Japanese Encephalitis Virus Infection Research Articles

Brain Microvascular Endothelial Cell-Derived HMGB1 Facilitates Monocyte Adhesion and Transmigration to Promote JEV Neuroinvasion.

Infection with Japanese encephalitis virus (JEV) induces high morbidity and mortality, including potentially permanent neurological sequelae. However, the mechanisms by which viruses cross the blood-brain barrier (BBB) and invade into the central nervous system (CNS) remain unclear. Here, we show that extracellular HMGB1 facilitates immune cell transmigration. Furthermore, the migration of immune cells into the CNS dramatically increases during JEV infection which may enhance viral clearance, but paradoxically expedite the onset of Japanese encephalitis (JE). In this study, brain microvascular endothelial cells (BMECs) were utilized for the detection of HMGB1 release, and leucocyte, adhesion, and the integrity of the BBB in vitro. Genetically modified JEV-expressing EGFP (EGFP-JEV) and the BBB model were established to trace JEV-infected immune cell transmigration, which mimics the process of viral neuroinfection. We find that JEV causes HMGB1 release from BMECs while increasing adhesion molecules. Recombinant HMGB1 enhances leukocyte-endothelium adhesion, facilitating JEV-infected monocyte transmigration across endothelia. Thus, JEV successfully utilizes infected monocytes to spread into the brain, expanding inside of the brain, and leading to the acceleration of JE onset, which was facilitated by HMGB1. HMGB1-promoted monocyte transmigration may represent the mechanism of JEV neuroinvasion, revealing potential therapeutic targets.

Activity of methanolic Andrographis paniculate crude extract and its purified andrographolide against Japanese-encephalitis virus

Infection of Japanese encephalitis virus (JEV) generates brain swelling, namely Japanese encephalitis (JE) in human. This virus is spread by Culex spp. mosquitoes and causes 30% case-fatality rate in Asia and Western Pacific regions annually. No specific cure for the disease is available. Andrographis paniculate methanolic extract and its purified andrographolide exhibit anti-inflammatory and antiviral properties. Our study is focused to antiviral activity of A. paniculate extracts and its elements against JEV in vitro. Half-maximal cytotoxicity (CC50) and the maximum non-toxic doses (MNTD) of A. paniculata in SK-NMC and LLC-MK2 are less than the CC50 and MNTD of andrographolide in SK-NMC and LLC-MK2 at 3 days-post treatment by MTT colorimetric assay. Both A. paniculata and andrographolide have antiviral effect by simultaneous treatment more than pre-treatment and post-treatment at 3 days-post treatment by time-of-addition assay. Antiviral action of these agents interfered virus attachment and had virucidal activity. The results indicated that A. paniculata and andrographolide have the potential antiviral activity against JEV.

C19orf66 Inhibits Japanese Encephalitis Virus Replication by Targeting -1 PRF and the NS3 Protein.

The Japanese encephalitis serogroup of the neurogenic Flavivirus has a specific feature that expresses a non-structural protein NS1' produced through a programmed -1 ribosomal frameshifting (-1 PRF). Herein, C19orf66, a novel member of interferon-stimulated gene (ISG) products, exhibited significant activity of antagonizing Japanese encephalitis virus (JEV) infection. Overexpression of C19orf66 in 293T cells significantly inhibited JEV replication, while knock-down of endogenous C19orf66 in HeLa cells and A549 cells significantly increased virus replication. Notably, C19orf66 had an inhibitory effect on frameshift production of JEV NS1'. The inhibition was more significant when C19orf66 and JEV NS1-NS2A were co-expressed in the 293T cells. Both C19orf66-209 and C19orf66-Zincmut did not significantly change the NS1' to NS1 ratio and had weaker antiviral effects than C19orf66. Similarly, C19orf66-209 and C19orf66-Zincmut had no significant effect on the expression of the JEV NS3 protein, whose expression was down-regulated by C19orf66 via the lysosome-dependent pathway. These findings suggest that C19orf66 may possess at least two different mechanisms of antagonizing JEV infection. This study identified C19orf66 as a novel interferon-stimulated gene product that can inhibit JEV replication by targeting -1 PRF and the NS3 protein. The study provides baseline information for the future development of broad-spectrum antiviral agents against JEV.

