Prophylactic Administration of Gut Microbiome Metabolites Abrogated Microglial Activation and Subsequent Neuroinflammation in an Experimental Model of Japanese Encephalitis.

Short-chain fatty acids (SCFAs) are gut microbial metabolic derivatives produced during the fermentation of ingested complex carbohydrates. SCFAs have been widely regarded to have a potent anti-inflammatory and neuro-protective role and have implications in several disease conditions, such as, inflammatory bowel disease, type-2 diabetes, and neurodegenerative disorders. Japanese encephalitis virus (JEV), a neurotropic flavivirus, is associated with life threatening neuro-inflammation and neurological sequelae in infected hosts. In this study, we hypothesize that SCFAs have potential in mitigating JEV pathogenesis. Postnatal day 10 BALB/c mice were intraperitoneally injected with either a SCFA mixture (acetate, propionate, and butyrate) or PBS for a period of 7 days, followed by JEV infection. All mice were observed for onset and progression of symptoms. The brain tissue was collected upon reaching terminal illness for further analysis. SCFA-supplemented JEV-infected mice (SCFA + JEV) showed a delayed onset of symptoms, lower hindlimb clasping score, and decreased weight loss and increased survival by 3 days (p < 0.0001) upon infection as opposed to the PBS-treated JEV-infected animals (JEV). Significant downregulation of inflammatory cytokines TNF-α, MCP-1, IL-6, and IFN-Υ in the SCFA + JEV group relative to the JEV-infected control group was observed. Inflammatory mediators, phospho-NF-kB (P-NF-kB) and iba1, showed 2.08 ± 0.1 and 3.132 ± 0.43-fold upregulation in JEV versus 1.19 ± 0.11 and 1.31 ± 0.11-fold in the SCFA + JEV group, respectively. Tissue section analysis exhibited reduced glial activation (JEV group─42 ± 2.15 microglia/ROI; SCFA + JEV group─27.07 ± 1.8 microglia/ROI) in animals that received SCFA supplementation prior to infection as seen from the astrocytic and microglial morphometric analysis. Caspase-3 immunoblotting showed 4.08 ± 1.3-fold upregulation in JEV as compared to 1.03 ± 0.14-fold in the SCFA + JEV group and TUNEL assay showed a reduced cellular death post-JEV infection (JEV-6.4 ± 1.5 cells/ROI and SCFA + JEV-3.7 ± 0.73 cells/ROI). Our study critically contributes to the increasing evidence in support of SCFAs as an anti-inflammatory and neuro-protective agent, we further expand its scope as a potential supplementary intervention in JEV-mediated neuroinflammation.

Intravenous immunoglobulin alleviates Japanese encephalitis virus-induced peripheral neuropathy by inhibiting the ASM/ceramide pathway.

Japanese encephalitis virus (JEV) infection is considered a global public health emergency. Severe peripheral neuropathy caused by JEV infection has increased disability and mortality rates in recent years. Because there are very few therapeutic options for JEV infection, prompt investigations of the ability of clinically safe, efficacious and globally available drugs to inhibit JEV infection and ameliorate peripheral neuropathy are urgently needed. In this study, we found that high doses of intravenous immunoglobulin, a function inhibitor of acid sphingomyelinase (FIASMA), inhibited acid sphingomyelinase (ASM) and ceramide activity in the serum and sciatic nerve of JEV-infected rats, reduced disease severity, reversed electrophysiological and histological abnormalities, significantly reduced circulating proinflammatory cytokine levels, inhibited Th1 and Th17 cell proliferation, and suppressed the infiltration of inflammatory CD4 + cells into the sciatic nerve. It also maintained the peripheral nerve-blood barrier without causing severe clinical side effects. In terms of the potential mechanisms, ASM was found to participate in immune cell differentiation and to activate immune cells, thereby exerting proinflammatory effects. Therefore, immunoglobulin is a FIASMA that reduces abnormal immune responses and thus targets the ASM/ceramide system to treat peripheral neuropathy caused by JEV infection.

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.

Methotrimeprazine is a neuroprotective antiviral in JEV infection via adaptive ER stress and autophagy.

