Regulation of Onecut2 by miR-9-5p in Japanese encephalitis virus infected neural stem/progenitor cells.

Japanese encephalitis virus (JEV) is one of the major neurotropic viral infections that is known to dysregulate the homeostasis of neural stem/progenitor cells (NSPCs) and depletes the stem cell pool. NSPCs are multipotent stem cell population of the central nervous system (CNS) which are known to play an important role in the repair of the CNS during insults/injury caused by several factors such as ischemia, neurological disorders, CNS infections, and so on. Viruses have evolved to utilize host factors for their own benefit and during JEV infection, host factors, including the non-coding RNAs such as miRNAs, are reported to be affected, thereby cellular processes regulated by the miRNAs exhibit perturbed functionality. Previous studies from our laboratory have demonstrated the role of JEV infection in dysregulating the function of neural stem cells (NSCs) by altering the cell fate and depleting the stem cell pool leading to a decline in stem cell function in CNS repair mechanism post-infection. JEV-induced alteration in miRNA expression in the NSCs is one of the major interest to us. In prior studies, we have observed an altered expression pattern of certain miRNAs following JEV infection. In this study, we have validated the role of JEV infection in NSCs in altering the expression of miR-9-5p, which is a known regulator of neurogenesis in NSCs. Furthermore, we have validated the interaction of this miRNA with its target, Onecut2 (OC2), in primary NSCs utilizing miRNA mimic and inhibitor transfection experiments. Our findings indicate a possible role of JEV mediated dysregulated interaction between miR-9-5p and its putative target OC2 in NSPCs. IMPORTANCE: MicroRNAs have emerged as key disease pathogenic markers and potential therapeutic targets. In this study, we solidify this concept by studying a key miRNA, miR-9-5p, in Japanese encephalitis virus infection of neural stem/progenitor cells. miRNA target Onecut2 has a possible role in stem cell pool biology. Here, we show a possible mechanistic axis worth investing in neurotropic viral biology.

Palmitoylation of hIFITM1 inhibits JEV infection and contributes to BBB stabilization.

Human brain microvascular endothelial cells (hBMECs) are the main component cells of the blood-brain barrier (BBB) and play a crucial role in responding to viral infections to prevent the central nervous system (CNS) from viral invasion. Interferon-inducible transmembrane protein 1 (IFITM1) is a multifunctional membrane protein downstream of type-I interferon. In this study, we discovered that hIFITM1 expression was highly upregulated in hBMECs during Japanese encephalitis virus (JEV) infection. Depletion of hIFITM1 with CRISPR/Cas9 in hBMECs enhanced JEV replication, while overexpression of hIFITM1 restricted the viruses. Additionally, overexpression of hIFITM1 promoted the monolayer formation of hBMECs with a better integrity and a higher transendothelial electrical resistance (TEER), and reduced the penetration of JEV across the BBB. However, the function of hIFITM1 is governed by palmitoylation. Mutations of palmitoylation residues in conserved CD225 domain of hIFITM1 impaired its antiviral capacity. Moreover, mutants retained hIFITM1 in the cytoplasm and lessened its interaction with tight junction protein Occludin. Taken together, palmitoylation of hIFITM1 is essential for its antiviral activity in hBMECs, and more notably, for the maintenance of BBB homeostasis.

Increased IL-6 Levels and the Upregulation of Iron Regulatory Biomarkers Contribute to the Progression of Japanese Encephalitis Virus Infection's Pathogenesis.

Integrated analysis of iron regulatory biomarkers and inflammatory response could be an important strategy for Japanese encephalitis viral (JEV) infection disease management. In the present study, the inflammatory response was assessed by measuring serum Interleukin-6 (IL-6) levels using ELISA, and the transcription levels of iron homeostasis regulators were analyzed via RT-PCR. Furthermore, inter-individual variation in the transferrin gene was analyzed by PCR-RFLP and their association with clinical symptoms, susceptibility, severity, and outcomes was assessed through binary logistic regression and classification and regression tree (CART) analysis. Our findings revealed elevated levels of IL-6 in serum as well as increased expression of hepcidin (HAMP), transferrin (TF), and transferrin receptor-1 (TFR1) mRNA in JEV infection cases. Moreover, we found a genetic variation in TF (rs4481157) associated with clinical symptoms of meningoencephalitis. CART analysis indicates that individuals with the wild-type TF genotype are more susceptible to moderate JEV infection, while those with the homozygous type are in the high-risk group to develop a severe JEV condition. In summary, the study highlights that JEV infection induces alteration in both IL-6 levels and iron regulatory processes, which play pivotal roles in the development of JEV disease pathologies.

