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.

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.

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 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.

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 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.

N-linked glycosylation of flavivirus E protein contributes to viral particle formation.

In the case of the Japanese encephalitis virus (JEV), the envelope protein (E), a major component of viral particles, contains a highly conserved N-linked glycosylation site (E: N154). Glycosylation of the E protein is thought to play an important role in the ability of the virus to attach to target cells during transmission; however, its role in viral particle formation and release remains poorly understood. In this study, we investigated the role of N-glycosylation of flaviviral structural proteins in viral particle formation and secretion by introducing mutations in viral structural proteins or cellular factors involved in glycoprotein transport and processing. The number of secreted subviral particles (SVPs) was significantly reduced in N154A, a glycosylation-null mutant, but increased in D67N, a mutant containing additional glycosylation sites, indicating that the amount of E glycosylation regulates the release of SVPs. SVP secretion was reduced in cells deficient in galactose, sialic acid, and N-acetylglucosamine modifications in the Golgi apparatus; however, these reductions were not significant, suggesting that glycosylation mainly plays a role in pre-Golgi transport. Fluorescent labeling of SVPs using a split green fluorescent protein (GFP) system and time-lapse imaging by retention using selective hooks (RUSH) system revealed that the glycosylation-deficient mutant was arrested before endoplasmic reticulum (ER)- Golgi transport. However, the absence of ERGIC-53 and ERGIC-L, ER-Golgi transport cargo receptors that recognize sugar chains on cargo proteins, does not impair SVP secretion. In contrast, the solubility of the N154A mutant of E or the N15A/T17A mutant of prM in cells was markedly lower than that of the wild type, and proteasome-mediated rapid degradation of these mutants was observed, indicating the significance of glycosylation of both prM and E in proper protein folding and assembly of viral particles in the ER.

Investigating RNA-RNA interactions through computational and biophysical analysis.

Numerous viruses utilize essential long-range RNA-RNA genome interactions, specifically flaviviruses. Using Japanese encephalitis virus (JEV) as a model system, we computationally predicted and then biophysically validated and characterized its long-range RNA-RNA genomic interaction. Using multiple RNA computation assessment programs, we determine the primary RNA-RNA interacting site among JEV isolates and numerous related viruses. Following in vitro transcription of RNA, we provide, for the first time, characterization of an RNA-RNA interaction using size-exclusion chromatography coupled with multi-angle light scattering and analytical ultracentrifugation. Next, we demonstrate that the 5' and 3' terminal regions of JEV interact with nM affinity using microscale thermophoresis, and this affinity is significantly reduced when the conserved cyclization sequence is not present. Furthermore, we perform computational kinetic analyses validating the cyclization sequence as the primary driver of this RNA-RNA interaction. Finally, we examined the 3D structure of the interaction using small-angle X-ray scattering, revealing a flexible yet stable interaction. This pathway can be adapted and utilized to study various viral and human long-non-coding RNA-RNA interactions and determine their binding affinities, a critical pharmacological property of designing potential therapeutics.

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.

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.

Inhibition of NADPH oxidase 2 enhances resistance to viral neuroinflammation by facilitating M1-polarization of macrophages at the extraneural tissues.

