Japanese Encephalitis Virus Infected Cells Research Articles

Japanese Encephalitis Virus-Infected Cells.

RNA virus infections have been a leading cause of pandemics. Aided by global warming and increased connectivity, their threat is likely to increase over time. The flaviviruses are one such RNA virus family, and its prototypes such as the Japanese encephalitis virus (JEV), Dengue virus, Zika virus, West Nile virus, etc., pose a significant health burden on several endemic countries. All viruses start off their life cycle with an infected cell, wherein a series of events are set in motion as the virus and host battle for autonomy. With their remarkable capacity to hijack cellular systems and, subvert/escape defence pathways, viruses are able to establish infection and disseminate in the body, causing disease. Using this strategy, JEV replicates and spreads through several cell types such as epithelial cells, fibroblasts, monocytes and macrophages, and ultimately breaches the blood-brain barrier to infect neurons and microglia. The neurotropic nature of JEV, its high burden on the paediatric population, and its lack of any specific antivirals/treatment strategies emphasise the need for biomedical research-driven solutions. Here, we highlight the latest research developments on Japanese encephalitis virus-infected cells and discuss how these can aid in the development of future therapies.

Japanese Encephalitis Virus exploits microRNA-155 to suppress the non-canonical NF-κB pathway in human microglial cells

Japanese Encephalitis Virus (JEV) is a single positive strand RNA virus, belongs to the Flaviviridae family. JEV is neurotropic in nature which accounts for 30–50% neurological, psychiatric sequelae and movement disorder, with 20–30% case fatality rate among children or elder population. JEV causes neuronal loss and microglial activation which leads to neuroinflammation. The microRNAs are the molecular switches, which regulate the gene expression post-transcriptionally. The microRNA-155 has been reported to be associated with CNS-related pathologies like, experimental autoimmune encephalitis, multiple sclerosis and amyotrophic lateral sclerosis. In the present study, we infected microglial cells with JEV, which resulted in the up-regulation of microRNA-155; quantified by real-time polymerase chain reaction. The gene target prediction databases revealed pellino 1 as a putative gene target for microRNA-155. The over-expression based studies of microRNA-155 mimics, scrambles, inhibitors, and cy3 negative control demonstrated the role of PELI1 in the regulation of the non-canonical NF-κB pathway via TRAF3. The luciferase assay showed the regulation of NF-κB promoter via microRNA-155 in JEV infected microglial cells. The suppression of NF-κB in JEV infected microglial cells led to the reduced expression of IL-6 and TNF-α. JEV exploits cellular microRNA-155 to suppress the expression of PELI1 in human microglial cells as a part of their immune evasion strategy.

Discordant Activity of Kaempferol Towards Dengue Virus and Japanese Encephalitis Virus.

Kaempferol, a plant-derived flavonoid, has been reported to have activity against Japanese encephalitis virus (JEV) in BHK-21 cells. To determine the broader utility of this compound, we initially evaluated the activity of kaempferol against JEV and dengue virus (DENV) in HEK293T/17 cells. Results showed no significant antiviral activity against either virus. We subsequently investigated the activity of kaempferol against both JEV and DENV in BHK-21 cells. Results showed a significant inhibition of JEV infection but, surprisingly, a significant enhancement of DENV infection. The effect of kaempferol on both host protein expression and transcription was investigated and both transcriptional and translational inhibitory effects were observed, although a more marked effect was observed on host cell protein expression. Markedly, while GRP78 was increased in DENV infected cells treated with kaempferol, it was not increased in JEV infected cells treated with kaempferol. These results show that cellular alteration induced by one compound can have opposite effects on viruses from the same family, suggesting the presence of distinct replication strategies for these two viruses.

