degeneration in the sciatic nerves. The electrophysiological recordings demonstrated the presence of demyelinating peripheral neuropathy with reduced nerve conduction velocity. The decreased amplitudes and the prolonged end latency revealed axonal-type motor neuropathy. Demyelination is predominant in the early stage, followed by axonal injury. The expression level of JEV-E protein and viral RNA was elevated in the injured sciatic nerves, suggesting that it may cause PNI at the early stage. Inflammatory cell infiltration and increased inflammatory cytokines indicated that neuroinflammation is involved in JEV-induced PNI. IMPORTANCE JEV is a neurotropic flavivirus belonging to the Flaviviridae family and causes high mortality and disability rates. It invades the central nervous system and induces acute inflammatory injury and neuronal death. Thus, JEV infection is a major global public health concern. Previously, motor dysfunction was mainly attributed to central nervous system damage. Our knowledge regarding JEV-induced PNI is vague and neglected. Therefore, a laboratory animal model is essential. Herein, we showed that C57BL/6 mice can be used to study JEV-induced PNI through multiple approaches. We also demonstrated that viral loads might be positively correlated with lesion severity. Therefore, inflammation and direct virus infection may be the putative mechanisms underlying JEV-induced PNI. The results of this study laid the foundation for further elucidation of the pathogenesis mechanisms of PNI caused by JEV.

Palmitoylation at Residue C221 of Japanese Encephalitis Virus NS2A Protein Contributes to Viral Replication Efficiency and Virulence.

Palmitoylation of viral proteins is crucial for host-virus interactions. In this study, we examined the palmitoylation of Japanese encephalitis virus (JEV) nonstructural protein 2A (NS2A) and observed that NS2A was palmitoylated at the C221 residue of NS2A. Blocking NS2A palmitoylation by introducing a cysteine-to-serine mutation at C221 (NS2A/C221S) impaired JEV replication in vitro and attenuated the virulence of JEV in mice. NS2A/C221S mutation had no effect on NS2A oligomerization and membrane-associated activities, but reduced protein stability and accelerated its degradation through the ubiquitin-proteasome pathway. These observations suggest that NS2A palmitoylation at C221 played a role in its protein stability, thereby contributing to JEV replication efficiency and virulence. Interestingly, the C221 residue undergoing palmitoylation was located at the C-terminal tail (amino acids 195 to 227) and is removed from the full-length NS2A following an internal cleavage processed by viral and/or host proteases during JEV infection. IMPORTANCE An internal cleavage site is present at the C terminus of JEV NS2A. Following occurrence of the internal cleavage, the C-terminal tail (amino acids 195 to 227) is removed from the full-length NS2A. Therefore, it was interesting to discover whether the C-terminal tail contributed to JEV infection. During analysis of viral palmitoylated protein, we observed that NS2A was palmitoylated at the C221 residue located at the C-terminal tail. Blocking NS2A palmitoylation by introducing a cysteine-to-serine mutation at C221 (NS2A/C221S) impaired JEV replication in vitro and attenuated JEV virulence in mice, suggesting that NS2A palmitoylation at C221 contributed to JEV replication and virulence. Based on these findings, we could infer that the C-terminal tail might play a role in the maintenance of JEV replication efficiency and virulence despite its removal from the full-length NS2A at a certain stage of JEV infection.

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.

Flavivirus recruits the valosin-containing protein-NPL4 complex to induce stress granule disassembly for efficient viral genome replication.

Flaviviruses are human pathogens that can cause severe diseases, such as dengue fever and Japanese encephalitis, which can lead to death. Valosin-containing protein (VCP)/p97, a cellular ATPase associated with diverse cellular activities (AAA-ATPase), is reported to have multiple roles in flavivirus replication. Nevertheless, the importance of each role still has not been addressed. In this study, the functions of 17 VCP mutants that are reportedly unable to interact with the VCP cofactors were validated using the short-interfering RNA rescue experiments. Our findings of this study suggested that VCP exerts its functions in replication of the Japanese encephalitis virus by interacting with the VCP cofactor nuclear protein localization 4 (NPL4). We show that the depletion of NPL4 impaired the early stage of viral genome replication. In addition, we demonstrate that the direct interaction between NPL4 and viral nonstructural protein (NS4B) is critical for the translocation of NS4B to the sites of viral replication. Finally, we found that Japanese encephalitis virus and dengue virus promoted stress granule formation only in VCP inhibitor-treated cells and the expression of NS4B or VCP attenuated stress granule formation mediated by protein kinase R, which is generally known to be activated by type I interferon and viral genome RNA. These results suggest that the NS4B-mediated recruitment of VCP to the virus replication site inhibits cellular stress responses and consequently facilitates viral protein synthesis in the flavivirus-infected cells.