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

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

Type I/type III IFN and related factors regulate JEV infection and BBB endothelial integrity.

BACKGROUND: Japanese encephalitis virus (JEV) remains a predominant cause of Japanese encephalitis (JE) globally. Its infection is usually accompanied by disrupted blood‒brain barrier (BBB) integrity and central nervous system (CNS) inflammation in a poorly understood pathogenesis. Productive JEV infection in brain microvascular endothelial cells (BMECs) is considered the initial event of the virus in penetrating the BBB. Type I/III IFN and related factors have been described as negative regulators in CNS inflammation, whereas their role in JE remains ambiguous. METHODS: RNA-sequencing profiling (RNA-seq), real-time quantitative PCR, enzyme-linked immunosorbent assay, and Western blotting analysis were performed to analyze the gene and protein expression changes between mock- and JEV-infected hBMECs. Bioinformatic tools were used to cluster altered signaling pathway members during JEV infection. The shRNA-mediated immune factor-knockdown hBMECs and the in vitro transwell BBB model were utilized to explore the interrelation between immune factors, as well as between immune factors and BBB endothelial integrity. RESULTS: RNA-Seq data of JEV-infected hBMECs identified 417, 1256, and 2748 differentially expressed genes (DEGs) at 12, 36, and 72 h post-infection (hpi), respectively. The altered genes clustered into distinct pathways in gene ontology (GO) terms and KEGG pathway enrichment analysis, including host antiviral immune defense and endothelial cell leakage. Further investigation revealed that pattern-recognition receptors (PRRs, including TLR3, RIG-I, and MDA5) sensed JEV and initiated IRF/IFN signaling. IFNs triggered the expression of interferon-induced proteins with tetratricopeptide repeats (IFITs) via the JAK/STAT pathway. Distinct PRRs exert different functions in barrier homeostasis, while treatment with IFN (IFN-ß and IFN-λ1) in hBMECs stabilizes the endothelial barrier by alleviating exogenous destruction. Despite the complex interrelationship, IFITs are considered nonessential in the IFN-mediated maintenance of hBMEC barrier integrity. CONCLUSIONS: This research provided the first comprehensive description of the molecular mechanisms of host‒pathogen interplay in hBMECs responding to JEV invasion, in which type I/III IFN and related factors strongly correlated with regulating the hBMEC barrier and restricting JEV infection. This might help with developing an attractive therapeutic strategy in JE.

Japanese Encephalitis virus infection in astrocytes modulate microglial function: Correlation with inflammation and oxidative stress.

BACKGROUND: Japanese Encephalitis Virus (JEV) is a neurotropic virus which has the propensity to infect neuronal and glial cells of the brain. Astrocyte-microglia crosstalk leading to the secretion of various factors plays a major role in controlling encephalitis in brain. This study focused on understanding the role of astrocytic mediators that further shaped the microglial response towards JEV infection. METHODS: After establishing JEV infection in C8D1A (mouse astrocyte cell line) and primary astrocyte enriched cultures (PAEC), astrocyte supernatant was used for preparation of conditioned media. Astrocyte supernatant was treated with UV to inactivate JEV and the supernatant was added to N9 culture media in ratio 1:1 for preparation of conditioned media. N9 microglial cells post treatment with astrocyte conditioned media and JEV infection were checked for expression of various inflammatory genes by qRT-PCR, levels of secreted cytokines in N9 cell supernatant were checked by cytometric bead array. N9 cell lysates were checked for expression of proteins - pNF-κß, IBA-1, NS3 and RIG-I by western blotting. Viral titers were measured in N9 supernatant by plaque assays. Immunocytochemistry experiments were done to quantify the number of infected microglial cells after astrocyte conditioned medium treatment. Expression of different antioxidant enzymes was checked in N9 cells by western blotting, levels of reactive oxygen species (ROS) was detected by fluorimetry using DCFDA dye. RESULTS: N9 microglial cells post treatment with JEV-infected astrocyte conditioned media and JEV infection were activated, showed an upsurge in expression of inflammatory genes and cytokines both at the transcript and protein levels. These N9 cells showed a decrease in quantity of viral titers and associated viral proteins in comparison to control cells (not treated with conditioned media but infected with JEV). Also, N9 cells upon conditioned media treatment and JEV infection were more prone to undergo oxidative stress as observed by the decreased expression of antioxidant enzymes SOD-1, TRX-1 and increased secretion of reactive oxygen species (ROS). CONCLUSION: Astrocytic mediators like TNF-α, MCP-1 and IL-6 influence microglial response towards JEV infection by promoting inflammation and oxidative stress in them. As a result of increased microglial inflammation and secretion of ROS, viral replication is lessened in conditioned media treated and JEV infected microglial cells as compared to control cells with no conditioned media treatment but only JEV infection.