Japanese Encephalitis Virus infection induces inflammation of swine testis through RIG-I—NF-ĸB signaling pathway

Japanese Encephalitis Virus (JEV) is an important zoonotic flavivirus transmitted by mosquitos. JEV infection in sows primarily manifests as a reproductive disease such as abortion and transient infertility while in infected boars, it can cause orchitis. Previous studies mainly focused on the pathogenesis of human encephalitis caused by JEV infection, while few concentrations have been made to unveil the potential mechanism of reproductive dysfunction in JEV-infected pigs. In this study, histopathological analysis and immunohistochemistry staining was performed on testis of JEV-infected boars, indicating that JEV could infect testicular cells and cause inflammatory changes in testis. In vitro assays reveal that primary swine testicular cells and swine testis (ST) cells are highly permissive to JEV and significant inflammatory response was shown during JEV infection. Mechanically, we found that JEV infection increases the expression of retinoic acid-inducible gene I (RIG-I) and activates transcription factor NF-κB. Production of pro-inflammatory cytokines was greatly reduced in JEV infected testicular cells after knockout of RIG-I or treatment with the NF-κB specific inhibitor. In addition, activation of NF-κB was also significantly suppressed upon RIG-I knockout. Taken together, our results reveal that JEV could infect boar testicles, and RIG-I—NF-κB signaling pathway is involved in JEV-induced inflammation in swine testicular cells.

Modulation of Type-I Interferon Response by hsa-miR-374b-5p During Japanese Encephalitis Virus Infection in Human Microglial Cells.

Japanese Encephalitis virus (JEV) is a neurotropic ssRNA virus, belonging to the Flaviviridae family. JEV is one of the leading causes of the viral encephalitis in Southeast-Asian countries. JEV primarily infects neurons however, the microglial activation has been reported to further enhance the neuroinflammation and promote neuronal death. The PI3K/AKT pathway has been reported to play an important role in type-I interferon response via IRF3. Phosphatase and tensin homolog (PTEN), a negative regulator of PI3K/AKT pathway, participates in microglial polarization and neuroinflammation. The microRNAs are small non-coding endogenously expressed RNAs, which regulate the gene expression by binding at 3′ UTR of target gene. The human microglial cells were infected with JEV (JaOArS982 strain) and up-regulation of microRNA; hsa-miR-374b-5p was confirmed by qRT-PCR. The genes in PI3K/AKT pathway, over-expression and knock-down studies of hsa-miR-374b-5p with and without JEV infection were analyzed through immuno blotting. The regulatory role of hsa-miR-374b-5p on the expression of type-I interferon was determined by luciferase assays. JEV infection modulated the expression of hsa-miR-374b-5p and PI3K/AKT pathway via PTEN. The over-expression of hsa-miR-374b-5p suppressed the PTEN while up-regulated the AKT and IRF3 proteins, whereas, the knockdown rescued the PTEN expression and suppressed the AKT and IRF3 proteins. The modulation of hsa-miR-374b-5p regulated the type-I interferon response during JEV infection. In present study, we have shown the modulation of PTEN by hsa-miR-374b-5p, which regulated the PI3K/AKT/IRF3 axis in JEV infected microglial cells.

PLVAP and GKN3 Are Two Critical Host Cell Receptors Which Facilitate Japanese Encephalitis Virus Entry Into Neurons

Japanese Encephalitis Virus (JEV), a globally important pathogen, belongs to the family Flaviviridae, is transmitted between vertebrate hosts by mosquitoes, principally by Culex tritaeniorhynchus. The E-glycoprotein of the virus mediates its attachment to the host cell receptors. In this study, we cloned and purified JEV E-glycoprotein in pET28a vector using E. coli BL21 (DE3) cells. A pull down assay was performed using plasma membrane fraction of BALB/c mouse brain and E-glycoprotein as a bait protein. 2-Dimensional Gel Electrophoresis based separation of the interacting proteins was analyzed by mass spectrometry. Among all the identified partners of E-glycoprotein, PLVAP (Plasmalemma vesicle associated protein) and GKN3 (Gastrokine3) showed significant up-regulation in both JEV infected mouse brain and neuro2a cells. In-silico studies also predicted significant interaction of these receptors with E-glycoprotein. Additionally, overexperssion and silencing of these receptors resulted in increase and reduction in viral load respectively, suggesting them as two critical cellular receptors governing JEV entry and propagation in neurons. In support, we observed significant expression of PLVAP but not GKN3 in post-mortem autopsied human brain tissue. Our results establish two novel receptor proteins in neurons in case of JEV infection, thus providing potential targets for antiviral research.