BACKGROUND: Macrophages play a pivotal role in the regulation of Japanese encephalitis (JE), a severe neuroinflammation in the central nervous system (CNS) following infection with JE virus (JEV). Macrophages are known for their heterogeneity, polarizing into M1 or M2 phenotypes in the context of various immunopathological diseases. A comprehensive understanding of macrophage polarization and its relevance to JE progression holds significant promise for advancing JE control and therapeutic strategies. METHODS: To elucidate the role of NADPH oxidase-derived reactive oxygen species (ROS) in JE progression, we assessed viral load, M1 macrophage accumulation, and cytokine production in WT and NADPH oxidase 2 (NOX2)-deficient mice using murine JE model. Additionally, we employed bone marrow (BM) cell-derived macrophages to delineate ROS-mediated regulation of macrophage polarization by ROS following JEV infection. RESULTS: NOX2-deficient mice exhibited increased resistance to JE progression rather than heightened susceptibility, driven by the regulation of macrophage polarization. These mice displayed reduced viral loads in peripheral lymphoid tissues and the CNS, along with diminished infiltration of inflammatory cells into the CNS, thereby resulting in attenuated neuroinflammation. Additionally, NOX2-deficient mice exhibited enhanced JEV-specific Th1 CD4 + and CD8 + T cell responses and increased accumulation of M1 macrophages producing IL-12p40 and iNOS in peripheral lymphoid and inflamed extraneural tissues. Mechanistic investigations revealed that NOX2-deficient macrophages displayed a more pronounced differentiation into M1 phenotypes in response to JEV infection, thereby leading to the suppression of viral replication. Importantly, the administration of H2O2 generated by NOX2 was shown to inhibit M1 macrophage polarization. Finally, oral administration of the ROS scavenger, butylated hydroxyanisole (BHA), bolstered resistance to JE progression and reduced viral loads in both extraneural tissues and the CNS, along with facilitated accumulation of M1 macrophages. CONCLUSION: In light of our results, it is suggested that ROS generated by NOX2 play a role in undermining the control of JEV replication within peripheral extraneural tissues, primarily by suppressing M1 macrophage polarization. Subsequently, this leads to an augmentation in the viral load invading the CNS, thereby facilitating JE progression. Hence, our findings ultimately underscore the significance of ROS-mediated macrophage polarization in the context of JE progression initiated JEV infection.

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.

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.

Neuroinvasive Flavivirus Pathogenesis Is Restricted by Host Genetic Factors in Collaborative Cross Mice, Independently of Oas1b.

Powassan virus (POWV) is an emerging tick-borne flavivirus that causes neuroinvasive diseases, including encephalitis, meningitis, and paralysis. Similar to other neuroinvasive flaviviruses, such as West Nile virus (WNV) and Japanese encephalitis virus (JEV), POWV disease presentation is heterogeneous, and the factors influencing disease outcome are not fully understood. We used Collaborative Cross (CC) mice to assess the impact of host genetic factors on POWV pathogenesis. We infected a panel of Oas1b-null CC lines with POWV and observed a range of susceptibility, indicating that host factors other than the well-characterized flavivirus restriction factor Oas1b modulate POWV pathogenesis in CC mice. Among the Oas1b-null CC lines, we identified multiple highly susceptible lines (0% survival), including CC071 and CC015, and two resistant lines, CC045 and CC057 (>75% survival). The susceptibility phenotypes generally were concordant among neuroinvasive flaviviruses, although we did identify one line, CC006, that was specifically resistant to JEV, suggesting that both pan-flavivirus and virus-specific mechanisms contribute to susceptibility phenotypes in CC mice. We found that POWV replication was restricted in bone marrow-derived macrophages from CC045 and CC057 mice, suggesting that resistance could result from cell-intrinsic restriction of viral replication. Although serum viral loads at 2 days postinfection were equivalent between resistant and susceptible CC lines, clearance of POWV from the serum was significantly enhanced in CC045 mice. Furthermore, CC045 mice had significantly lower viral loads in the brain at 7 days postinfection than did CC071 mice, suggesting that reduced central nervous system (CNS) infection contributes to the resistant phenotype of CC045 mice. IMPORTANCE Neuroinvasive flaviviruses, such as WNV, JEV, and POWV, are transmitted to humans by mosquitoes or ticks and can cause neurologic diseases, such as encephalitis, meningitis, and paralysis, and they can result in death or long-term sequelae. Although potentially severe, neuroinvasive disease is a rare outcome of flavivirus infection. The factors that determine whether someone develops severe disease after a flavivirus infection are not fully understood, but host genetic differences in polymorphic antiviral response genes likely contribute to the outcome of infection. We evaluated a panel of genetically diverse mice and identified lines with distinct outcomes following infection with POWV. We found that resistance to POWV pathogenesis corresponded to reduced viral replication in macrophages, more rapid clearance of virus in peripheral tissues, and reduced viral infection in the brain. These susceptible and resistant mouse lines will provide a system for investigating the pathogenic mechanisms of POWV and identifying polymorphic host genes that contribute to resistance.

Novel antiviral discoveries for Japanese encephalitis virus infections through reporter virus-based high-throughput screening.