Pathobiology of Japanese encephalitis virus infection

Japanese encephalitis virus (JEV) is a flavivirus, spread by the bite of carrier Culex mosquitoes. The subsequent disease caused is Japanese encephalitis (JE), which is the leading global cause of virus-induced encephalitis. The disease is predominant in the entire Asia-Pacific region with the potential of global spread. JEV is highly neuroinvasive with symptoms ranging from mild fever to severe encephalitis and death. One-third of JE infections are fatal, and half of the survivors develop permanent neurological sequelae. Disease prognosis is determined by a series of complex and intertwined signaling events dictated both by the virus and the host. All flaviviruses, including JEV replicate in close association with ER derived membranes by channelizing the protein and lipid components of the ER. This leads to activation of acute stress responses in the infected cell-oxidative stress, ER stress, and autophagy. The host innate immune and inflammatory responses also enter the fray, the components of which are inextricably linked to the cellular stress responses. These are especially crucial in the periphery for dendritic cell maturation and establishment of adaptive immunity. The pathogenesis of JEV is a combination of direct virus induced neuronal cell death and an uncontrolled neuroinflammatory response. Here we provide a comprehensive review of the JEV life cycle and how the cellular stress responses dictate the pathobiology and resulting immune response. We also deliberate on how modulation of these stress pathways could be a potential strategy to develop therapeutic interventions, and define the persisting challenges.

Japanese encephalitis virus infection in meerkats (Suricata suricatta)

Japanese encephalitis virus (JEV) infection has been recognized as a serious disease in humans. Wildlife animal infections due to JEV have not been well described. This study identified JEV infection in two deceased meerkats in Thailand, with clinical signs of neurological disease. Histopathology of brains revealed severe lymphoplasmacytic necrotizing meningoencephalitis, while similar inflammation was observed in the lung and liver. Partial JEV sequences were identified from the formalin-fixed paraffin-embedded-derived brain sections of two meerkats and were found to be genetically similar to a JEV strain detected in China but not from a local strain. Using immunohistochemistry, the virus was identified in neurons and glial cells, and also found in bronchial glands, Kupffer's cells in liver, lymphocytes in the spleen and pancreatic acini, which suggests extraneural infection. Transmission electron microscopy confirmed the presence of spheroid viral particles in the lungs. These findings may suggest that infection of extraneural organs in meerkats is similar to that described in JEV-infected humans. In conclusion, this study identified the first JEV infection in meerkats as an interesting case study. The JEV should be considered as an important differential diagnosis in meerkats with encephalitis. Further surveillance on JEV infection in meerkats and other wildlife species in a large cohort is needed in the future study.

Proteomic analyses identify intracellular targets for Japanese encephalitis virus nonstructural protein 1 (NS1)

Japanese encephalitis is a zoonotic disease caused by Japanese encephalitis virus (JEV). JEV nonstructural protein 1 (NS1) is involved in many crucial biological events during viral infection and immune suppression. To investigate the role of JEV NS1 in virus-infected cells, the molecules with which it interacts intracellularly were screened with a pull-down assay and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The interaction between heterogeneous nuclear ribonucleoprotein K (hnRNP K), vimentin and NS1 were verified with coimmunoprecipitation and confocal assays. Our results show that JEV NS1 interacts with vimentin, hnRNP K and colocalizes with cellular vimentin and hnRNP K. Furthermore, our results demonstrate that the expression of vimentin and hnRNP K were up-regulated in both NS1-transfected and JEV-infected cells. Knocking down vimentin and hnRNP K reduced viral replication while conversely, over-expression of vimentin and hnRNP K improved viral replication, suggesting an important role for this protein in the viral life cycle. Also, We found that vimentin also interacted with hnRNP K after overexpression of NS1 or JEV infection. These findings provide insight into the molecular mechanism of JEV replication and highlight the key role the NS1 in JEV infection.