Diphenyleneiodonium enhances oxidative stress and inhibits Japanese encephalitis virus induced autophagy and ER stress pathways

Diphenyleneiodonium (DPI) and N-acetyl-l-cysteine (NAC), two widely used anti-oxidants, were employed to evaluate the role of oxidative stress in Japanese encephalitis virus (JEV) induced autophagy, stress responses and replication. DPI and NAC exerted opposite effects on ROS levels in JEV infected mouse neuronal cells (Neuro2a), mouse embryonic fibroblasts (MEFs) and human epithelial cells (HeLa). While NAC effectively quenched ROS, DPI enhanced ROS levels, suggesting that DPI induces oxidative stress in JEV infected cells. DPI treatment of JEV infected Neuro2a cells further blocked autophagy induction and activation of all three arms of the ER stress pathway, and, inhibited virus particle release. Autophagy induction in JEV infection has been previously shown to be linked to the activation of XBP1 and ATF6 ER stress sensors. Our data suggests that DPI mediated block of autophagy is a result of inhibition of ER stress responses and is not associated with an anti-oxidative effect. Since DPI has a wide inhibitory potential for all Flavin dependent enzymes, it is likely that the signalling pathways for ER stress and autophagy during JEV infection are modulated by DPI sensitive enzymes.

Integration analysis of miRNA and mRNA expression profiles in swine testis cells infected with Japanese encephalitis virus

To elucidate the role of microRNAs (miRNA) in the regulation of gene expression in Japanese encephalitis virus (JEV) infected swine testis (ST) cells, we analyzed miRNA and mRNA expression profiles of JEV infected ST cells by high-throughput sequencing technology as compared to uninfected controls. The results showed that 104 known miRNAs and 9 new miRNA candidates were differentially expressed in ST cells after JEV infection. We identified 396 differentially expressed mRNAs. Bioinformatics analysis identified 435 known miRNA–mRNA interaction pairs and 94 novel miRNA–mRNA interaction pairs involving miRNAs inversely correlated with the expression of their predicted target mRNAs. The known miRNAs inversely correlated with their target genes were involved in the biological processes of immunity, cytokine production, inflammation, and apoptosis. Selected miRNA–mRNA interactions were validated by luciferase reporter assay. Overall, our findings indicate that miRNAs may play critical roles in the pathogenesis of JEV infection.

Inhibition of ERK and proliferation in NK cell lines by soluble HLA-E released from Japanese encephalitis virus infected cells

Productive infection of human endothelial cells with Japanese encephalitis virus (JEV), a single stranded RNA virus induces shedding of sHLA-E. We show here that sHLA-E that is released upon infection with this flavivirus can inhibit IL-2 and PMA mediated ERK 1/2 phosphorylation in two NK cell lines, Nishi and NKL. Virus infected or IFN-γ treated cell culture supernatants containing sHLA-E were found to partially inhibit IL-2 mediated induction of CD25 molecules on NKL cells. It was also found that sHLA-E could inhibit IL-2 induced [3H]-thymidine incorporation suggesting that, similar to cell surface expressed HLA-E, sHLA-E could also inhibit NK cell responses. Hence JEV-induced shedding of sHLA-E needs further investigation to better understand immune responses in JEV infections since it may have a role in viral evasion of NK cell responses.