Japanese encephalitis virus (JEV) pathogenesis is driven by a combination of neuronal death and neuroinflammation. We tested 42 FDA-approved drugs that were shown to induce autophagy for antiviral effects. Four drugs were tested in the JE mouse model based on in vitro protective effects on neuronal cell death, inhibition of viral replication, and anti-inflammatory effects. The antipsychotic phenothiazines Methotrimeprazine (MTP) & Trifluoperazine showed a significant survival benefit with reduced virus titers in the brain, prevention of BBB breach, and inhibition of neuroinflammation. Both drugs were potent mTOR-independent autophagy flux inducers. MTP inhibited SERCA channel functioning, and induced an adaptive ER stress response in diverse cell types. Pharmacological rescue of ER stress blocked autophagy and antiviral effect. MTP did not alter translation of viral RNA, but exerted autophagy-dependent antiviral effect by inhibiting JEV replication complexes. Drug-induced autophagy resulted in reduced NLRP3 protein levels, and attenuation of inflammatory cytokine/chemokine release from infected microglial cells. Our study suggests that MTP exerts a combined antiviral and anti-inflammatory effect in JEV infection, and has therapeutic potential for JE treatment.

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.

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.

Antiviral effect of melatonin on Japanese encephalitis virus infection involves inhibition of neuronal apoptosis and neuroinflammation in SH-SY5Y cells.

Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, causes high mortality rates in humans and it is the most clinically important and common cause of viral encephalitis in Asia. To date, there is no specific treatment for JEV infection. Melatonin, a neurotropic hormone, is reported to be effective in combating various bacterial and viral infections. However, the effects of melatonin on JEV infection have not yet been studied. The investigation tested the antiviral effects of melatonin against JEV infection and elucidated the possible molecular mechanisms of inhibition. Melatonin inhibited the viral production in JEV-infected SH-SY5Y cells in a time- and dose-dependent manner. Time-of-addition assays demonstrated a potent inhibitory effect of melatonin at the post-entry stage of viral replication. Molecular docking analysis revealed that melatonin negatively affected viral replication by interfering with physiological function and/or enzymatic activity of both JEV nonstructural 3 (NS3) and NS5 protein, suggesting a possible underlying mechanism of JEV replication inhibition. Moreover, treatment with melatonin reduced neuronal apoptosis and inhibited neuroinflammation induced by JEV infection. The present findings reveal a new property of melatonin as a potential molecule for the further development of anti-JEV agents and treatment of JEV infection.

4-Phenyl-butyric Acid Inhibits Japanese Encephalitis Virus Replication via Inhibiting Endoplasmic Reticulum Stress Response.

Japanese encephalitis virus (JEV) infection causes host endoplasmic reticulum stress (ERS) reaction, and then induces cell apoptosis through the UPR pathway, invading the central nervous system and causing an inflammation storm. The endoplasmic reticulum stress inhibitor, 4-phenyl-butyric acid (4-PBA), has an inhibitory effect on the replication of flavivirus. Here, we studied the effect of 4-PBA on JEV infection both in vitro and vivo. The results showed that 4-PBA treatment could significantly decrease the titer of JEV, inhibit the expression of the JEV NS3 protein (in vitro, p < 0.01) and reduce the positive rate of the JEV E protein (in vivo, p < 0.001). Compared to the control group, 4-PBA treatment can restore the weight of JEV-infected mice, decrease the level of IL-1ß in serum and alleviate the abnormalities in brain tissue structure. Endoplasmic reticulum stress test found that the expression level of GRP78 was much lower and activation levels of PERK and IRE1 pathways were reduced in the 4-PBA treatment group. Furthermore, 4-PBA inhibited the UPR pathway activated by NS3, NS4b and NS5 RdRp. The above results indicated that 4-PBA could block JEV replication and inhibit ER stress caused by JEV. Interestingly, 4-PBA could reduce the expression of NS5 by inhibiting transcription (p < 0.001), but had no effect on the expression of NS3 and NS4b. This result may indicate that 4-PBA has antiviral activity independent of the UPR pathway. In summary, the effect of 4-PBA on JEV infection is related to the inhibition of ER stress, and it may be a promising drug for the treatment of Japanese encephalitis.

Development of antiviral carbon quantum dots that target the Japanese encephalitis virus envelope protein.