High-throughput sequencing-based Detection of Japanese encephalitis virus and its effect on micro ribonucleic acid.

it was to explore the mechanism of Japanese encephalitis virus (JEV) and micro ribonucleic acid (miRNA) under high-throughput sequencing. 20 experimental mice, with good growth status and no disease infection, were selected. The cells used in the experiment included mouse microglial cell line (BV2), mouse neuroblastoma cell line (NA), and mouse brain endothelial cell line (bEnd.3). JEV titration was performed with JEV-infected cells, ribonucleic acid (RNA) in the cells was extracted, and finally the miRNA high-throughput sequencing data was analyzed. Agarose gel electrophoresis showed that the 28S and 18S electrophoresis bands were bright. Among the miRNAs detected in mouse brain tissues, 2986 were down-regulated and 1251 were up-regulated. Among miRNAs detected in NA cells, 4238 the decreasing expression and 2356 were expressed increasingly. In reducing miRNA expression, 1 multiplicity of infection (MOI) of P3 strain infection was more significant than 0.1 MOI. 10 miRNAs with significantly decreasing expression were miR-466d-3p, miR-381-3p, miR-540-3p, miR-466a-3p, miR-467a-3p, miR-574-5p, miR-199a-5p, miR-467a-5p, miR-674-5p, and miR-376b-3p. These were all obviously down-regulated in JEV-infected BV2, NA, and bEnd.3 neurons. High-throughput sequencing of JEV-infected mouse brain tissues and mouse neuronal cells found that JEV infection led to down-regulation of overall miRNA expression in host cells.

H3K27me3 of Rnf19a promotes neuroinflammatory response during Japanese encephalitis virus infection.

Histone methylation is an important epigenetic modification that affects various biological processes, including the inflammatory response. In this study, we found that infection with Japanese encephalitis virus (JEV) leads to an increase in H3K27me3 in BV2 microglial cell line, primary mouse microglia and mouse brain. Inhibition of H3K27me3 modification through EZH2 knockdown and treatment with EZH2 inhibitor significantly reduces the production of pro-inflammatory cytokines during JEV infection, which suggests that H3K27me3 modification plays a crucial role in the neuroinflammatory response caused by JEV infection. The chromatin immunoprecipitation-sequencing (ChIP-sequencing) assay revealed an increase in H3K27me3 modification of E3 ubiquitin ligases Rnf19a following JEV infection, which leads to downregulation of Rnf19a expression. Furthermore, the results showed that Rnf19a negatively regulates the neuroinflammatory response induced by JEV. This is achieved through the degradation of RIG-I by mediating its ubiquitination. In conclusion, our findings reveal a novel mechanism by which JEV triggers extensive neuroinflammation from an epigenetic perspective.

Characterization of the complete mitochondrial genome of Culex vishnui (Diptera: Culicidae), one of the major vectors of Japanese encephalitis virus.

