Susceptibility of naïve and differentiated PC12 cells to Japanese encephalitis virus infection (original) (raw)
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Immunomodulatory Cytokines Determine the Outcome of Japanese Encephalitis Virus Infection in Mice
Japanese encephalitis virus (JEV) induces an acute infection of the central nervous system, the pathogenic mechanism of which is not fully understood. To investigate host response to JEV infection, 14-day-old mice were infected via the extraneural route, which resulted in encephalitis and death. Mice that received JEV immune splenocyte transfer were protected from extraneural JEV infection. Pathology and gene expression profiles were then compared in brains of mice that either succumbed to JEV infection or were protected from infection by JEV immune cell transfer. Mice undergoing progressive JEV infection had increased expression of proinflammatory cytokines, chemokines, and signal transducers associated with the interferon (IFN) pathway. In contrast, mice receiving immune cell transfer had increased production of the Th2 cytokine IL-4, and of IL-10, with subdued expression of IFN-g. We observed IL-10 to be an important factor in determining clinical outcome in JEV infection. Data obtained by microarray analysis were further confirmed by quantitative RT-PCR. Together, these data suggest that JEV infection causes an unregulated inflammatory response that can be countered by the expression of immunomodulatory cytokines in mice that survive lethal infection. J. Med. Virol. 82:304– 310, 2010. 2009 Wiley-Liss, Inc. KEY WORDS: Japanese encephalitis; microarray; adoptive transfer; splenocytes INTRODUCTION Japanese encephalitis (JE) is a flaviviral disease of great medical importance. Japanese encephalitis virus (JEV) infection occurs throughout most of Asia and is also spreading to new geographical areas like Australia. Children are at greatest risk of infection in endemic areas [Ruben and Gajanana, 1997]. Factors governing the establishment of JEV infection in the central nervous system (CNS) are poorly understood. The course of disease in humans can effectively be replicated in laboratory rodents. Young mice are susceptible to extraneural inoculation with JEV and succumb to infection soon after the establishment of infection in the CNS [Johnson et al., 1985; Hase et al., 1990]. The adoptive transfer ofJEVimmunesplenocytes into these young mice has been shown to confer protection against subsequent lethal challenge with JEV, thus demonstrating a role for cellular immunity in JEV infection [Biswas et al., 2009]. Differential expression profiling of genes using high throughput system enables the identification of critical genes responsible for modulation of infection and a subsequent understanding of the cellular and molecular pathways associated with disease progression. Most gene profiling studies in flavivirus infection have been carried out using permissive cell lines. Microarray analysis of human glioblastoma cells infected with West Nile Virus revealed the differential expression of 173 cellular genes, of which 23 were identified that might play a role in cellular neuro-degeneration [Koh and Ng, 2005]. Apoptosis was identified as the mechanism of cell death induced by JEV replication in several neuronal and non-neuronal cell lines [Liao et al., 1997]. In addition, activation of tumor necrosis factor receptor 1 (TNFR1) and signaling through the TNFR-associated death domain (TRADD) following JEV infection was assumed to trigger downstream apoptotic cascades in mouse and human neuroblastoma cell lines [Swarup et al., 2007a
Pathogenesis and Host Immune Response during Japanese Encephalitis Virus Infection
2021
Japanese Encephalitis Virus (JEV) is a mosquito borne flavivirus infection. Transmission of JEV starts with the infected mosquito bite where human dermis layer act as the primary site of infection. Once JEV makes its entry into blood, it infects monocytes wherein the viral replication peaks up without any cell death and results in production of TNF-α. One of the most characteristics pathogenesis of JEV is the breaching of blood brain barrier (BBB). JEV propagation occurs in neurons that results in neuronal cell death as well as dissemination of virus into astrocytes and microglia leading to overexpression of proinflammatory cytokines. JEV infection results in host cells mediated secretion of various types of cytokines including type-1 IFN along with TNF-α and IFN-γ. Molecule like nitrous oxide (NO) exhibits antiviral activities against JEV infection and helps in inhibiting the viral replication by blocking protein synthesis and viral RNA and also in virus infected cells clearance. I...