Infection of Human Endothelial Cells by Japanese Encephalitis Virus: Increased Expression and Release of Soluble HLA-E

Japanese encephalitis virus (JEV) is a single stranded RNA virus that infects the central nervous system leading to acute encephalitis in children. Alterations in brain endothelial cells have been shown to precede the entry of this flavivirus into the brain, but infection of endothelial cells by JEV and their consequences are still unclear. Productive JEV infection was established in human endothelial cells leading to IFN-β and TNF-α production. The MHC genes for HLA-A, -B, -C and HLA-E antigens were upregulated in human brain microvascular endothelial cells, the endothelial-like cell line, ECV 304 and human foreskin fibroblasts upon JEV infection. We also report the release/shedding of soluble HLA-E (sHLA-E) from JEV infected human endothelial cells for the first time. This shedding of sHLA-E was blocked by an inhibitor of matrix metalloproteinases (MMP). In addition, MMP-9, a known mediator of HLA solubilisation was upregulated by JEV. In contrast, human fibroblasts showed only upregulation of cell-surface HLA-E. Addition of UV inactivated JEV-infected cell culture supernatants stimulated shedding of sHLA-E from uninfected ECV cells indicating a role for soluble factors/cytokines in the shedding process. Antibody mediated neutralization of TNF-α as well as IFNAR receptor together not only resulted in inhibition of sHLA-E shedding from uninfected cells, it also inhibited HLA-E and MMP-9 gene expression in JEV-infected cells. Shedding of sHLA-E was also observed with purified TNF-α and IFN-β as well as the dsRNA analog, poly (I:C). Both IFN-β and TNF-α further potentiated the shedding when added together. The role of soluble MHC antigens in JEV infection is hitherto unknown and therefore needs further investigation.

Antiviral Activity of Baicalein and Quercetin against the Japanese Encephalitis Virus

Japanese encephalitis (JE), a mosquito-borne viral disease, is endemic to the entire east and southeast Asia, and some other parts of the world. Currently, there is no effective therapeutic available for JE; therefore, finding the effective antiviral agent against JEV replication is crucial. In the present study, the in vitro antiviral activity of baicalein and quercetin, two purportedly antiviral bioflavonoids, was evaluated against Japanese encephalitis virus (JEV) replication in Vero cells. Anti-JEV activities of these compounds were examined on different stages of JEV replication cycle. The effects of the compounds on virus replication were determined by foci forming unit reduction assay (FFURA) and quantitative RT-PCR. Baicalein showed potent antiviral activity with IC50 = 14.28 μg/mL when it was introduced to the Vero cells after adsorption of JEV. Quercetin exhibited weak anti-JEV effects with IC50 = 212.1 μg/mL when the JEV infected cells were treated with the compound after virus adsorption. However, baicalein exhibited significant effect against JEV adsorption with IC50 = 7.27 μg/mL while quercetin did not show any anti-adsorption activity. Baicalein also exhibited direct extracellular virucidal activity on JEV with IC50 = 3.44 μg/mL. However, results of quantitative RT-PCR experiments confirmed the findings from FFURA. This study demonstrated that baicalein should be considered as an appropriate candidate for further investigations, such as the study of molecular and cellular mechanism(s) of action and in vivo evaluation for the development of an effective antiviral compound against Japanese encephalitis virus.

The Cellular Antiviral Protein Viperin Is Attenuated by Proteasome-Mediated Protein Degradation in Japanese Encephalitis Virus-Infected Cells

Viperin is identified as an antiviral protein induced by interferon (IFN), viral infections, and pathogen-associated molecules. In this study, we found that viperin is highly induced at the RNA level by Japanese encephalitis virus (JEV) and Sindbis virus (SIN) and that viperin protein is degraded in JEV-infected cells through a proteasome-dependent mechanism. Promoter analysis revealed that SIN induces viperin expression in an IFN-dependent manner but that JEV by itself activates the viperin promoter through IFN regulatory factor-3 and AP-1. The overexpression of viperin significantly decreased the production of SIN, but not of JEV, whereas the proteasome inhibitor MG132 sustained the protein level and antiviral effect of viperin in JEV-infected cells. Knockdown of viperin expression by RNA interference also enhanced the replication of SIN, but not that of JEV. Our results suggest that even though viperin gene expression is highly induced by JEV, it is negatively regulated at the protein level to counteract its antiviral effect. In contrast, SIN induces viperin through the action of IFN, and viperin exhibits potent antiviral activity against SIN.

