Japanese encephalitis virus (JEV), a mosquito-borne flavivirus is the causative agent of Japanese encephalitis (JE). The disease affects mostly children and around 30000– 50000 cases of JE and up to 15000 deaths are reported annually. No anti-viral drugs have been discovered against JE so far, but advances in our knowledge of the molecular biology of flaviviruses is propelling flaviviral drug research at an expeditious pace. Since JEV has a small genome which encodes for only ten proteins, there is dearth of potential drug targets. Researchers are now focusing on cellular interactomes, a complex and dynamic molecular biosystem which identifies host proteins which interact with either viral proteins or viral genomes, leading to the generation of an astronomical number of potential drug targets involving common cellular pathways that are required for the life cycle of different viruses. Such studies can pave way for the development of ‘broad-spectrum’, ‘silver-bullet’ anti-viral drugs for the treatment of multiple viral diseases. The cellular interactomes can be studied by Genomics tools such as microarray. Systematic profiling of genes involved in virus infection by RNAi, transcriptome sequencing, microRNA profiling and yeast two-hybrid system has allowed us to assess global gene expression changes providing an unprecedented view on the host-side of the virus–host interactions. Advent of these tools has led to identification of plethora anti-viral genes. For example, over expression of IFN-stimulated gene15 (ISG15) results in inhibition of JEV leading to significant reduction of viral titers. Chemokine profiling of JEV-infected cells by microarray can provide possible therapeutic modalities that can mitigate the morbidity associated with JEV infection. Functional classification of interferon-stimulated genes (ISG) identified using innovative methods have been the stepping stone for identification of many anti-viral genes, among them are few Broadly acting effectors like IRF1, C6orf150, HPSE, RIG-I, MDA5 and IFITM3 and some more targeted antiviral specific like DDX60, IFI44L, IFI6, IFITM2, MAP3K14, MOV10, NAMPT, OASL, RTP4, TREX1 and UNC84B. In this study, we have identified a B16F10 murine melanoma cell line that is resistant to JEV infection. DNA microarray analysis of JEV-susceptible and resistant B16F10 cell lines gave us interesting insights into JEV-induced host gene expression changes. Real time PCR validation of microarray data indicates that a number of virus and interferon inducible genes are expressed constitutively at high levels in this JEV-resistant cell line. Further, several of the mouse genes induced by JEV in B16F10 cell line were also upregulated in JEV-infected mouse brain. To understand the significance of these host gene expression changes, we attempted to generate stable murine cell lines constitutively expressing select JEV-inducible genes and study the JEV infection pattern in these cell lines. One of the JEV-inducible genes encoding thymidylate kinase (Tyki), a mitochondrial protein involved in the sysnthesis of nucleoside diphosphates, when overexpressed in NIH3T3 cells confers resistance to JEV infection as evident from reduced JEV-induced cytopathic effects and significant reduction in viral titer. Since TYKI has two distinct domains: the N-terminal domain with unknown function and the C-terminal domain with the nucleoside monophosphate kinase function, suggest that TYKI may be a bifunctional protein with other biological functions in addition to its UMP-CMP kinase activity. In order to examine whether N-terminal domain is responsible for antiviral activity of the protein, a stable cell line constitutively expressing N-terminal domain of gene was made, but the overexpression of N-terminal domain didn't confer any antiviral immunity. Thus signifying importance of kinase activity in confering antiviral immunity. Our studies indicate for the first time that Tyki may have a role in host resistance to JEV and understanding the mechanism of action Tyki may pave way for novel anti-JEV therapy. Stable cell lines constitutively expressing other JEV-inducible genes (Atf3, Gimap3, Rtp4, Glipr2, Tmem140 and Garg49) couldn't be generated. Therefore, to study the effect of overexpression of these genes on JEV infection, expression vectors encoding these genes were transfected individually to human 293T cells by nucleofection, then infected with JEV and viral titres were examined by plaque assay. Nucleofection was opted as a method of choice since it is the only non-viral method, which transfects DNA directly enter the nucleus. In contrast, other commonly used non-viral transfection methods rely on cell division for the transfer of DNA into the nucleus. Nucleofection of vectors encoding different JEV-inducible genes followed by JEV infection and assay of viral titer led to identification of one more anti-viral gene and three pro-viral genes. Garg49, an interferon and JEV inducible mitochondrial gene was identified as antiviral gene. Further studies led to the identification of GARG49 as a mitochondrial protein. Three genes, Atf3, encoding a cAMP responsive element binding protein family transcription factor, Glipr2, encoding a Glioma related pathogenesis protein and Gimap3, encoding an outer mitochondrial membrane GTPase were identified as pro viral genes. Overexpression of Tmem140, encoding a transmembrane protein and Rtp4, encoding a golgi chaperone did not significantly affect JEV titer. Conclusions: . A JEV-resistant B16F10 murine melanoma cell line was identified and several JEV-inducible genes were found to be expressed constitutively at high levels in this cell line. .We demonstrate for the first time that Tyki/Ump-Cmpk2 encoding a mitochondrial nucleoside monophosphate kinase has an anti-JEV function and the C-terminal domain is essential for anti-viral activity. .Garg49/Ifit3 encodes an interferon and JEV-inducible mitochondrial protein and it has an anti-JEV function. . Activating transcription factor 3 (ATF3), GTPase, IMAP family member 3 (GIMAP3) and GLI pathogenesis-related 2 (GLIPR2) are pro-viral proteins which facilitate virus multiplication resulting in enhanced JEV titer.