Culex mosquitoes (Diptera: Culicidae) can transmit a variety of arthropod-borne viruses (arboviruses), causing human and animal diseases. Cx. vishnui, Cx. pseudovishnui, and Cx. tritaeniorhynchus are three representative species in Culex vishnui subgroup, which are widely distributed in southeast Asia, and they have been proved as the main vectors transmitting Japanese encephalitis virus (JEV) that could cause human infectious mosquito-borne disease across Asia. However, the epidemiology, biology, and even molecular information of those mosquitos remain poorly understood, and only the mitochondrial genome (mitogenome) of Cx. tritaeniorhynchus has been reported in these species. In the present study, we sequenced and annotated the complete mitogenome sequence of Cx. vishnui which was 15,587 bp in length, comprising 37 genes. Comparisons of nucleotide and amino acid sequences between Cx. vishnui and Cx. tritaeniorhynchus revealed that most genes within Culex vishnui subgroup were conserved, except atp8, nad1, atp6, and nad6, with differences of 0.4 (rrnS) - 15.1% (tRNAs) and 0 (nad4L) - 9.4% (atp8), respectively, interestingly suggesting the genes nad4L and rrnS were the most conserved but atp8 gene was the least. The results based on nucleotide diversity also supported a relatively uniform distribution of the intraspecific differences in Cx. vishnui and Cx. tritaeniorhynchus with only one highly pronounced peak of divergence centered at the control region. Phylogenetic analyses using concatenated amino acid sequences of 13 protein-coding genes supported the previous taxonomic classification of the family Culicidae and the monophyly of tribes Aedini, Culicini, Mansoniini, and Sabethini. The present study revealed detailed information on the subgroup Culex vishnui, reanalyzed the relationships within the family Culicidae, provided better markers to identify and distinguish Culex species, and offered more markers for studying the molecular epidemiology, population genetics, and molecular phylogenetics of Cx. vishnui.

The relationship between MMP-2 rs243865, MMP-9 rs398242 and CXCL-12 rs1801157 gene polymorphisms with Japanese encephalitis disease and disease outcome in North Indian population.

BACKGROUND & OBJECTIVES: Japanese encephalitis virus (JEV) is one of the most important causes of acute and uncontrolled inflammatory disease in Asia. Matrix metalloproteinases (MMPs) and chemokines play a detrimental role in the host response to JE disease, aetiology, and disease outcome. Evidently, MMPs are widely circulated in the brain and regulate various process including microglial activation, inflammation, blood-brain barrier disruption as well as affects central nervous system (CNS). The present study was to assess the association of single nucleotide polymorphisms of MMP-2, MMP-9 and chemokine (CXCL-12/SDF1-3') in the north Indian population. METHODS: We performed case-control study comprising of 125 patients and 125 healthy controls in north Indian population. Genomic DNA was extracted from whole blood and gene polymorphism have been determined by PCR-RFLP method. RESULTS: MMP-2, MMP-9 and CXCL-12 gene was not significantly associated with JE disease, but homozygous (T/T) genotype of MMP-2 was statically associated with disease outcome (p=0.05, OR=0.110). A/G and G/G genotype of CXCL-12 was significantly associated with severity of disease. (p=0.032, OR=5.500, p=0.037, OR= 9.167). The serum level of MMP-2 was observed significantly increased in JE patients with homozygous (T/T) genotype whereas increased MMP-9 level was associated with heterozygous genotype. INTERPRETATION & CONCLUSION: MMP-2, MMP-9 and CXCL-12 gene polymorphism were not associated with JE susceptibility, but MMP-2 may be contributed to disease protection. CXCL-12 was associated with disease severity. In our concern this is the first report from northern India.

Interactions of host miRNAs in the flavivirus 3´UTR genome: From bioinformatics predictions to practical approaches.

The genus Flavivirus of the Flaviviridae family includes important viruses, such as Dengue, Zika, West Nile, Japanese encephalitis, Murray Valley encephalitis, tick-borne encephalitis, Yellow fever, Saint Louis encephalitis, and Usutu viruses. They are transmitted by mosquitoes or ticks, and they can infect humans, causing fever, encephalitis, or haemorrhagic fever. The treatment resources for these diseases and the number of vaccines available are limited. It has been discovered that eukaryotic cells synthesize small RNA molecules that can bind specifically to sequences present in messenger RNAs to inhibit the translation process, thus regulating gene expression. These small RNAs have been named microRNAs, and they have an important impact on viral infections. In this review, we compiled the available information on miRNAs that can interact with the 3' untranslated region (3'UTR) of the flavivirus genome, a conserved region that is important for viral replication and translation.