Japanese encephalitis (JE) caused by JE virus (JEV), remains a global public health concern. Currently, there is no specific antiviral drug approved for the treatment of JE. While vaccines are available for prevention, they may not cover all at-risk populations. This underscores the urgent need for prophylaxis and potent anti-JEV drugs. In this context, a high-content JEV reporter system expressing Nanoluciferase (Nluc) was developed and utilized for a high-throughput screening (HTS) of a commercial antiviral library to identify potential JEV drug candidates. Remarkably, this screening process led to the discovery of five drugs with outstanding antiviral activity. Further mechanism of action analysis revealed that cepharanthine, an old clinically approved drug, directly inhibited virus replication by blocking GTP binding to the JEV RNA-dependent RNA polymerase. Additionally, treatment with cepharanthine in mice models alleviated JEV infection. These findings warrant further investigation into the potential anti-JEV activity of cepharanthine as a new therapeutic approach for the treatment of JEV infection. The HTS method employed here proves to be an accurate and convenient approach that facilitates the rapid development of antiviral drugs.

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.

N6-methyladenosine modification positively regulate Japanese encephalitis virus replication.

N6-methyladenosine (m6A) is present in diverse viral RNA and plays important regulatory roles in virus replication and host antiviral innate immunity. However, the role of m6A in regulating JEV replication has not been investigated. Here, we show that the JEV genome contains m6A modification upon infection of mouse neuroblast cells (neuro2a). JEV infection results in a decrease in the expression of m6A writer METTL3 in mouse brain tissue. METTL3 knockdown by siRNA leads to a substantial decrease in JEV replication and the production of progeny viruses at 48 hpi. Mechanically, JEV triggered a considerable increase in the innate immune response of METTL3 knockdown neuro2a cells compared to the control cells. Our study has revealed the distinctive m6A signatures of both the virus and host in neuro2a cells infected with JEV, illustrating the positive role of m6A modification in JEV infection. Our study further enhances understanding of the role of m6A modification in Flaviviridae viruses.

Nucleotide at position 66 of NS2A in Japanese encephalitis virus is associated with the virulence and proliferation of virus.

Most wild strains of Japanese encephalitis virus (JEV) produce NS1' protein, which plays an important role in viral infection and immune escape. The G66A nucleotide mutation in NS2A gene of the wild strain SA14 prevented the ribosomal frameshift that prevented the production of NS1' protein, thus reduced the virulence. In this study, the 66th nucleotide of the NS2A gene of SA14 was mutated into A, U or C, respectively. Both the G66U and G66C mutations cause the E22D mutation of the NS2A protein. Subsequently, the expression of NS1' protein, plaque size, replication ability, and virulence to mice of the three mutant strains were examined. The results showed that the three mutant viruses could not express NS1' protein, and their proliferation ability in nerve cells and virulence to mice were significantly reduced. In addition, the SA14(G66C) was less virulent than the other two mutated viruses. Our results indicate that only when G is the 66th nucleotide of NS2A, the JEV can produce NS1' protein, which affects the virulence.

Modelling the risk of Japanese encephalitis virus in Victoria, Australia, using an expert-systems approach.

Predictive models for vector-borne diseases (VBDs) are instrumental to understanding the potential geographic spread of VBDs and therefore serve as useful tools for public health decision-making. However, predicting the emergence of VBDs at the micro-, local, and regional levels presents challenges, as the importance of risk factors can vary spatially and temporally depending on climatic factors and vector and host abundance and preferences. We propose an expert-systems-based approach that uses an analytical hierarchy process (AHP) deployed within a geographic information system (GIS), to predict areas susceptible to the risk of Japanese encephalitis virus (JEV) emergence. This modelling approach produces risk maps, identifying micro-level risk areas with the potential for disease emergence. The results revealed that climatic conditions, especially the minimum temperature and precipitation required for JEV transmission, contributed to high-risk conditions developed during January and March of 2022 in Victora. Compared to historical climate records, the risk of JEV emergence was increased in most parts of the state due to climate. Importantly, the model accurately predicted 7 out of the 8 local government areas that reported JEV-positive cases during the outbreak of 2022 in Victorian piggeries. This underscores the model's potential as a reliable tool for supporting local risk assessments in the face of evolving climate change.