Japanese encephalitis virus infects neural progenitor cells and decreases their proliferation

Japanese encephalitis virus (JEV), a common cause of encephalitis in humans, especially in children, leads to substantial neuronal injury. The survivors of JEV infection have severe cognitive impairment, motor and behavioral disorders. We hypothesize that depletion of neural progenitor cells (NPCs) by the virus culminates in neurological sequelae in survivors of Japanese encephalitis (JE). We utilized both in vivo model of JEV infection and in vitro neurosphere cultures to study progressive JEV infection. Cellular infection and cell death was determined by flow cytometry. BrdU administration in animals and in neurospheres was used to determine the proliferative ability of NPCs. JEV leads to massive loss of actively proliferating NPC population from the subventricular zone (SVZ). The ability of JEV infected subventricular zone cells to form neurospheres is severely compromised. This can be attributed to JEV infection in NPCs, which however do not result in robust death of the resilient NPC cells. Instead, JEV suppresses the cycling ability of these cells, preventing their proliferation. JEV primarily targets at a critical postnatal age and severely diminishes the NPC pool in SVZ, thus impairing the process of recovery after the insult. This arrested growth and proliferation of NPCs might have an effect on the neurological consequences in JE survivors.

Processing of Japanese encephalitis virus non-structural proteins: NS2B-NS3 complex and heterologous proteases.

Processing of Japanese encephalitis (JE) virus non-structural (NS) proteins expressed by recombinant vaccinia viruses was analysed to characterize the responsible viral protease. Analysis of the processing of polyprotein NS2A-2B-3' containing the N-terminal 322 amino acids of NS3 revealed products consistent with cleavages at the predicted intergenic junctions as well as at one or possibly two sites within NS2A. Cleavage at the alternate site(s) containing the cleavage sequence motif within NS2A could possibly explain the production of the NS1' protein in JE virus-infected cells. Polyprotein NS2A-d2B-3' containing a large deletion within NS2B was cleavage-defective, despite the presence of the proposed NS3 protease domain. Cleavage of NS2A-d2B-3' was restored if NS2B or NS2A-2B was supplied in trans, providing evidence that NS2B is strictly required for NS3 proteolytic activity. NS2B- or NS3-specific sera raised against the bacterial TrpE fusion protein co-precipitated NS2B and NS3 or NS3' from the lysate of JE virus or recombinant virus-infected cells. Thus both protease components are associated as a complex, presumably representing the active JE virus protease. JE virus and the analogous dengue 4 (DEN-4) protease components were employed to examine the activity of heterologous proteases. The defective cleavage of JE virus NS2A-d2B-3' was complemented by heterologous DEN-4 NS2B, whereas the defective cleavage of DEN-4 NS2A-d2B-3' was not corrected by heterologous JE virus NS2B. This suggests that the heterologous JE virus NS2B-DEN-4 NS3 protease is not active, despite the considerable sequence conservation of NS2B and NS3 between the two viruses. The cleavage activity was restored by replacement of the C-terminal 80 amino acids of JE virus NS2B with the corresponding DEN-4 sequence, consistent with the notion that the C-terminal region contains amino acid residues for interaction with DEN-4 NS3.

Glycoprotein detection in nitrocellulose transfers of electrophoretically separated protein mixtures using concanavalin A and peroxidase: Application to arenavirus and flavivirus proteins

Glycoproteins in nitrocellulose transfers of electrophoretically separated mixtures of cellular and viral proteins are rapidly and sensitively located by sequential incubation with the lectin concanavalin A and the enzymatically active glycoprotein horseradish peroxidase. The bound enzyme is located by incubation with a substrate which is converted to a highly insoluble colored product. The specificity of the method is demonstrated by the abolition of concanavalin A binding in the presence of α-methyl mannoside. The method is capable of detecting as little as 60 ng of a purified model glycoprotein after electrophoresis. It has been applied to the analysis of the glycoproteins of purified Lassa virus and of the virus-specific glycoproteins in Japanese encephalitis virus-infected cells.