Cytotoxic T Lymphocytes (CTL) are known to play an important role in clearing infectious virus from infected hosts in a variety of viral infections. Depending on the type of virus and mode of virus entry both class I and class II restricted CTL can contribute to protection from virus-induced disease. Although CD8 positive CTL are associated with virus elimination and control in many viral infections, elimination of neurotropic viruses from the Central Nervous system (CNS) is more complex due to the lowered expression of MHC antigens on neuronal cells. This failure to constitutively express high levels of MHC antigens by neurons could serve as an advantage to avoid damage to this differentiated and non-renewable tissue. However, abnormal induction of MHC antigens in the CNS mediated by CD4 positive lymphocytes or by astrocytes have also been shown to cause destructive inflammation in the CNS. The present study deals with CTL responses against one such neurotropic virus called Japanese Encephalitis Virus (JEV). JEV is a positive-stranded RNA virus that belongs to the flavivirus group, a group that is among the most important agents causing human encephalitis worldwide. Although passive transfer of monoclonal antibodies against this virus has been shown to confer protection of mice from lethal challenge with virus, neither the presence of CTL against this virus nor its role in conferring protection has been reported so far. Understanding the CTL responses against these viruses acquired importance in light of recent reports that neurovirulence of JEV and yellow fever viruses can be enhanced by the administration of virus specific antibodies. Hence this study was undertaken to examine the possibility of raising CTL specific to JEV. The specificity of the CTL raised, their therapeutic value and the ability of different lymphocyte subsets to mediate protection in vivo are dealt with in this study. Generation of CTL against JEV The generation of CTL against JEV in BALB/c mice, requires MHC defined cell lines that not only support virus infection but are also histocompatible. Several cell lines were initially examined for their ability to support JEV infection as a prc-rcquisitc before their utilization in in vivo and in vitro stimulation protocols aimed at generating JEV-specific CTL. Virus infection was monitored by immunofluorescence using JEV envelope-specific monoclonal antibodies as well as by titration of virus produced from infected cells by plaque assays. These different cell lines that were characterised for their ability to support JEV infection were then utilised to generate and monitor antiviral CTL. Several in vivo immunisation protocols were examined initially find out which of these infected cells prime BALB/c mice efficiently for generation of virus-specific CTL upon secondary stimulation in vitro with infected syngeneic cells. Immunisation of mice with infected cells per se was preferred over free virus since this was thought to facilitate priming against some viral non-structural proteins preferentially found on infected cells in addition to other viral structural proteins. It was observed that not only infected syngeneic and allogeneic cells but also infected xenogeneic cells prime BALB/c mice for the generation of JEV- specific CTL upon secondary restimulation in vitro. An optimal protocol was standardised for the generation of CTL against JEV. This included primary in vivo immunisation of mice followed by secondary in vitro restimulation of splenocytes with infected syngeneic cells. Either immunisation alone or in vitro stimulation of naive splenocytes alone was unsuccessful. The effector cells generated specifically lysed JEV-infccted P388D1 targets but not uninfected P388D1 or YAC-1 targets suggesting that the lysis on infected targets is not mediated by Natural Killer activity. Specificity and MHC restriction of anti JEV Effectors Cell depletion studies using complement mediated lysis were performed to examine the phenotype of the cells mediating virus specific lysis of infected targets. Depletion of Lyt 2.2+ or Thy 1+ but not L3T4+ sub-populations of effector cells inhibited lysis of infected targets showing that the effectors mediating virus-specific lysis were Lyt-2+ T cells. Examination of target specificities and MHC restriction of the antiviral CTL generated showed that although infected xenogeneic cells were used for immunisation, the effector cells recognised only infected syngeneic (P388D1, Sp2/0) and semisyngeneic (Neuro 2a, YAC-1) cells. Virus-specific recognition was found to be class I Kd and class I Dd restricted. These effector cells were also found to recognise cells infected with a closely related flavivirus, West Nile Virus (WNV) suggesting that they were crossreactive to some degree. Based on the consensus motif that has been established for H-2Kd associated peptides, several nonamers were predicted as possible CTL epitopes by scanning the deduced amino acid sequences of three strains of JEV and WNV. Among several predicted nonamers, three peptides were examined for their ability to reconstitute lysis of uninfected targets by polyclonal anti JEV CTL populations. Results demonstrate that peptides