Precise localization and dynamic distribution of Japanese encephalitis virus in the rain nuclei of infected mice.

Japanese encephalitis virus (JEV) is a pathogen that causes severe vector-borne zoonotic diseases, thereby posing a serious threat to human health. Although JEV is potentially neurotropic, its pathogenesis and distribution in the host have not been fully elucidated. In this study, an infected mouse model was established using a highly virulent P3 strain of JEV. Immunohistochemistry and in situ hybridization, combined with anatomical imaging of the mouse brain, were used to dynamically localize the virus and construct three-dimensional (3D) images. Consequently, onset of mild clinical signs occurred in some mice at 3.5 d post JEV infection, while most mice displayed typical neurological signs at 6 d post-infection (dpi). Moreover, brain pathology revealed typical changes associated with non-suppurative encephalitis, which lasted up to 8 d. The earliest detection of viral antigen was achieved at 3 dpi in the thalamus and medulla oblongata. At 6 dpi, the positive viral antigen signals were mainly distributed in the cerebral cortex, olfactory area, basal ganglia, thalamus, and brainstem regions in mice. At 8 dpi, the antigen signals gradually decreased, and the localization of JEV tended to concentrate in the cerebrum and thalamus, while no viral antigen was detected in the brain at 21 dpi. In this model, the viral antigen was first expressed in the reticular thalamic nucleus (Rt), and the virus content is relatively stable. The expression of the viral antigen in the hippocampal CA2 region, the anterior olfactory nucleus, and the deep mesencephalic nucleus was high and persistent. The 3D images showed that viral signals were mostly concentrated in the parietal cortex, occipital lobe, and hippocampus, near the mid-sagittal plane. In the early stages of infection in mice, a large number of viral antigens were detected in denatured and necrotic neurons, suggesting that JEV directly causes neuronal damage. From the time of its entry, JEV is widely distributed in the central nervous system thereby causing extensive damage.

Association of interleukin-6 (174 G/C) and interleukin-12B (1188 A/C) gene polymorphism with expression and risk of Japanese encephalitis disease in North Indian population

BackgroundJapanese encephalitis is an acute inflammatory disease caused by Japanese encephalitis virus (JEV). In this study we aim to determine the association of IL-6 (174) and IL-12B (1188A/C) gene polymorphisms with JEV susceptibility, disease severity and outcomes in north Indian population. MethodsThis study was performed an equal number of cases and control individuals (125). Gene polymorphism has been analyzed by PCR-RFLP and expression by ELISA. ResultsHomozygous(C/C) genotypes of IL-12B were significantly associated with protection in JE infection (p = 0.008, OR = 0.368) whereas IL-6 was not associated with JEV infection (p = 0.269, OR = 1.245). The C allele of IL-6 was associated with protection in JE disease and G/C genotype was associated with outcomes with recovered individuals. ConclusionIL-12B gene polymorphism leads to increase level of IL-12B in JE patients, which can contribute to JE susceptibility and disease severity. IL-6 polymorphism has not been associated with susceptibility of JE. Overall, this is the first information from northern India shows association of IL-6 and IL-12B polymorphisms with JE disease.

Japanese Encephalitis Virus Infected Human Monocyte-Derived Dendritic Cells Activate a Transcriptional Network Leading to an Antiviral Inflammatory Response.