Japanese encephalitis is a mosquito-borne disease caused by the Japanese encephalitis virus (JEV) that is prevalent in Asia and the Western Pacific. Currently, there is no effective treatment for Japanese encephalitis. Curcumin (Cur) is a compound extracted from the roots of Curcuma longa, and many studies have reported its antiviral and anti-inflammatory activities. However, the high cytotoxicity and very low solubility of Cur limit its biomedical applications. In this study, Cur carbon quantum dots (Cur-CQDs) were synthesized by mild pyrolysis-induced polymerization and carbonization, leading to higher water solubility and lower cytotoxicity, as well as superior antiviral activity against JEV infection. We found that Cur-CQDs effectively bound to the E protein of JEV, preventing viral entry into the host cells. In addition, after continued treatment of JEV with Cur-CQDs, a mutant strain of JEV was evolved that did not support binding of Cur-CQDs to the JEV envelope. Using transmission electron microscopy, biolayer interferometry, and molecular docking analysis, we revealed that the S123R and K312R mutations in the E protein play a key role in binding Cur-CQDs. The S123 and K312 residues are located in structural domains II and III of the E protein, respectively, and are responsible for binding to receptors on and fusing with the cell membrane. Taken together, our results suggest that the E protein of flaviviruses represents a potential target for the development of CQD-based inhibitors to prevent or treat viral infections.

Discovery and synthesis of 1,2,4-oxadiazole derivatives as novel inhibitors of Zika, dengue, Japanese encephalitis, and classical swine fever virus infections.

Zika virus (ZIKV), an arbovirus of the Flaviviridae family, has emerged as a significant public health concern owing to its association with congenital abnormalities and severe neurological sequelae. Thus, there is an urgent need to develop effective therapeutic approaches to efficiently treat ZIKV infections. This study used phenotypic screening to identify a series of 1,2,4-oxadiazole derivatives that possess antiviral activity against ZIKV infection. Subsequently, 28 new derivatives were designed, synthesized, and evaluated for this purpose. Among these compounds, 4-(5-phenyl-1,2,4-oxadiazol-3-yl)-N-(pyridin-3-ylmethyl)aniline (5d) had potent antiviral activity against ZIKV infections. Furthermore, a structure-activity relationship analysis indicated that a benzyl substitution on the aniline nitrogen of this compound improved potency while augmenting its drug-like properties. In addition, 5d exhibited antiviral activity against various viruses of Flaviviridae family of worldwide public health importance, such as dengue, Japanese encephalitis and classical swine fever viruses, indicating its potential as a lead compound for generating 1,2,4-oxadiazole derivatives with broad-spectrum anti-flaviviral properties.

Lessons Learned from the Japanese Encephalitis Vaccine Introduction in India That Supported the Introduction of Ivermectin-Diethylcarbamazine-Albendazole for Lymphatic Filariasis Elimination.

We used the introduction of the Japanese encephalitis (JE) vaccine in India as an example to understand more fully the process of introducing any new clinical product in India. We discuss the key decision-making points as well as the many activities involved in introducing a new clinical product in India's public health program. We write from our experience in supporting the government of India to introduce new products successfully-namely, vaccines-to India's health system. In India, the process begins with identifying the public health problem (e.g., an outbreak of JE), deciding to take action, prioritizing where action is needed, securing a supply and price of the intervention (the vaccine; in this case, the live, attenuated SA 14-14-2 vaccine), and determining how to ensure effective rollout of the intervention (the vaccination program). Reflecting on the experience of the JE vaccination program helped to inform the introduction of the triple-drug therapy of ivermectin, diethylcarbamazine, and albendazole in India as a new treatment protocol for lymphatic filariasis.

Antiviral drug research for Japanese encephalitis: an updated review.

Japanese encephalitis (JE) caused by the Japanese encephalitis virus (JEV) is one of Asia's most common viral encephalitis. JEV is a flavivirus, common in rural and sub-urban regions of Asian countries. Although only 1% of JEV-infected individuals develop JE, there is a 20-30% chance of death among these individuals and possible neurological sequelae post-infection. No licensed anti-JE drugs are currently available, despite extensive efforts to develop them. Literature search was performed using databases such as PubMed Central, Google Scholar, Wiley Online Library, etc. using keywords such as Japanese encephalitis virus, antiviral drugs, antiviral drug screening, antiviral drug targets, etc. From around 230 papers/abstracts and research reviews retrieved and reviewed for this study, approximately 180 most relevant and important ones have been cited. Different approaches in drug testing and various antiviral drug targets explored so far have been thoroughly searched from the literature and compiled, besides addressing the future perspectives of the antiviral drug development strategies. Although the development of effective anti-JE drugs is an urgent issue, only supportive care is currently available. Recent advancements in understanding the biology of infection and new drug targets have been promising improvements. Despite hindrances such as the unavailability of a proper drug delivery system or a treatment regimen irrespective of the stage of infection, several promising anti-JE candidate molecules are in different phases of clinical trials. Nonetheless, efficient therapy against JEV is expected to be achieved with drug combinations and a highly targeted drug delivery system soon.