Protein synthesis in japanese encephalitis virus-infected cells

We studied the effects of actinomycin D, cycloheximide, and puromycin on virus-specified protein synthesis in Japanese encephalitis (JE) virus-infected chick embryo and LLC-MK 2 (rhesus monkey kidney) cells. To prove that the radioactively labeled proteins we had previously identified in infected chick cells were virus-specified, we showed that they electrophoretically comigrated with radioactively labeled proteins from infected, but not from uninfected, LLC-MK 2 cells. We found that the maximum value for the ratio of protein synthesis in infected chick cells to uninfected cells occurred when the cells were treated with actinomycin D and were pulse-inhibited with cycloheximide. Alone, actinomycin D treatment decreased the background radioactivity of high molecular weight in electropherograms of infected chick cells and allowed virus-specified proteins to be prominent. Cycloheximide pulse-inhibition of infected, actinomycin D-treated cells decreased total cellular protein synthesis and slightly decreased the background radioactivity in electropherograms without changing the distribution of radioactivity among virus-specified proteins. Neither drug treatment decreased the yield of infectious virus. These results differ in some respects from the related results of Trent and Qureshi (1971). In contrast to our results with cycloheximide, pulse-inhibition of infected chick embryo cells with puromycin inhibited the synthesis of polypeptides NV-5, NV-4, and NV-1 (virus-specified nonstructural, nonglycosylated proteins) to a greater extent than that of V-3, NV-3, NV-2, and V-2 (virus-specified glycosylated and/or structural proteins). It also generally inhibited the synthesis of large proteins relative to small ones. We then studied the effects of puromycin and cycloheximide in LLC-MK 2 cells. In contrast to our results in chick embryo cells, pulse-inhibition of infected LLC-MK 2 cells with either drug (in the continuous presence of actinomycin D) did not alter the pattern of virus-specified proteins in electropherograms from that obtained without pulse-inhibition. Treatment with continuous levels of either drug (in the presence of actinomycin D) did, however, alter the protein pattern by differentially inhibiting the synthesis of nonstructural, nonglycosylated proteins. By labeling infected cells with one of eight different amino acids, we were unable to find an unusually enriched (or lowered) amino acid content that was common to all nonstructural, nonglycosylated proteins. A possible explanation for the differential inhibition is that the virus directs the formation of functionally different polyribosomes, messengers, or initiation factors which vary in their susceptibility to low levels of inhibitors of protein synthesis.

Japanese encephalitis virus replication: a procedure for the selective isolation and characterization of viral RNA species.

The viral RNA species synthesized in a porcine kidney cell line, PS(Y-15), by Japanese encephalitis virus (JEV) are described. Virus titers on these cells ranged between 106 to 107 p.f.u./ml at the end of 2 to 3 days incubation at 35° C. Actinomycin D (AD) could not be used to unmask JEV RNA synthesis since it inhibited virus replication at concentrations necessary to substantially reduce host cell RNA synthesis. Treatment of cells with 1 μg AD/ml and removal prior to infection permitted good JEV replication, and at the same time strongly suppressed synthesis of 28 S and 18 S cellular ribosomal RNA. The problem of separating viral RNA from non-ribosomal RNA that was still being synthesized by AD pretreated cells was resolved by the isolation of the cytoplasmic membrane fraction of infected cells. RNA extracted from the membranes of infected AD pretreated cells and analyzed for sedimentation characteristics on sucrose gradients has four RNA species not found in uninfected cells.They are: (1) 45 S single stranded RNA believed to be the infectious RNA found in the virion; (2) a 27 S RNA single stranded RNA; (3) a 20 S ribonuclease resistant RNA believed to be double stranded and (4) an 8 S RNA species. The RNA species found in JEV infected cells, except for the 8 S form, have been found in group A arboviruses. The procedure described utilizing AD pretreatment of host cells and the separation of the cellular cytoplasmic fraction may well have value for the study of the biosynthetic events involved in the replication of other animal viruses that are inhibited by AD.