BackgroundMicroglial cells, which are resident macrophages of the central nervous system, play important roles in immune responses and pathogenesis. Japanese encephalitis virus (JEV) is a neurotropic virus that infects microglial cells in brain. Several microRNAs including miR-155 and miR-146a play an important role in defining the microglia inflammatory profile. In this study, we have investigated the effect of miR-155 and miR-146a modulation on JEV infection as well as innate immune responses in human microglial cells.MethodsIn vitro studies were performed in JEV-infected human microglial CHME3 cells. miR-155 or miR-146a were overexpressed and total RNA and protein were extracted following JEV-infection. Expression of genes involved in innate immune responses was studied by PCR array, quantitative real-time PCR (qPCR), western blot and Fluorescence activated cell sorter (FACS). JEV replication was monitored by studying the viral RNA by qPCR, protein by western blot, and titres by plaque assay.ResultsOverexpression of miR-155 in CHME3 cells resulted in significantly reduced JEV replication whereas miR-146a overexpression had an insignificant effect. Additionally, interferon regulatory factor 8 (IRF8) and complement factor H (CFH) were induced during JEV infection; however, this induction was attenuated in miR-155 overexpressing cells following JEV infection. Further, JEV-induced NF-κB regulated downstream gene expression was attenuated. Interestingly, an increased level of CD45, a negative regulator of microglia activation and a reduced phosphorylated-Signal Transducers and Activators of Transcription (p-STAT1) expression was observed in miR-155 overexpressing cells upon JEV infection.ConclusionInduction of miR-155 in human microglial cells may negatively modulate JEV-induced innate immune gene expression and may have a beneficial role in limiting JEV replication in human microglial cells.

Microglia cells in the brain play essential role during Japanese Encephalitis Virus (JEV) infection and may lead to change in microRNA (miRNA) and mRNA profile. These changes may together control disease outcome. Using Affymetrix microarray platform, we profiled cellular miRNA and mRNA expression at multiple time points during viral infection in human microglial (CHME3) cells. In silico analysis of microarray data revealed a phased pattern of miRNAs expression, associated with JEV replication and provided unique signatures of infection. Target prediction and pathway enrichment analysis identified anti correlation between differentially expressed miRNA and the gene expression at multiple time point which ultimately affected diverse signaling pathways including Notch signaling pathways in microglia. Activation of Notch pathway during JEV infection was demonstrated in vitro and in vivo. The expression of a subset of miRNAs that target multiple genes in Notch signaling pathways were suppressed and their overexpression could affect JEV induced immune response. Further analysis provided evidence for the possible presence of cellular competing endogenous RNA (ceRNA) associated with innate immune response. Collectively, our data provide a uniquely comprehensive view of the changes in the host miRNAs induced by JEV during cellular infection and identify Notch pathway in modulating microglia mediated inflammation.

MicroRNAs (miRNAs) released from the activated microglia upon neurotropic virus infection may exacerbate the neuronal damage. Here, we identified let‐7a and let‐7b (let‐7a/b) as one of the essential miRNAs over‐expressed upon Japanese Encephalitis virus (JEV) infection and released in the culture supernatant of the JEV‐infected microglial cells through extracellular vesicles. The let‐7a/b was previously reported to modulate inflammation in microglial cells through Toll‐like receptor 7 (TLR7) pathways; although their role in accelerating JEV pathogenesis remain unexplored. Therefore, we studied the role of let‐7a/b in modulating microglia‐mediated inflammation during JEV infection and investigated the effect of let‐7a/b‐containing exosomes on primary neurons. To this end, we examined let‐7a/b and NOTCH signaling pathway in TLR7 knockdown (KD) mice. We observed that TLR7 KD or inhibition of let‐7a/b suppressed the JEV‐induced NOTCH activation possibly via NF‐κB dependent manner and subsequently, attenuated JEV‐induced TNFα production in microglial cells. Furthermore, exosomes secreted from let‐7a/b over‐expressed microglia when transferred to uninfected mice brain induced caspase activation. Exosomes secreted from virus‐infected or let‐7a/b over‐expressed microglia when co‐incubated with mouse neuronal (Neuro2a) cells or primary cortical neurons also facilitated caspase activation leading to neuronal death. Thus, our results provide evidence for the multifaceted role of let‐7a/b miRNAs in JEV pathogenesis. Let‐7a/b can interact with TLR7 and NOTCH signaling pathway and enhance TNFα release from microglia. On the other hand, the exosomes secreted by JEV‐infected microglia can activate caspases in uninfected neuronal cells which possibly contribute to bystander neuronal death. Cover Image for this issue: doi: .