A comprehensive understanding of the human immune response to virus infection is imperative for developing effective therapies, antivirals, and vaccines. Dendritic cells (DCs) are among the first cells to encounter the virus and are also key antigen-presenting cells that link the innate and adaptive immune system. In this study, we focus on the human immune response to the mosquito-borne Japanese encephalitis virus (JEV), which is the leading cause of virus-induced encephalitis in south-east Asia and has the potential to become a global pathogen. We describe the gene regulatory circuit of JEV infection in human monocyte-derived DCs (moDCs) along with its functional validation. We observe that JEV can productively infect human moDCs leading to robust transcriptional activation of the interferon and NF-κB-mediated antiviral and inflammatory pathways. This is accompanied with DC maturation and release of pro-inflammatory cytokines and chemokines TNFα, IL-6, IL-8, IL-12, MCP-1. and RANTES. JEV-infected moDCs activated T-regulatory cells (Tregs) in allogenic mixed lymphocyte reactions (MLR) as seen by upregulated FOXP3 mRNA expression, suggestive of a host response to reduce virus-induced immunopathology. The virus also downregulated transcripts involved in Peroxisome Proliferator Activated Receptor (PPAR) signalling and fatty acid metabolism pathways suggesting that changes in cellular metabolism play a crucial role in driving the DC maturation and antiviral responses. Collectively, our data describe and corroborate the human DC transcriptional network that is engaged upon JEV sensing.

Type I IFN signaling limits hemorrhage-like disease after infection with Japanese encephalitis virus through modulating a prerequisite infection of CD11b+Ly-6C+ monocytes

BackgroundThe crucial role of type I interferon (IFN-I, IFN-α/β) is well known to control central nervous system (CNS) neuroinflammation caused by neurotrophic flaviviruses such as Japanese encephalitis virus (JEV) and West Nile virus. However, an in-depth analysis of IFN-I signal-dependent cellular factors that govern CNS-restricted tropism in JEV infection in vivo remains to be elucidated.MethodsViral dissemination, tissue tropism, and cytokine production were examined in IFN-I signal-competent and -incompetent mice after JEV inoculation in tissues distal from the CNS such as the footpad. Bone marrow (BM) chimeric models were used for defining hematopoietic and tissue-resident cells in viral dissemination and tissue tropism.ResultsThe paradoxical and interesting finding was that IFN-I signaling was essentially required for CNS neuroinflammation following JEV inoculation in distal footpad tissue. IFN-I signal-competent mice died after a prolonged neurological illness, but IFN-I signal-incompetent mice all succumbed without neurological signs. Rather, IFN-I signal-incompetent mice developed hemorrhage-like disease as evidenced by thrombocytopenia, functional injury of the liver and kidney, increased vascular leakage, and excessive cytokine production. This hemorrhage-like disease was closely associated with quick viral dissemination and impaired IFN-I innate responses before invasion of JEV into the CNS. Using bone marrow (BM) chimeric models, we found that intrinsic IFN-I signaling in tissue-resident cells in peripheral organs played a major role in inducing the hemorrhage-like disease because IFN-I signal-incompetent recipients of BM cells from IFN-I signal-competent mice showed enhanced viral dissemination, uncontrolled cytokine production, and increased vascular leakage. IFN-I signal-deficient hepatocytes and enterocytes were permissive to JEV replication with impaired induction of antiviral IFN-stimulated genes, and neuron cells derived from both IFN-I signal-competent and -incompetent mice were vulnerable to JEV replication. Finally, circulating CD11b+Ly-6C+ monocytes infiltrated into the distal tissues inoculated by JEV participated in quick viral dissemination to peripheral organs of IFN-I signal-incompetent mice at an early stage.ConclusionAn IFN-I signal-dependent model is proposed to demonstrate how CD11b+Ly-6C+ monocytes are involved in restricting the tissue tropism of JEV to the CNS.

Novel Japanese encephalitis virus NS1-based vaccine: Truncated NS1 fused with E. coli heat labile enterotoxin B subunit.