Journal of Virology, 2011
Interferon is a principal component of the host antiviral defense system. In this study, abortive focus formation by Japanese encephalitis virus (JEV) in primate cells was accompanied by early interferon induction, while productive focus formation in porcine cells was associated with a late interferon response. Neutralization antibodies against interferon relieved the restricted infection in primate cells, and increasingly larger foci were generated as treatment with exogenous interferon was delayed, thereby establishing a solid correlation between interferon response and viral dissemination. However, delayed interferon induction in JEV-infected porcine cells occurred in the absence of active inhibition by the virus. We further demonstrated that JEV mediates interferon activation through double-stranded RNA and cytosolic pattern recognition receptors. Immunofluorescence and subcellular fractionation studies revealed that double-stranded RNA is concealed in intracellular membranes at...
Japanese Encephalitis Virus Generated Neurovirulence, Antigenicity, and Host Immune Responses
ISRN Virology, 2013
In response to a JE virus attack, infected body cells start secretion of different cytokines and activate innate immune response. Virus starts neuronal invasion by entering into nerve cells and inflecting the central nervous system. It avoids exposure of body's natural immunity and generates neurotrophic effects. Virus causes acute susceptibility to CNS and establishes encephalitis syndrome that results in very high fatality in children. In survivors, JEV inhibits the growth and proliferation of NCPs and imposes permanent neuronal disorders like cognitive, motor, and behavioral impairments. However, body cells start TCR mediated interactions, to recognize viral antigens with class I MHC complex on specific target cells, and operate mass killing of virus infected cells by increased CTL activity. Thus, both cell mediated and antibody interactions plays a central role in protection against JEV. In the present review article virus generated neurovirulence, antigenicity, and host immune responses are described in detail. More emphasis is given on diagnosis, clinical care, and active immunization with well-designed potential antiflavivirus vaccines. Further, for achieving an elite success against JEV, global eradication strategies are to be needed for making vaccination program more responsible and effective in endemic areas.
Journal of Virology, 2013
The cellular entry of JEV is poorly characterized in terms of molecular requirements and pathways. Here we present a systematic study of the internalization mechanism of JEV in fibroblasts and neuroblastoma cells. To verify the roles of distinct pathways of cell entry, we used fluorescently labeled virus particles, a combination of pharmacological inhibitors, RNA interference (RNAi), and dominant-negative (DN) mutants of regulatory proteins involved in endocytosis. Our study demonstrates that JEV infects fibroblasts in a clathrin-dependent manner, but it deploys a clathrin-independent mechanism to infect neuronal cells. The clathrin-independent pathway requires dynamin and plasma membrane cholesterol. Virus binding to neuronal cells leads to rapid actin rearrangements and an intact and dynamic actin cytoskeleton, and the small GTPase RhoA plays an important role in viral entry. Immunofluorescence analysis of viral colocalization with endocytic markers showed that JEV traffics through Rab5-positive early endosomes and that release of the viral nucleocapsid occurs at the level of the early and not the late endosomes. FIG 3 JEV entry and infection in neuronal cells are not inhibited by chlorpromazine treatment. (A) Neuro2a, SH-SY5Y, and Vero cells were pretreated for 30 min with chlorpromazine at the indicated concentrations and given a pulse of Alexa 488-Tf for 10 min. Cells were fixed, and transferrin uptake was quantified by flow cytometry. The averages Ϯ SDs of measured geometric means of internalized Tf in control and chlorpromazine-treated cells are shown. (B) Neuro2a, SH-SY5Y, and Vero cells were pretreated for 30 min with 50 M chlorpromazine and infected with JEV (MOI, 0.4) in the presence of inhibitor. Infection was scored as described in Materials and Methods. Student's t test was used to generate P values. *, P Ͻ 0.05. (C) Neuro2a cells were infected with JEV (MOI, 10) in the presence of 50 M chlorpromazine for 1 h, and the amount of virus endocytosed was estimated by qRT-PCR of JEV positive-strand RNA. (D) Virus titers (mean Ϯ SD) in culture supernatants at 24 hpi (MOI, 0.4) were calculated by plaque assay.