Changes in circulating microRNAs (miRNAs) in the cerebrospinal fluid (CSF) have been associated with different neurological diseases. Here, we presented results of a pilot study aimed at determining the feasibility of detecting miRNAs in the CSF of Japanese Encephalitis virus (JEV) infected individuals with acute encephalitis syndrome (AES). We demonstrated the circulating miRNA profile in CSF of acute encephalitis patients infected with JEV. Using a quantitative real-time PCR-based miRNA array, we examined the level of 87 miRNAs expressed in human exosomes isolated from CSF. Subsequently, correlation between cytokine level and miRNAs expression in CSF samples was examined. In this study, we identified and validated the upregulated expression of three miRNAs, miR-21-5p, miR-150-5p, and miR-342-3p that were specifically circulated in CSF of acute encephalitis patients infected with JEV. CSF miR-21-5p, miR-150-5p, and miR-342-3p expressions were also elevated in infected mice brain. However, the expression pattern of these miRNAs differed in neuronal cells, microglial cells, and the exosome derived from JEV-infected cell culture supernatant. Interestingly, neuronal cells infected with vaccine strain (SA-14-14) did not lead to any upregulation of these three miRNAs. Further, miR-150-5p expression was found to be negatively correlated(r = -0.5279, p = 0.016) with TNFα level. Pathway analysis of putative target genes of these miRNAs indicated involvement of TGF-β, NGF, axon guidance, and MAPK signaling pathways in JEV/AES patients. This study for the first time represents the circulating miRNA in CSF of AES patients and identified the upregulated miRNAs in JEV-infected patients and offers the basis for future investigation.

Artificial microRNA (amiRNA)-mediated inhibition of viral replication has recently gained importance as a strategy for antiviral therapy. In this study, we evaluated the benefit of using the amiRNA vector against Japanese encephalitis virus (JEV). We designed three single amiRNA sequences against the consensus sequence of 3′ untranslated region (3′UTR) of JEV and tested their efficacy against cell culture-grown JEV Vellore strain (P20778) in neuronal cells. The binding ability of three amiRNAs on 3′UTR region was tested in vitro in HEK293T cells using a JEV 3′UTR tagged with luciferase reporter vector. Transient transfection of amiRNAs was nontoxic to cells as evident from the MTT assay and caused minimal induction in interferon-stimulated gene expression. Furthermore, our result suggested that transient expression of two amiRNAs (amiRNA #1 and amiRNA #2) significantly reduced intracellular viral RNA and nonstructural 1 (NS1) protein, as well as diminished infectious viral particle release up to 95% in the culture supernatant as evident from viral plaque reduction assay. Overall, our results indicated that RNA interference based on amiRNAs targeting viral conserved regions at 3′UTR was a useful approach for improvements of nucleic acid inhibitors against JEV.

21Extracellular microRNAs (miRNAs) are essential for the cell to cell communication in the 22 healthy and diseased brain. MicroRNAs released from the activated microglia upon 23 neurotropic virus infection may exacerbate CNS damage. Here, we identified let-7a and let-7b 24 (let-7a/b) as the overexpressed miRNAs in Japanese Encephalitis virus (JEV) infected 25 microglia and assessed their role in JEV pathogenesis. We measured the let-7a/b expressions 26 in JEV infected post-mortem human brains, mice brains and in mouse microglial N9 cells by the 27 qRT-PCR and in situ hybridization assay. The interaction between let-7a/b and NOTCH 28 signaling pathway further examined in Toll-like receptor 7 knockdown (TLR7 KD) mice to 29 assess the functions. Exosomes released from JEV infected or let-7a/b mimic transfected N9, and 30 HEK-293 cells were isolated and evaluated their function. We observed an upregulation of let-31 7a/b in the infected brains as well as in microglia. Knockdown of TLR7 or Inhibition of let-32 7a/b suppressed the JEV induced NOTCH activation possibly via NF-κB dependent manner 33 and subsequently, attenuated JEV induced TNFα production in microglial cells. Further, 34 exosomes secreted from JEV-infected microglial cells specifically contained let-7a/b. 35Exosomes overexpressed with let-7a/b were injected into BALB/c mice as well as co-incubated 36 with mouse neuronal (Neuro2a) cells, or primary cortical neuron resulted in caspase activation 37 leading to neuronal damage in the brain. Thus, our results provide evidence for the 38 multifaceted role of let-7a/b miRNAs and unravel the exosomes mediated mechanism for JEV 39 induced pathogenesis. 40 41 42 43MicroRNAs (miRNAs) are a class of regulatory non-coding RNAs that regulate many biological 44 processes within a cell by direct binding to mRNA, thus influence protein abundance (1). Recent 45 evidence demonstrates that miRNAs can also affect RNA virus replication and pathogenesis 46 through virus-mediated changes in the host transcriptome (2-9). Furthermore, microRNAs have 47 been detected extracellularly and circulating in the blood, CSF and other body fluids (10). These 48 exosomal microRNAs are thought to be released from cells as encapsulated in the vesicles, 49 called as exosomes. These miRNAs containing exosomes can be taken up by specific target cells 50 within the same or in remote tissues where they can exert their repressive function (11, 12). 51These characteristics make extracellular microRNAs ideal candidates for intercellular 52 communication over short and long distances (13). 53In the brain, the exosomes mediated neuron microglia communication recently discussed in few 54 reports (14-16). Most of the cells in the CNS, including neurons, astrocytes, oligodendrocytes, 55 and microglia shed exosomes. These extracellular vesicles are secreted by brain cells under both 56 normal and pathological conditions and transfer specific signal to the neighboring cells. Transfer 57 of miRNAs via exosome may act as a principal mediator for neuroinflamma...

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