derived from NS1 and NS3 but not NS5 protein of JEV were able to partially reconstitute lysis of uninfected targets by effectors when pulsed with the appropriate peptide. Protective ability of the CTL raised against JEV To examine whether anti-JEV effectors raised in vitro could confer protection from intracerebral challenge with JEV, these effectors were adoptively transferred into adult BALB/c mice intracerebrally along with 10 x LDJ0 dose of JEV. More than 55% of these animals were protected from death and survived beyond 100 days after JEV challenge demonstrating that adoptively transferred anti-JEV effectors could indeed confer protection from lethal challenge with JEV. However, adoptive transfer of effectors by either intravenous or intraperitoneal routes did not protect adult mice from the lethal effects of intracerebral challenge with JEV. In contrast to adult mice, newborn mice were not protected from death by the adoptively transferred effector cells. This was also supported by experiments where a correlation was observed with the increasing age of mice and the success of protection conferred by the adoptively transferred effector cells. To establish the identity of cell subsets responsible for protection, Lyt 2, L3T4 or Thy 1 positive cells were specifically depleted from the polyclonal CTL by multiple cycles of complement mediated lysis and the remaining cells were adoptively transferred intracerebrally along with 10 x LD of JEV. These results demonstrate that both Lyt 2 and L3T4 positive T cells present in the effector population were necessary to confer protection of adult mice. Examination of virus-specific neutralising antibodies in the sera of protected and unprotected mice revealed that presence of L3T4 positive cells in the adoptively transferred population increases virus-specific neutralising antibodies. However presence of neutralising antibodies alone was not sufficient to confer protection. The protection required both Lyt-2 and L3T4 positive cells together. These studies could in the long term throw some light on similar observations about age dependant susceptibility to JEV in humans.

Chapter 1: Role of JEV NS1 in protective immunity and in immunopathology. Previous studies from our laboratory revealed T cell immunodominance of non structural proteins NS3 and NS1 during natural JEV infections in humans where as the structural protein E, which is a good target for neutralizing antibody response is a poor inducer of T cells. Flavivirus NS1 is also known to induce humoral immune response. Several studies in different flaviviruses have indicated a role for NS1-specific immune responses in protection against flaviviruses. Paradoxically, studies also pointed to the contribution of NS1 in pathology and immune modulation. We screened serum samples from 72 convalescent JE patients for the presence of anti-NS1 antibodies by ELISA and radioimmunoprecipitation and found NS1 reactivity in 45 samples. These antibodies to NS1 are capable of inducing complement mediated cytolysis of cells expressing NS1 on the surface. Additionally, we demonstrated twenty two fold reduction in the infectious virus produced at 48h in SW-13 cells in the presence of human complement and NS1 antiserum compared to control serum, suggesting that complement mediated cytolytic activity of anti NS1 antibody helps the host in controlling the virus propagation. Chapter 2: Comparison of immune responses to JEV structural proteins Capsid and Envelope in human volunteers vaccinated with inactivated JE vaccine and naturally exposed to live JEV. We compared the CMI responses to structural proteins E and C in human volunteers vaccinated with commercially available killed JE vaccine and in humans naturally exposed to live JEV. The results revealed that structural proteins E and C are inherently poor inducers of T cells even in killed vaccine preparation, where there is no competition from immunodominant non structural proteins. Therefore inclusion of nonstructural proteins NS1 and NS3 along with neutralizing antibody inducing envelope should improve memory and efficacy of a JE vaccine. Chapter 3: Construction and testing in the mouse model of experimental recombinant poxvirus vaccines expressing prM, E, NS1, and NS3 of JEV. Guided by the information on immune responses to JEV in the JE endemic human cohort and volunteers vaccinated with killed JE vaccine, we designed experimental vaccines as recombinant vaccinia viruses expressing NS1, NS3, prM, and E proteins of JEV (vNS1NS3prME) or NS1, NS3, prM, and C-terminally truncated E (vNS1NS3prMΔE) and studied the immune responses elicited by these vaccines in mice. Our data showed that a recombinant vaccinia virus expressing prM, ΔE, NS1, and NS3 of JEV is superior to killed JE vaccine in eliciting long lived neutralizing antibodies as well as NS1 and NS3-specific cytotoxic T lymphocytes (CTL) in addition to NS1-specific cytolytic antibodies, resulting in long lasting and enhanced protection from lethal JEV infection in mice. Our results thus identified all B and T cell antigens whose inclusion in a live-vectored vaccine would provide a vaccine with far superior efficacy over the inactivated JE vaccine.