Novel insights into host responses to Japanese Encephalitis Virus infection: Reanalysis of public transcriptome and microRNAome datasets.

PURPOSE: Japanese encephalitis (JE), caused by the Japanese encephalitis virus (JEV), is the principal cause of viral encephalitis in South-East Asian and Western Pacific countries; accounting for 68,000 cases, and up to 20,400 fatalities, annually across the world. Despite being a high-risk condition, there is no specific treatment for JE. Given rapid additions in genomics databases and the power of data reanalysis in addressing critical medical questions, the present study was designed to identify novel host factors that might have potential roles in JEV infection. METHODS: We extracted microarray and RNA-Seq data sets from NCBI-GEO and compared mock and JEV-infected samples. Raw data from all the studies were re-analyzed to identify host factors associated with JEV replication. RESULTS: We identified several coding and non-coding host factors that had no prior known role in viral infections. Of these, the coding transcripts: Myosin Heavy Chain 10 (MYH10), Progestin and AdipoQ Receptor Family Member 8 (PAQR8), and the microRNAs: hsa-miR-193b-5p, hsa-miR-3714 and hsa-miR-513a-5p were found to be novel host factors deregulated during JEV infection. MYH10 encodes a conventional non-muscle myosin, and mutations in MYH10 have been shown to cause neurological defects. PAQR8 has been associated with epilepsy, which exhibits symptoms similar to JEV infection. JE is a neuro-degenerative disease, and the known involvement of MYH10 and PAQR8 in neurological disorders strongly indicates potential roles of these host factors in JEV infection. Additionally, we observed that MYH10 and PAQR8 had a significant negative correlation with Activating transcription factor 3 (ATF3), which is a previously validated modulator of JEV infection. ATF3 is a transcription factor that binds to the promotors of genes encoding other transcription factors or interferon-stimulated genes and negatively regulates host antiviral responses during JE. CONCLUSION: Our findings demonstrate the significance of data reanalysis in the identification of novel host factors that may become targets for diagnosis/ therapy against viral diseases of major concern, such as, JE. The deregulated coding and non-coding transcripts identified in this study need further experimental analysis for validation.

Nucleotide-Binding Oligomerization Domain 1 (NOD1) Positively Regulates Neuroinflammation during Japanese Encephalitis Virus Infection.

Japanese encephalitis virus (JEV) is a neurotropic flavivirus that invades the central nervous system and causes neuroinflammation and extensive neuronal cell death. Nucleotide-binding oligomerization domain 1 (NOD1) is a type of pattern recognition receptor that plays a regulatory role in both bacterial and nonbacterial infections. However, the role of NOD1 in JEV-induced neuroinflammation remains undisclosed. In this study, we evaluated the effect of NOD1 activation on the progression of JEV-induced neuroinflammation using a human astrocytic cell line and NOD1 knockout mice. The results showed that JEV infection upregulated the mRNA and protein expression of NOD1, ultimately leading to an enhanced neuroinflammatory response in vivo and in vitro. Inhibition of NOD1 in cultured cells or mice significantly abrogated the inflammatory response triggered by JEV infection. Moreover, compared to the wild-type mice, the NOD1 knockout mice showed resistance to JEV infection. Mechanistically, the NOD1-mediated neuroinflammatory response was found to be associated with increased expression or activation/phosphorylation of downstream receptor-interacting protein 2 (RIPK2), mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK), Jun N-terminal protein kinase (JNK), and NF-κB signaling molecules. Thus, NOD1 targeting could be a therapeutic approach to treat Japanese encephalitis. IMPORTANCE Neuroinflammation is the main pathological manifestation of Japanese encephalitis (JE) and the most important factor leading to morbidity and death in humans and animals infected by JEV. An in-depth understanding of the basic mechanisms of neuroinflammation will contribute to research on JE treatment. This study proved that JEV infection can activate the NOD1-RIPK2 signal cascade to induce neuroinflammation through the proven downstream MAPK, ERK, JNK, and NF-κB signal pathway. Thus, our study unveiled NOD1 as a potential target for therapeutic intervention for JE.