Archives of Virology, 2003
The analysis of cell-mediated immune responses in virus-exposed but healthy individuals may contribute to define the features of the T cell response associated with resistance. We report, for the first time, on adaptive T cell responses to 5 largest of the 10 proteins that together constitute 76% of the coding potential of the Japanese encephalitis virus (JEV) genome in a naturally exposed healthy JEimmune human cohort. Fixed and sonified whole cell preparations of insect cells individually expressing recombinant prM, E, NS1, NS3 and NS5 proteins of JEV were used in vitro to stimulate lymphocytes from individuals who had experienced subclinical JEV infections. NS3-specific memory T cells were detected in up to 86% of the JEV-infected cohort whereas prM, E and NS1 each elicited reactions in approximately 45% among individuals tested, suggesting that NS3 is an important target for JEV-specific cell-mediated immune responses. Responses to NS5, the largest viral protein were in contrast the poorest, seen in only 13% of the cohort. Moreover, NS3 stimulated interferon-γ production in both CD4 + and CD8 + T cells indicating that a Th1 immune response to the NS3 protein may be a critical determinant of immune control of JEV infection.
2018
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. Japanese Encephalitis Virus belonging to the family Flaviviridae, is a major cause of epidemic encephalitis worldwide especially in eastern and southeastern Asia covering a population of approximately 3 billion 1. Nearly 1% of the virus infected human population develops encephalitis symptoms; out of which 20–30% cases are fatal while 30–50% survivors develop consequential neurological damage 2. Like all other viruses, JEV infection in a host requires interaction of viral attachment proteins and cellular membrane proteins. The 52 kDa JEV–E glycoprotein mediates viral attachment to the host cell membrane proteins followed by membrane fusion. E glycoprotein has three antigenic domains 3. Domain I (DI) contains 9 beta-barrels present in between domain II (DII) and globular domain III (DIII). Domain III is found to be present at the C-terminus of the protein and is connected to DI by a short peptide linker. DIII mediates the main interaction of the E-glycoprotein with the host cell membrane. Immunoglobulin like domain DIII is reported to play a major role in membrane protein interaction in cases of Dengue and Tick borne encephalitis viruses 4,5. The other flaviviruses like West Nile Virus and Dengue have been shown to enter host cells via clathrin mediated endocytosis 6,7. Several studies show clathrin mediated endocytic internalization in case of JE virus too 8. Lipid rafts are reported to be important in JEV entry into neural stem/progenitor cells through activation of Phosphoinositide 3′ Kinase/Akt signaling 9. However, clathrin independent mechanisms are also responsible for JEV entry into fibroblasts and neuroblastoma cells 10. Heat shock protein 70 (HSP70) has been shown to be Published: xx xx xxxx OPEN
Specific tropism of Japanese encephalitis virus for developing neurons in primary rat brain culture
Archives of Virology, 1993
Among all the neural cells in fetal rat brain culture developing neurons showed the highest rate of infection by Japanese encephalitis virus (JEV). JEV specifically bound to these cells as measured by immuno-staining. These results indicate that developing neurons are the major target of JEV, and that the initial specific binding of virus to these cells may be one of the reasons for the neurotropism of JEV. Japanese encephalitis virus (JEV) is a prototypic member of the family Flaviviridae; the virus causes severe, even lethal encephalitis in humans. Pathological
Virology, 1989
Neurons are the major target cell of Japanese encephalitis virus (JEV). Rats intracerebrally inoculated with JEV show an age-dependent pattern of resistance to infection in which resistance is closely associated with neuronal maturation. However, the mechanisms underlying this association remain poorly understood, because there is no reliable and convenient cell culture system that mimics the in vivo properties of JEV infection of immature and mature neurons. The aim of the present study was to examine JEV infection in immortalized CSM14.1 rat neuronal cells, which can be induced to differentiate into neurons by culture under non-permissive conditions. JEV infection of undifferentiated CSM14.1 cells was more efficient than that of differentiated cells, resulting in the production of more progeny virus. An infectious virus recovery assay detected more internalized virions in undifferentiated cells. On the other hand, JEV infection of differentiated cells induced more rapid and stronger expression of interferon (IFN)-β gene, along with lower levels of JEV RNA. Taken together, these results show that the initial phase of viral infection and the later IFN response play a role in the viral susceptibility of undifferentiated and differentiated CSM14.1 cells. Since CSM14.1 cells became more resistant to JEV infection as they matured, this culture system can be used as an in vitro model for studying the age-dependent resistance of neurons to JEV infection.