Japanese encephalitis virus (JEV) is one of the major causes of encephalitis in Asia. JEV causes serious inflammation of the brain, which may lead to permanent brain damage and has a high mortality rate. Almost 3 billion people live in JE endemic areas and JEV causes an estimated 20,000 cases of disease and 6000 deaths per year. JEV is a positive stranded RNA virus belonging to the Flavivirus genus of the family Flaviviridae. The genome of JEV is about 11 kb long and codes for a polyprotein which is cleaved by both host and viral encoded proteases to form 3 structural and 7 non-structural proteins. JEV transmission occurs through a zoonotic cycle involving mosquitoes and vertebrate amplifying hosts, chiefly pigs and ardeid birds. Humans are infected when bitten by an infected mosquito and are dead end hosts. The role of humoral and cell mediated immune responses during JEV infection have been studied by several groups. While the humoral responses play a central role in protection against JEV, the cell mediated immune responses contributing to this end are not fully understood. The MHC molecules have been known to play predominant roles in host responses to viral infections and the consequences of virus infection on the expression of MHC molecules are varied. The expression of MHC-I molecules is known to decrease upon infection with many viruses such as HIV, MCMV, HCMV, Adv, and EBV. In contrast, infection with flavivirus such as West Nile Virus (WNV) has been shown to increase the cell surface expression of both MHC-I and MHC-II molecules. It has been reported previously that WNV infection increases the cell surface expression of adhesion molecules such as ICAM-1, VCAM-1 as well as E-Selectin and these changes were mediated directly by WNV and not by soluble cytokines. In contrast to classical MHC-I molecules, the nonclassical MHC-I molecules do not belong to a single group of structurally and functionally homologous proteins and normally have lower cell surface expression. Earlier studies have shown that the expression of nonclassical MHC-I molecules were induced during infection with JHM strain of mouse hepatitis virus (MHV). However, the functional significance of this induction is unclear. Expression of nonclassical MHC-I molecules upon flaviviral infection is not very well understood. In this thesis, evidence is presented that JEV infection induces the expression of both classical and nonclassical MHC-I molecules on primary mouse brain astrocytes, mouse embryonic fibroblasts (MEFs) and H6 (hepatoma cell). The levels of adhesion molecules as well as molecules involved in antigen processing and presentation were also analyzed and our results clearly demonstrate that JEV infection induces their expression on astrocytes, MEFs and H6. The role of NF-κB and type-I IFNs in the induction of classical and nonclassical MHC-I molecules as well as molecules involved in antigen processing and presentation were also analyzed and our results demonstrated that type-I IFN mediated signaling is responsible for the induction of these molecules during JEV infection. Chapter 1 discusses the innate and adaptive immune system, the role of classical and nonclassical MHC molecules in the initiation of immune response and diverse strategies adapted by different viruses to evade the immune response. It also includes a detailed discussion about the IFN and NF-κB signaling pathways and their modulation by viral infection. Finally, the genome organization, epidemiology, transmission cycle, pathogenesis and pathology, clinical features, humoral as well as cell mediated immune response to JEV infection and the current vaccine status to JEV infection are briefly discussed. Chapter 2 describes the general materials and methods used in this study. It includes the details of the reagents and cell lines used in the experiments. It also discusses the various techniques such as RT-PCR, FACS analysis, EMSA and ELISA. Chapter 3 focusses on the validation of different knockout MEFs used in the study as well as confirming the purity of primary astrocyte cultures established from pub brains. The susceptibility of various cells to JEV infection has also been investigated. Our results confirmed the authenticity of all the cells and the purity of primary astrocyte cultures used in the study. Our results also indicated that all the cells used in the study are susceptible to JEV infection. Chapter 4 discusses the expression of MHC and related genes involved in immune response upon JEV infection of primary mouse brain astrocytes, MEFs and H6. Chapter 4 demonstrates for the first time that JEV infection induces the expression of nonclassical MHC-I or class Ib molecules namely Qa-1, Qb1 and T10 in addition to the induction of classical MHC-I molecules. In contrast to WNV, there was no increase in the cell surface expression of MHC-II molecules upon JEV infection of primary mouse brain astrocytes. JEV infection also induces the expression of adhesion molecules as well as molecules involved in antigen processing and presentation namely Tap1, Tap2, Tapasin, Lmp2, Lmp7 and