BackgroundCurrent vaccines against Japanese encephalitis virus (JEV) of flaviviruses have some disadvantages, such as the risk of virulent reversion. Non-structural protein NS1 is conserved among flaviviruses and confers immune protection without the risk of antibody-dependent enhancement (ADE). Therefore, NS1 has become a promising vaccine candidate against flaviviruses.MethodsA NS1-based vaccine (LTB-NS1∆63) with a truncated NS1 protein (NS1∆63) fused to E. coli heat-labile enterotoxin B subunit (LTB) was expressed in E.coli and explored for its ability to induce immune responses. Safety of LTB-NS1∆63 was assessed by determining its toxicity in vitro and in vivo. Protective capability of LTB-NS1∆63 and its-induced antisera was evaluated in the mice challenged with JEV by analyzing mortality and morbidity.FindingsLTB-NS1∆63 induced immune responses to a similar level as LTB-NS1, but more robust than NS1∆63 alone, particularly in the context of oral immunization of mice. Oral vaccination of LTB-NS1∆63 led to a higher survival rate than that of NS1∆63 or live-attenuated JEV vaccine SA14–14–2 in the mice receiving lethal JEV challenge. LTB-NS1∆63 protein also significantly decreases the morbidity of JEV-infected mice. In addition, passive transfer of LTB-NS1∆63-induced antisera provides a protection against JEV infection in mice.InterpretationNS1∆63 bears JEV NS1 antigenicity. Besides, LTB-NS1∆63 could serve as a novel protein-based mucosa vaccine targeting JEV and other flaviviruses.FundingThis work was supported by the National Natural Science Foundation, Jiangxi Province Science and Technology Committee, Education Department of Jiangxi Province.

Japanese B Encephalitis.

RNA viruses are not only reported for viral pandemics but also as important agents for emerging/re-emerging diseases. Japanese encephalitis virus (JEV) is reported to cause epidemics of encephalitis in Southeast Asia, India, Korea, China, and Indonesia. In addition, several reports show that JEV has spread to new populations beyond these geographical regions. The disease mostly affects children with a mortality rate up to 30%. In peridomestic settings, pigs are reported as amplifiers of JEV transmission and aquatic birds as maintenance hosts of the virus. The Culex mosquito is the vector for transmission of JEV. This virus is a member of the family Flaviviridae and has a single-stranded positive-sense RNA virus. Five different genotypes (G-I to G-V) of JEV have been reported. Four different kinds of vaccines have been produced to prevent JEV infection. However, there is no FDA-approved antiviral drug available for JEV.How to cite this article: Mehta A, Singh R, Mani VE, Poddar B. Japanese B Encephalitis. Indian J Crit Care Med 2021;25(Suppl 2):S171–S174.

Molecular epidemiology of mosquito-borne viruses at the China\u2013Myanmar border: discovery of a potential epidemic focus of Japanese encephalitis

BackgroundMosquito-based arbovirus surveillance can serve as an early warning in evaluating the status of mosquito-borne virus prevalence and thus prevent local outbreaks. Although Tengchong County in Yunnan Province—which borders Myanmar—is abundant and diverse in mosquitoes, very few mosquito-based arbovirus investigations have been conducted in the recent decade. Herein, this study aims to evaluate the presence and the diffusion of mosquito-borne pathogens, currently prevalent in this region.MethodsWe collected 9486 mosquitoes, representing eight species, with Culex tritaeniorhynchus and Anopheles sinensis as the dominant species, during high mosquito activity seasons (July–October) in Tengchong, in 2018. Samples collected from 342 pools were tested using reverse-transcription PCR to determine the species, distribution, and infection rates of virus and parasite, and further analyze their genotypes, phylogenetic relationships, infection rate, and potential pathogenicity.ResultsFifteen Japanese encephalitis virus (JEV) strains from Cx. tritaeniorhynchus pools were detected. Seven strains of insect-specific flaviviruses (ISFVs), including two Aedes flavivirus (AeFV) and Yunnan Culex flavivirus strains each, one Culex theileri flavivirus, Yamadai flavivirus (YDFV) and Anopheles-associated flavivirus (AAFV) strains each were detected in Aedes albopictus, Cx. tritaeniorhynchus, Cx. vagans, Cx. pseudovihnui, and An. sinensis pools, respectively. The whole-genome was successfully amplified in one strain of JEV and AeFV each. Phylogenetic analysis using the E gene placed all the newly detected JEV strains into the GI-b genotype. They showed highly nucleotide identities, and were most closely related to the strain detected in Tengchong in 2010. The comparison of the E protein of JEV strains and vaccine-derived strain, showed six amino residue differences. The bias-corrected maximum likelihood estimation values (and 95% confidence interval) for JEV in Cx. tritaeniorhynchus collected in Tengchong in 2018 were 2.4 (1.4–3.9).ConclusionsA potential Japanese encephalitis epidemic focus with the abundance of host mosquitoes and high JEV infection rate was observed in Tengchong. In addition, at least five species of ISFVs co-circulate in this area. This study highlights the importance of widespread and sustained mosquito-based arbovirus surveillance in local areas to prevent the transmission of JEV, and other emerging/re-emerging mosquito-borne pathogens.Graphic