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.

Efficacy and safety of intravenous high-dose immunoglobulin in treatment of the severe form of Japanese encephalitis.

PURPOSE: To explore the efficacy and safety of intravenous immunoglobulin (IVIG) in the treatment of severe Japanese encephalitis (JE). METHODS: A retrospective study of 124 children diagnosed with the severe or very severe form of JE was undertaken. There were 62 cases in the IVIG group and control group. The efficacy, safety, and tolerability of IVIG were evaluated 3 days, as well as 1, 2, and 3 weeks after IVIG, respectively, and the prognosis was assessed at 6 months. RESULTS: Cox regression survival analysis suggested that the IVIG group reached the criteria for efficacious treatment faster than that in the control group. The duration of unconsciousness and the number of days of seizures, a dull response to light, the pyramidal sign, and meningeal-irritation sign in the IVIG group were shorter than those in the control group (p < 0.05). The number of complications occurring in the IVIG group (including gastrointestinal bleeding and pneumonia) was less than that in the control group (p < 0.05). Cox regression survival analysis suggested that age (p = 0.003) and imaging abnormalities (p = 0.042) had an effect on the efficacy of IVIG treatment. The Liverpool Outcome Score at 6 months showed that the prognosis of the IVIG group was better than that of the control group (p < 0.05). IVIG treatment was safe and tolerable. CONCLUSION: IVIG showed good efficacy, safety, and tolerance for treatment of the severe form of JE. The age and imaging abnormalities of patients affect the efficacy of IVIG treatment.

Mechanistic insights into the Japanese encephalitis virus RNA dependent RNA polymerase protein inhibition by bioflavonoids from Azadirachta indica.

Japanese encephalitis (JE) virus is a flavivirus causing encephalitis causing neurological damage. RNA-dependent-RNA-polymerase (RdRp) is responsible for genome replication making it excellent anti-viral target. In this study, the crystal structure of JE RdRp (jRdRp) and bioflavonoids reported in Azadirachta indica were retrieved from specific databases. Structure-based virtual screening was employed using MTiOpenScreen server and top four compounds selected with the most negative docking scores. Conformations were redocked using AutoDock Vina; these complexes showed mechanistic interactions with Arg474, Gly605, Asp668, and Trp800 residues in the active site of jRdRp, i.e., guanosine-5'-triphosphate. Furthermore, 100 ns classical molecular dynamics simulation and binding free energy calculation showed stability of docked bioflavonoids in the active jRdRp pocket and significant contribution of van-der-Waals interactions for docked complex stability during simulation. Therefore, this study predicted the anti-viral activity of Gedunin, Nimbolide, Ohchinin acetate, and Kulactone against jRdRp and can be considered for further antiviral drug development.

Berbamine inhibits Japanese encephalitis virus (JEV) infection by compromising TPRMLs-mediated endolysosomal trafficking of low-density lipoprotein receptor (LDLR).

Japanese encephalitis virus (JEV), a member of the Flavivirus genus, is an important pathogen that causes human and animal infectious diseases in Asia. So far, no effective antiviral agents are available to treat JEV infection. Here, we found that LDLR is a host factor required for JEV entry. Berbamine significantly decreases the level of LDLR at the plasma membrane by inducing the secretion of LDLR via extracellular vesicles (EVs), thereby inhibiting JEV infection. Mechanistically, berbamine blocks TRPMLs (Ca2+ permeable non-selective cation channels in endosomes and lysosomes) to compromise the endolysosomal trafficking of LDLR. This leads to the increased secretion of LDLR via EVs and the concomitant decrease in its level at the plasma membrane, thereby rendering cells resistant to JEV infection. Berbamine also protects mice from the lethal challenge of JEV. In summary, these results indicate that berbamine is an effective anti-JEV agent by preventing JEV entry.