Zinc Finger Protein ZFP36L1 Inhibits Flavivirus Infection by both 5'-3' XRN1 and 3'-5' RNA-Exosome RNA Decay Pathways.

Zinc-finger protein 36, CCCH type-like 1 (ZFP36L1), containing tandem CCCH-type zinc-finger motifs with an RNA-binding property, plays an important role in cellular RNA metabolism mainly by RNA decay pathways. Recently, we demonstrated that human ZFP36L1 has potent antiviral activity against influenza A virus infection. However, its role in the host defense response against flaviviruses has not been addressed. Here, we demonstrate that ZFP36L1 functions as a host innate defender against flaviviruses, including Japanese encephalitis virus (JEV) and dengue virus (DENV). Overexpression of ZFP36L1 reduced JEV and DENV infection, and ZFP36L1 knockdown enhanced viral replication. ZFP36L1 destabilized the JEV genome by targeting and degrading viral RNA mediated by both 5'-3' XRN1 and 3'-5' RNA-exosome RNA decay pathways. Mutation in both zinc-finger motifs of ZFP36L1 disrupted RNA-binding and antiviral activity. Furthermore, the viral RNA sequences specifically recognized by ZFP36L1 were mapped to the 3'-untranslated region of the JEV genome with the AU-rich element (AUUUA) motif. We extend the function of ZFP36L1 to host antiviral defense by directly binding and destabilizing the viral genome via recruiting cellular mRNA decay machineries. IMPORTANCE Cellular RNA-binding proteins are among the first lines of defense against various viruses, particularly RNA viruses. ZFP36L1 belongs to the CCCH-type zinc-finger protein family and has RNA-binding activity; it has been reported to bind directly to the AU-rich elements (AREs) of a subset of cellular mRNAs and then lead to mRNA decay by recruiting mRNA-degrading enzymes. However, the antiviral potential of ZFP36L1 against flaviviruses has not yet been fully demonstrated. Here, we reveal the antiviral potential of human ZFP36L1 against Japanese encephalitis virus (JEV) and dengue virus (DENV). ZFP36L1 specifically targeted the ARE motif within viral RNA and triggered the degradation of viral RNA transcripts via cellular degrading enzymes 5'-3' XRN1 and 3'-5' RNA exosome. These findings provide mechanistic insights into how human ZFP36L1 serves as a host antiviral factor to restrict flavivirus replication.

Melatonin inhibits Japanese encephalitis virus replication and neurotoxicity via calcineurin-autophagy pathways.

BACKGROUND: Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus that has no specific treatment except for supportive medical care. JEV is a neurotropic virus that affects the nervous system and triggers inflammation in the brain. METHODS: Melatonin is used as a sleep-inducing agent in neurophysiology and may serve as a protective agent against neurological and neurodegenerative diseases. Herein, we investigated the effects of melatonin and the critical roles of the serine/threonine protein phosphatase calcineurin during JEV infection in SK-N-SH neuroblastoma cells. RESULTS: Melatonin treatment decreased JEV replication and JEV-mediated neurotoxicity. Calcineurin activity was increased by JEV infection and inhibited by melatonin treatment. Through calcineurin regulation, melatonin decreased the JEV-mediated neuroinflammatory response and attenuated JEV-induced autophagy. CONCLUSIONS: Calcineurin inactivation has a protective effect in JEV-infected neuronal cells, and melatonin is a novel resource for the development of anti-JEV agents.

Japanese encephalitis virus induces apoptosis by activating the RIG-1 signaling pathway.