Lmp10. Chapter 5 demonstrates that JEV infection induces NF-κB activation in astrocytes and MEFs. Studies using MEFs deficient in classical and alternate pathways of NF-κB activation indicate that JEV activates the classical pathway of NF-κB activation and is dependent on canonical lKKβ/IKK2 activity. JEV infection of astrocytes, MEFs and H6 induces the production of type-I IFNs. To determine the mechanism of type-I IFN induction during JEV infection, MEFs deficient in NF-κB signaling and IFN signaling were used. Results indicate that type-I IFN production in MEFs occurs by both NF-κB dependent and independent mechanisms. In contrast, the production of IFN-α was completely abrogated in IFNAR-\- MEFs whereas IFN-β production was greatly reduced. Production of type-I IFNs in IFNGR-\- MEFs is also reduced upon JEV infection but the reason for this is unclear. Chapter 6 demonstrates that JEV induced expression of classical MHC-I molecules occurs by type-I IFN mediated signaling. This result is in contrast to WNV infection, in which both NF-κB and type-I IFNs are involved in the induction of classical MHC-I molecules. Type-I IFNs were also shown to be involved in the induction of nonclassical MHC molecules namely, Qa-1 and Qb1 during JEV infection. In contrast, the expression of T10, another nonclassical MHC molecule occurs independent of type-I IFN signaling. The expression of molecules involved in antigen processing and presentation namely, Tap1, Tap2, Lmp2 and Lmp7 was type-I IFN-mediated, whereas the expression of Tapasin and Lmp10 was mediated by both type-I IFN dependent and independent mechanisms. The expression of VCAM-1 was dependent on NF-κB mediated signaling. Chapter 7 precisely describes the underlying mechanism of induction of MHC and various other related molecules and their significance during JEV infection. In addition, it also includes a working model for the induction of these molecules during JEV infection. In summary, this is the first study in which the mechanism of JEV mediated induction of classical as well as nonclassical MHC molecules has been studied in detail. This study clearly demonstrated that type-I IFNs are involved in the induction of classical and nonclassical MHC-I molecules during JEV infection. The functional significance of this JEV mediated induction of classical MHC-I molecules is unclear, but it has been proposed that this is to escape from the action of NK cells. The absence of MHC-II induction during JEV infection could be important because it may lead to the initiation of an immune response which is different from that induced during other viral infections which induce the expression of MHC-II molecules. In contrast to classical MHC-I molecules, the functional and biological significance of nonclassical MHC-I molecules are poorly studied. Nonclassical MHC-I molecules play an important role in bridging adaptive and innate immune response. So the nonclassical MHC molecules induced during JEV infection may play an important role in the initiation of immune response during JEV infection. The role these nonclassical MHC-I molecules in antigen presentation during JEV infection is not known. These nonclassical antigens are also recognized by NK and γδT cells, thus the expression of nonclassical MHC-I molecules during JEV infection might also confer a protective role.

Expanding urbanization, climate change, and population growth contribute to increased transmission and spread of arthropod-borne viruses (arboviruses), many of which cause severe disease in humans. Pathogenic arboviruses include dengue, Zika, tick-borne encephalitis, and sindbis viruses, which together threaten more than half the global population. Thus, there is a constant need for safe, specific, and sensitive molecular tests to identify early-stage infections for accurate diagnosis and molecular epidemiological data for disease prevention and control. The study tested the biosafety of using FTA™ cards when working with pathogenic arboviruses by conducting an infectivity assay using sindbis virus. Conditions for RNA extraction and storage of arboviruses on FTA were analyzed by measuring viral RNA (vRNA) stability using a SYBR-Green, Pan-Flavi RT-qPCR method composed of degenerate primers able to detect a variety of flaviviruses. Data from a Pan-Flavi RT-qPCR study comprising of 222 clinical blood and serum samples collected from a 2018 dengue virus outbreak in Hanoi (Vietnam) was analyzed to establish applicability of FTA for molecular epidemiology and diagnosis. Results showed that sindbis virus infectivity was inhibited by FTA-adsorption. FTA-adsorbed arboviruses were extracted with the highest yield using Trizol extraction and were preserved at storage at 4-20ºC for up to 30 days. The results showed that clinical blood samples acquired higher yields of vRNA for molecular testing than serum samples and that it may be possible to perform sequencing for genomic analysis. The study suggests that FTA cards may facilitate the storage and transportation of adsorbed arboviruses for downstream molecular epidemiological and diagnostic tests.