Mechanism through Which Retrocyclin Targets Flavivirus Multiplication.

Currently, there are no approved drugs for the treatment of flavivirus infection. Accordingly, we tested the inhibitory effects of the novel θ-defensin retrocyclin-101 (RC-101) against flavivirus infection and investigated the mechanism underlying the potential inhibitory effects. First, RC-101 robustly inhibited both Japanese encephalitis virus (JEV) and Zika virus (ZIKV) infections. RC-101 exerted inhibitory effects on the entry and replication stages. Results also indicated that the nonstructural protein NS2B-NS3 serine protease might serve as a potential viral target. Furthermore, RC-101 inhibited protease activity at the micromolar level. We also demonstrated that with respect to the glycoprotein E protein of flavivirus, the DE loop of domain III (DIII), which is the receptor-binding domain of the E protein, might serve as another viral target of RC-101. Moreover, a JEV DE mutant exhibited resistance to RC-101, which was associated with deceased binding affinity of RC-101 to DIII. These findings provide a basis for the development of RC-101 as a potential candidate for the treatment of flavivirus infection. IMPORTANCE Retrocyclin is an artificially humanized circular θ-defensin peptide, containing 18 residues, previously reported to possess broad antimicrobial activity. In this study, we found that retrocyclin-101 inhibited flavivirus (ZIKV and JEV) infections. Retrocyclin-101 inhibited NS2B-NS3 serine protease activity, suggesting that the catalytic triad of the protease is the target. Moreover, retrocyclin-101 bound to the DE loop of the E protein of flavivirus, which prevented its entry.

RNA Interference Screening Reveals Requirement for Platelet-Derived Growth Factor Receptor Beta in Japanese Encephalitis Virus Infection.

Mosquito-borne Japanese encephalitis virus (JEV) causes serious illness worldwide and is associated with high morbidity and mortality. To identify potential host therapeutic targets, a high-throughput receptor tyrosine kinase small interfering RNA library screening was performed with recombinant JEV particles. Platelet-derived growth factor receptor beta (PDGFRß) was identified as a hit after two rounds of screening. Knockdown of PDGFRß blocked JEV infection and transcomplementation of PDGFRß could partly restore its infectivity. The PDGFRß inhibitor imatinib, which has been approved for the treatment of malignant metastatic cancer, protected mice against JEV-induced lethality by decreasing the viral load in the brain while abrogating the histopathological changes associated with JEV infection. These findings demonstrated that PDGFRß is important in viral infection and provided evidence for the potential to develop imatinib as a therapeutic intervention against JEV infection.

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

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

Computational identification of natural product leads that inhibit mast cell chymase: an exclusive plausible treatment for Japanese encephalitis.

A recent research has identified chymase, a mast cell-specific protease as an exclusive novel therapeutic target to prevent Japanese encephalitis virus (JEV) induced encephalitis. Interestingly, JEV activates mast cell specific chymase during its penetration through blood brain barrier (BBB) which eventually guide to viral encephalitis. Hence, in this study, natural chemical entities (NCE) from multiple databases (MPD3, TIPDB and MTDP) were virtually screened for their binding affinity as chymase inhibitors, a promising negotiator for prolong survival against JEV tempted encephalitis. Merged computational programs, Maestro software, QikProp, ProTox and Gromacs were applied to screen the NCEs against target receptor (PDB: 4KP0). Three hits (C00008437, C00014417 and 8141903) were identified after employing a series of sieves such as High Throughput Virtual Screening (HTVS), Standard precision (SP) and Xtra precision (XP) molecular docking simulations followed by desired pharmacokinetic-toxicity profile predictions and molecular dynamics (MD) examinations. Maestro simulations resulted in best three binding energy scores as -11.992 kcal/mol (first ranked; C00008437), -11.673 kcal/mol (second ranked; C00014417) and -11.456 kcal/mol (third ranked; 8141903), respectively. The top three hits revealed an ideal range of pharmacokinetic and toxicity descriptors values. In addition, MD simulations enabled us to confirm top hits higher selectivity toward chymase receptor. In conclusion, this might potentially represent remarkable novel classes with an effective chymase mediated treatment to combat JEV induced encephalitis, which need to justify with further detail studies.