Differential miRNA Expression Profiling Reveals Correlation of miR125b-5p with Persistent Infection of Japanese Encephalitis Virus.

MicroRNAs (miRNAs) play versatile roles in multiple biological processes. However, little is known about miRNA’s involvement in flavivirus persistent infection. Here, we used an miRNA array analysis of Japanese encephalitis virus (JEV)-infected cells to search for persistent infection-associated miRNAs in comparison to acute infection. Among all differentially expressed miRNAs, the miR-125b-5p is the most significantly increased one. The high level of miR-125b-5p in persistently JEV-infected cells was confirmed by Northern analysis and real-time quantitative polymerase chain reaction. As soon as the cells established a persistent infection, a significantly high expression of miR-125b-5p was readily observed. Transfecting excess quantities of a miR-125b-5p mimic into acutely infected cells reduced genome replication and virus titers. Host targets of miR125b-5p were analyzed by target prediction algorithms, and six candidates were confirmed by a dual-luciferase reporter assay. These genes were upregulated in the acutely infected cells and sharply declined in the persistently infected cells. The transfection of the miR125b-5p mimic reduced the expression levels of Stat3, Map2k7, and Triap1. Our studies indicated that miR-125b-5p targets both viral and host sequences, suggesting its role in coordinating viral replication and host antiviral responses. This is the first report to characterize the potential roles of miR-125b-5p in persistent JEV infections.

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 (PDGFRβ) was identified as a hit after two rounds of screening. Knockdown of PDGFRβ blocked JEV infection and transcomplementation of PDGFRβ could partly restore its infectivity. The PDGFRβ 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 PDGFRβ is important in viral infection and provided evidence for the potential to develop imatinib as a therapeutic intervention against JEV infection.

Valosin-containing protein/p97 plays critical roles in the Japanese encephalitis virus life cycle.

Host factors provide critical support for every aspect of the virus life cycle. We recently identified the valosin-containing protein (VCP)/p97, an abundant cellular ATPase with diverse cellular functions, as a host factor important for Japanese encephalitis virus (JEV) replication. In cultured cells, using siRNA-mediated protein depletion and pharmacological inhibitors, we show that VCP is crucial for replication of three flaviviruses: JEV, Dengue, and West Nile viruses. An FDA-approved VCP inhibitor, CB-5083, extended survival of mice in the animal model of JEV infection. While VCP depletion did not inhibit JEV attachment on cells, it delayed capsid degradation, potentially through the entrapment of the endocytosed virus in clathrin-coated vesicles (CCVs). Early during infection, VCP-depleted cells showed an increased colocalization of JEV capsid with clathrin, and also higher viral RNA levels in purified CCVs. We show that VCP interacts with the JEV nonstructural protein NS5 and is an essential component of the virus replication complex. The depletion of the major VCP cofactor UFD-1 also significantly inhibited JEV replication. Mechanistically, thus, VCP affected two crucial steps of the JEV life cycle - nucleocapsid release and RNA replication. Our study establishes VCP as a common host factor with a broad antiviral potential against flaviviruses.ImportanceJEV is the leading cause of viral encephalitis epidemics in South-east Asia, affecting majorly children with high morbidity and mortality. Identification of host factors is thus essential for the rational design of anti-virals that are urgently need as therapeutics. Here we have identified the VCP protein as one such host-factor. This protein is highly abundant in cells and engages in diverse functions and cellular pathways by its ability to interact with different co-factors. Using siRNA mediated protein knockdown, we show that this protein is essential for release of the viral RNA into the cell so that it can initiate replication. The protein plays a second crucial role for the formation of the JEV replication complex. FDA-approved drugs targeting VCP show enhanced mouse survival in JE model of disease, suggesting that this could be a druggable target for flavivirus infections.

