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 3-4 weeks old C57BL/6 mice. The blood-brain barrier leakage was evident in JEV-S3 infected mice, where viral antigens and RNA were consistently demonstrated in the brain and infiltration of activated immune cells as evidenced by an increased level of 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 IFN-γ, Il-6, TNF-α, and TGF-β 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 the virus pathogenesis in the adolescent mouse model.

Japanese encephalitis (JE) is a clinical manifestation of the brain inflammation caused by JE virus (JEV). This virus imparts permanent neurological damage, thus imposing a heavy burden on public health and society. Neuro-inflammation is the hallmark of JEV infection. The prolonged pro-inflammatory response is due primarily to microglial activation, which eventually leads to severe encephalitis. A continual effort is going on in the scientific community toward an understanding of cellular and molecular factors that are involved in JEV neuro-invasion and inflammatory processes. This review not only gives a comprehensive update on the recent advances on understanding virus structure and mechanisms of pathogenesis but also briefly discusses crucial unresolved issues. We also highlight challenging areas of research that might open new avenues for controlling virus-induced neuro-inflammation.

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.

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.

Dengue virus (DENV) infection causes dengue fever in humans, which can lead to thrombocytopenia showing a marked reduction in platelet counts, and dengue hemorrhagic fever. The virus may cause thrombocytopenia either by destroying the platelets or by interfering with their generation via the process of megakaryopoiesis. MEG-01 is the human megakaryoblastic leukemia cell line that can be differentiated in vitro by phorbol-12-myristate-13-acetate (PMA) treatment to produce platelet-like-particles (PLPs). We have studied DENV infection of MEG-01 cells to understand its effect on megakaryopoiesis and the generation of PLPs. We observed that DENV could infect only naive MEG-01 cells, and differentiated cells were refractory to virus infection/replication. However, DENV-infected MEG-01 cells, when induced for differentiation with PMA, supported an enhanced viral replication. Following the virus infection, the MEG-01 cells showed a marked reduction in the surface expression of platelet markers (CD41, CD42a, and CD61), a decreased polyploidy, and significantly reduced PLP counts. DENV infection caused an enhanced Notch signaling in MEG-01 cells where the virus envelope protein was shown to interact with TAL-1, a host protein important for megakaryopoiesis. These observations provide new insight into the role of DENV in modulating the megakaryopoiesis and platelet production process.