Differential expression of circulating microRNAs in serum: Potential biomarkers to track Japanese encephalitis virus infection.

Japanese encephalitis is one of the serious vector-borne viral encephalitis diseases found worldwide and poses a major threat to public health. Most Japanese encephalitis virus (JEV) infections are subclinical; only 1: 250 to 1:1000 infected persons develop clinical presentations. Delay in proper diagnosis of JE affects the timeliness of treatment initiation and increases the mortality rate in patients. Therefore, there is an extreme need to develop potential biomarkers, which might improve the diagnosis and can become the basis for development of new therapeutics. The microRNAs (miRNAs/or miRs) are small noncoding RNAs of 17-24 nucleotides that are known to regulate about 60% of human genes. Although miRNAs have been found to regulate various aspects of innate and adaptive immune responses, less information on circulating miRNAs in JE is known. The study of JEV infected human serum miRNAs will provide novel information for the diagnosis of JE as well as for the improvement of disease outcome. Total RNA, including miRNA, was extracted from serum followed by the complementary DNA (cDNA) synthesis by using sequence-specific primers. cDNA was amplified using target-specific TaqMan MicroRNA Assay. Real-time polymerase chain reaction data was normalized using both exogenous (cel-miR-39) and endogenous (hsa-miR-93) controls. We have found significantly altered expression of miR-155 and miR-21 in serum of JEV infected patients as compared to healthy controls, revealing their role as a a noninvasive biomarker in JE. A significant correlation between miRNAs and JE was observed that offers the basis for miRNAs to serve as a new component to develop possible therapeutic strategies for JE in near future.

Association of single nucleotide polymorphisms in the CD209, MMP9, TNFA and IFNG genes with susceptibility to Japanese encephalitis in children from North India.

Japanese encephalitis (JE), an acute encephalitis syndrome disease caused by infection with JE virus (JEV), is an important mosquito borne disease in developing countries. The clinical outcomes of JEV infection show inter individual differences. Only in a minor percent of the infected subjects, the disease progresses into acute encephalitis syndrome. Single nucleotide polymorphisms in the host immune response related genes are known to affect susceptibility to JE. In the present study, 238 JE cases and 405 healthy controls (HCs) without any known history of encephalitis were investigated for SNPs in the CD209 MX1, TLR3, MMP9, TNFA and IFNG genes which are important in the immune response against JEV by PCR based methods. The results revealed higher frequencies of heterozygous genotypes of CD209 rs4804803, MMP9 rs17576, TNFA rs1800629 and IFNG rs2430561 in JE cases compared to HCs. These SNPs were associated with JE in an over-dominant genetic model (Odds ratio with 95% CI 1.51 (1.09-2.10) for CD209 rs4804803, 1.52 (1.09-2.11) for MMP9 rs17576, and 1.55 (1.12-2.15) for IFNG rs2430561). The association of G/A genotype of TNFA rs1800629 with JE was confirmed in a larger sample size. The results suggest the association of CD209 rs4804803, MMP9 rs17576, IFNG rs2430561 and TNFA rs1800629 polymorphisms with susceptibility to JE.

Pro-inflammatory and anti-inflamatory cytokine genes polymorphisms and susceptibility to Japanese encephalitis disease in the North Indian population.