Temperate conditions restrict Japanese encephalitis virus infection to the mid-gut and prevents systemic dissemination in Culex pipiens mosquitoes

Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, is the main cause of viral encephalitis in Asia. However, with changing climate JEV has the potential to emerge in novel temperate regions. Here, we have assessed the vector competence of the temperate mosquito Culex pipiens f. pipiens to vector JEV genotype III at temperatures representative of those experienced, or predicted in the future during the summer months, in the United Kingdom. Our results show that Cx. pipiens is susceptible to JEV infection at both temperatures. In addition, at 25 °C, JEV disseminated from the midgut and was recovered in saliva samples, indicating the potential for transmission. At a lower temperature, 20 °C, following an incubation period of fourteen days, there were reduced levels of JEV dissemination and virus was not detected in saliva samples. The virus present in the bodies of these mosquitoes was restricted to the posterior midgut as determined by microscopy and viable virus was successfully recovered. Apart from the influence on virus dissemination, mosquito mortality was significantly increased at the higher temperature. Overall, our results suggest that temperature is a critical factor for JEV vector competence and infected-mosquito survival. This may in turn influence the vectorial capacity of Cx. pipiens to vector JEV genotype III in temperate areas.

Potential Role of Birds in Japanese Encephalitis Virus Zoonotic Transmission and Genotype Shift.

Japanese encephalitis (JE) is a vaccine-preventable disease caused by the Japanese encephalitis virus (JEV), which is primarily prevalent in Asia. JEV is a Flavivirus, classified into a single serotype with five genetically distinct genotypes (I, II, III, IV, and V). JEV genotype III (GIII) had been the most dominant strain and caused numerous outbreaks in the JEV endemic countries until 1990. However, recent data shows the emergence of JEV genotype I (GI) as a dominant genotype and it is gradually displacing GIII. The exact mechanism of this genotype displacement is still unclear. The virus can replicate in mosquito vectors and vertebrate hosts to maintain its zoonotic life cycle; pigs and aquatic wading birds act as an amplifying/reservoir hosts, and the humans and equines are dead-end hosts. The important role of pigs as an amplifying host for the JEV is well known. However, the influence of other domestic animals, especially birds, that live in high abundance and close proximity to the human is not well studied. Here, we strive to briefly highlight the role of birds in the JEV zoonotic transmission, discovery of birds as a natural reservoirs and amplifying host for JEV, species of birds susceptible to the JEV infection, and the proposed effect of JEV on the poultry industry in the future, a perspective that has been neglected for a long time. We also discuss the recent in vitro and in vivo studies that show that the newly emerged GI viruses replicated more efficiently in bird-derived cells and ducklings/chicks than GIII, and an important role of birds in the JEV genotype shift from GIII to GI.

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.

Axl Alleviates Neuroinflammation and Delays Japanese Encephalitis Progression in Mice.

Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus, which causes the most commonly diagnosed viral encephalitis named Japanese encephalitis (JE) in the world with an unclear pathogenesis. Axl, a receptor tyrosine kinase from TAM family, plays crucial role in many inflammatory diseases. We have previously discovered that Axl deficiency resulted in more severe body weight loss in mice during JEV infection, which we speculate is due to the anti-inflammatory effect of Axl during JE. Currently, the role of Axl in regulating the neuroinflammation and brain damage during JE has not been investigated yet. In this study, by using Axl deficient and heterozygous control mice, we discovered that Axl deficient mice displayed accelerated JE progression and exacerbated brain damage characterized by increased neural cell death, extended infiltration of inflammatory cells, and enhanced production of pro-inflammatory cytokines, in comparison to control mice. Additionally, consistent with our previous report, Axl deficiency had no impact on the infection and target cell tropism of JEV in brain. Taken together, our results suggest that Axl plays an anti-inflammatory and neuroprotective role during the pathogenesis of JE.