Japanese encephalitis virus (JEV) infection can cause brain tissue lesions characterized by neuronal death, and apoptosis is involved in JEV-induced neuronopathy. In the present study, mouse microglia were infected with JEV, and pyknosis with dark-staining nuclei of infected cells was detected using Hoechst 33342 staining. TUNEL staining showed that JEV infection promoted the apoptosis of BV2 cells, and the apoptosis rate was significantly increased at 24-60 hours postinfection (hpi) (P < 0.01) and was the highest at 36 h (P < 0.0001). Western blot results showed that the expression of the Bcl-2 protein in JEV-infected cells was downregulated significantly at 60 hpi (P < 0.001), whereas that of the Bax protein was observably upregulated at 60 hpi (P < 0.001). At the same time, the level of cytochrome c (Cyt c) was significantly increased (P < 0.001), and the expression levels of two apoptosis-related proteins, namely, cleaved caspase-3 (P < 0.01) and caspase-9 (P < 0.001), were elevated significantly. Immunofluorescence staining showed that the amount of Cyt c increased with time after infection. After BV2 cells were infected with JEV, the expression of RIG-1 increased significantly from 24 hpi to 60 h (P < 0.001). The expression of MAVS increased significantly at 24 h (P < 0.001) and decreased gradually from 24 h to 60 hpi. The expression of TBK1 and NF-κB (p65) was not significantly changed. The expression of p-TBK1 and p-NF-κB (p-p65) increased significantly within 24 h (P < 0.001) and decreased from 24 to 60 hpi. The expression levels of IRF3 and p-IRF3 peaked at 24 hpi (P < 0.001) and decreased gradually from 24 to 60 hpi. However, the expression levels of JEV proteins showed no significant change at 24 and 36 hpi but were markedly elevated at 48 and 60 hpi. Interference with the expression of the RIG-1 protein in BV2 cells resulted in a dramatic increase in the expression of the anti-apoptotic protein Bcl-2 (P < 0.05), whereas the pro-apoptotic protein Bax, cleaved caspase-9, and especially cleaved caspase-3 were downregulated (P < 0.05), and viral protein expression was notably reduced (P < 0.05). These results indicate that JEV induces apoptosis through mitochondrial-dependent apoptosis pathways, interfering with the expression of RIG-1 in BV2 cells can inhibit viral replication and inhibit apoptosis.

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.

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

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

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.

Autophagy inhibitors alleviate Japanese encephalitis virus-induced cerebral inflammation in mice.

Japanese encephalitis (JE) is a zoonotic epidemic disease caused by Japanese encephalitis virus (JEV), and currently, no medicines are available to treat this disease. Autophagy modulators play an important role in the treatment of tumors, heart disease, and some viral diseases. The aim of this study was to investigate the effects of autophagy modulators on JEV infection and the host response in mice. The experimental mice were grouped as follows: DMEM (control), JEV, JEV+rapamycin (JEV+Rapa), JEV+wortmannin (JEV+Wort), JEV+chloroquine (JEV+CQ), Rapa, Wort, and CQ. The control group was treated with DMEM. The mice in other groups were infected with 105 PFU of JEV, and Rapa, Wort, and CQ were administered 2 h prior to JEV challenge and then administered daily for 10 consecutive days. All mice were monitored for neurological signs and survival. The damage of subcellular structures in the mouse brain was evaluated by transmission electron microscopy. The distribution of virus in the mouse brain was determined by RNAScope staining and immunohistochemical staining. The neuroinflammatory responses in the brain were examined via quantitative real-time PCR, and the signal pathways involved in neuroinflammation were identified by Western blot. The mice in the JEV+Wort and JEV+CQ groups showed milder neurological symptoms, less damage to the mitochondria in the brain tissue, and a higher survival rate than those in the JEV+Rapa and JEV groups. Compared with the JEV+Rapa and JEV groups, the distribution of JEV in the brain of mice in the JEV+Wort and JEV+CQ groups was lower, and the inflammatory response was weaker. No significant difference was observed in the expression of the PI3K/AKT/NF-κB pathway in mouse brain among the different groups. Our study suggests that the autophagy inhibitors Wort and CQ reduce JEV infection and weaken the inflammatory response, which does not depend on the PI3K/AKT/NF-κB pathway in mouse brain.

The Japanese Encephalitis Virus NS1' Protein Inhibits Type I IFN Production by Targeting MAVS.

Japanese encephalitis virus (JEV) is a mosquito-borne Flavivirus that causes severe neurologic disease in humans. NS1' is a NS1-related protein only reported in the Japanese encephalitis serogroup members of Flavivirus It is produced through programmed -1 ribosomal frameshift in NS2A. Our previous study demonstrated that JEV NS1' could antagonize type I IFN (IFN-I) production, but the mechanism is still unclear. In the current study, we found that JEV NS1' inhibits the expression of MAVS, and knockdown of MAVS hampers inhibition of IFN-ß induction by NS1', suggesting that JEV NS1' inhibits IFN-I production by targeting MAVS. This finding is further supported by the result of the in vivo assay that showed the similar mortality caused by NS1'-deficient virus and its wild type virus in MAVS-deficient mice. Based on our previous sequencing results of noncoding RNA in JEV-infected cells, microRNA-22 (miR-22) was identified to be a key regulator for MAVS expression during JEV infection. Furthermore, we demonstrated that JEV NS1' could induce the expression of miR-22 by increasing the binding of transcriptional factors, CREB and c-Rel, to the promoter elements of miR-22. Taken together, our results reveal a novel mechanism by which JEV NS1' antagonizes host MAVS by regulating miR-22, thereby inhibiting the IFN-I production and facilitating viral replication.

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.