BACKGROUND: Japanese encephalitis virus (JEV) is the major cause of viral encephalitis in many regions of Asia. Cytokines, including pro-inflammatory and anti-inflammatory are key regulators playing a detrimental role in the host response to JE infection, pathogenesis and disease outcome. Evidently, the host's cytokine response is genetically determined, representing the complexity of interindividual differences regarding immune response to viral infection. The current study assesses the association of single nucleotide polymorphisms of classical interleukin IL-1ß and IL-10 with JEV susceptibility and disease severity in north Indian population. METHODS: We performed a case-control study using 85 JE patients and 85 healthy controls. Polymorphisms in the IL-1ß (-511 C/T) and IL-10 (-1082 A/G) genes were genotyped using PCR-RFLP. All continuous variables were expressed as mean ± standard deviation, and categorical variables were expressed in percentage. RESULTS: The mRNA level of IL-1ß and IL-10 were found significantly increased in JE patients. In severe JE patients, IL-1ß mRNA level was significantly higher with heterozygous (C/T) and homozygous (C/C) genotype compared to wild (T/T) genotype and mRNA level of IL-10 was higher in heterozygous genotype (A/G) compared to wild genotype (A/A). The C/T and C/C genotypes of IL-1ß were significantly associated with higher risk of JE infection (p < 0.05, OR = 7.25 and 4.40) whereas, the A/G genotype of IL-10 was associated with a reduced risk of JEV infection (p < 0.05, OR = 0.30). The C allele of IL-1ß was associated with fever and neck stiffness (p < 0.05) and CT genotype was associated with disease severity and worse outcomes in JE patients. Along with this, IL-10 polymorphism was found associated with fever, and AG genotype was found to be associated with worse disease outcomes such as neurological sequelae (p < 0.05). CONCLUSION: Mutant allele and genotype at IL-1ß (-511 C/T) and IL-10 (-1082 A/G) gene polymorphism show increased expression of IL-1ß and IL-10 in JE patients which contribute to disease severity as well as adverse outcomes of disease. Overall this is the first report from northern India, which shows the association of IL-1ß and IL-10 polymorphisms with JEV infection.

Proteomic landscape of Japanese encephalitis virus-infected fibroblasts.

Advances in proteomics have enabled a comprehensive understanding of host-pathogen interactions. Here we have characterized Japanese encephalitis virus (JEV) infection-driven changes in the mouse embryonic fibroblast (MEF) proteome. Through tandem mass tagging (TMT)-based mass spectrometry, we describe changes in 7.85 % of the identified proteome due to JEV infection. Pathway enrichment analysis showed that proteins involved in innate immune sensing, interferon responses and inflammation were the major upregulated group, along with the immunoproteasome and poly ADP-ribosylation proteins. Functional validation of several upregulated anti-viral innate immune proteins, including an active cGAS-STING axis, was performed. Through siRNA depletion, we describe a crucial role of the DNA sensor cGAS in restricting JEV replication. Further, many interferon-stimulated genes (ISGs) were observed to be induced in infected cells. We also observed activation of TLR2 and inhibition of TLR2 signalling using TLR1/2 inhibitor CU-CPT22-blocked production of inflammatory cytokines IL6 and TNF-α from virus-infected N9 microglial cells. The major proteins that were downregulated by infection were involved in cell adhesion (collagens), transport (solute carrier and ATP-binding cassette transporters), sterol and lipid biosynthesis. Several collagens were found to be transcriptionally downregulated in infected MEFs and mouse brain. Collectively, our data provide a bird's-eye view into how fibroblast protein composition is rewired following JEV infection.

Development and characterization of an animal model of Japanese encephalitis virus infection in adolescent C57BL/6 mouse.

A mouse-adapted isolate of Japanese encephalitis virus (JEV), designated as JEV-S3, was generated by serially passaging the P20778 strain of the virus in 3- to 4-week-old C57BL/6 mice. Blood-brain barrier leakage was evident in JEV-S3-infected mice, in which viral antigens and RNA were consistently demonstrated in the brain, along with infiltration of activated immune cells, as evidenced by an increased CD45+CD11b+ cell population. Histopathology studies showed the presence of perivascular cuffing, haemorrhage and necrotic foci in the virus-infected brain, conforming to the pathological changes seen in the brain of JEV-infected patients. Mass spectrometry studies characterized the molecular events leading to brain inflammation in the infected mice. Notably, a significant induction of inflammatory cytokines, such as IFNγ, IL6, TNFα and TGFß, was observed. Further, genome sequencing of the JEV-S3 isolate identified the mutations selected during the mouse passage of the virus. Overall, we present an in-depth characterization of a robust and reproducible mouse model of JEV infection. The JEV-S3 isolate will be a useful tool to screen antivirals and study virus pathogenesis in the adolescent mouse model.

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.

BACKGROUND: Japanese 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. METHODS: This study was performed an equal number of cases and control individuals (125). Gene polymorphism has been analyzed by PCR-RFLP and expression by ELISA. RESULTS: Homozygous(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. CONCLUSION: IL-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.

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.