<p>Flaviviruses are enveloped, positive-strand RNA viruses that are spread by mosquitoes and ticks and can cause serious disease in humans. Flavivirus virions undergo extensive structural changes during their life cycle, including during maturation and fusion. Flaviviruses are initially assembled at the endoplasmic reticulum in a non-infectious, immature state, and then traffic to the trans-Golgi network, where a pH drop triggers a structural rearrangement of glycoproteins prM and E on the virus surface from 60 trimers to 90 dimers. A host protease, furin, then cleaves prM which makes the transition irreversible. Upon exiting the host cell, pr disassociates from the virus and the infectious, mature virus is able to enter a new cell. <br></p><p><br></p> <p> </p> <p>In Chapter 1, an overview of flaviviruses is presented, including a brief history of their discovery and interaction with humans, followed by what is known about their life cycle and the maturation process. The structure of a mature flavivirus is then described, including the symmetrical arrangement of glycoproteins on the virion surface, the lipid membrane, and the nucleocapsid core, followed by an introduction of the structural proteins that assemble into the virion. The structure of the immature flavivirus is then described. The chapter concludes with a description of the dynamics and heterogeneity observed for flaviviruses.</p><p><br></p> <p> </p> <p>The conformational rearrangements that occur during flavivirus maturation remain unclear. The structures of immature and mature flaviviruses determined with cryo-electron microscopy (cryo-EM) demonstrated that flaviviruses are icosahedral particles with 180 copies of glycoproteins on their surface. Icosahedral viruses typically have a quasi-equivalent arrangement of glycoproteins, but flaviviruses lack quasi-equivalence and instead the three subunits within an asymmetric unit occupy different chemical environments. Although the subunits are the same proteins, the unique environment of each subunit can be exploited for tracking subunits during conformational rearrangements. For example, the unique labeling of a subunit can be used to identify it in the immature and mature virion.</p><p><br></p> <p> </p> <p>In Chapter 2, the maturation process was studied by developing tools to differentially label protein subunits and trap potential intermediates of maturation. The tools included heavy-atom compounds and antibody Fabs, which were used to probe Kunjin virus (KUNV), an Australian subtype of West Nile virus (WNV). One heavy-atom compound, potassium tetranitroplatinate(II), was found to derivatize immature KUNV, likely at sites on both E and prM. Higher-resolution studies will be required to determine if the compound differentially labeled the three subunits. The other tool developed was the E16 Fab. E16 Fab, originally isolated from a mouse immunized with WNV E and found to bind to two out of three subunits on mature WNV, was used to differentially label subunits in immature KUNV. Based on poor epitope accessibility on immature KUNV, E16 Fab was hypothesized to trap an intermediate state of maturation. In the cryo-EM reconstruction of E16 Fab bound to immature KUNV it was found that the virion had localized distorted density and apparent non-uniform binding of the E16 Fab. Based on this result it was proposed that flaviviruses had imperfect icosahedral symmetry. <br></p><p><br></p> <p> </p> <p>The structural asymmetry of immature and mature flaviviruses was investigated in Chapter 3. Icosahedral symmetry has always been imposed during cryo-EM reconstructions of flaviviruses, as it led to stable convergence of orientations. When reconstructions of immature KUNV and ZIKV were performed without imposing symmetry, the reconstructions showed that the flaviviruses had an eccentric nucleocapsid core, which was positioned closer to the membrane at one pole. At the opposite pole, the glycoprotein and inner leaflet densities were weak and distorted. Furthermore, there were protrusions from the core that contacted the transmembrane helices of the glycoproteins. In the asymmetric reconstruction of mature KUNV, the core was positioned concentric with the glycoprotein shell, in contrast to the immature virion, indicating that maturation alters the interactions between the core and the glycoproteins. The asymmetric reconstructions suggested that there is variable contact between the core and glycoproteins during assembly, which may be due to membrane curvature restrictions in the budding process. </p> <p> </p> <p><br></p><p>In Chapter 4, extracellular vesicles (EVs) that were released during dengue virus (DENV) infection were characterized by mass spectrometry. EVs may play a significant role in flavivirus infection, as they have been shown to transport both viral proteins and infectious RNA. EVs likely represent alternative modes of virus transmission and aid in immune evasion. However, previous studies on EVs are controversial because EVs are potential contaminated during assays by co-purifying virions and other particulates. The identification of EV biomarkers would greatly reduce contamination because biomarkers would enable isolation of pure EVs by affinity purification. Therefore, a strategy was developed to isolate EVs and profile them with proteomics. The four proteins cystatin-A, filamin B, fibrinogen beta chain, and endothelin converting enzyme 1 were found to be statistically enriched in the DENV sample and represent potential EV biomarkers. </p> <p> </p>

Flaviviruses (genus Flavivirus, family Flaviviridae) includes a number of medically and veterinary important arboviruses. Most of them are transmitted by mosquitoes, such as West Nile virus, yellow fever virus, dengue virus or Zika virus. In the last decade, there has been discovered number of flaviviruses which have not known vertebrate host. They are called insect-specific flaviviruses (ISFs). These viruses have been detected in dipteran insects (mostly in mosquitoes) from all over the world. It seems they induce no pathogenic changes even in insect and they are not able to infect any vertebrate cells. However in some recent studies has been shown that some ISFs could influence the replication of other medically important flaviviruses in mosquito cells. In addition, they probably represent an ancestrial lineage of the family Flaviviridae and further studies focused on them could help to clarify which characteristics of flaviviruses enable them to infect vertebrates. There is not a lot of information about the ecology of ISFs and it is suggested that most ISFs have not been discovered yet. Main goal of this thesis was to detect ISFs in mosquitoes in the Czech Republic, where have not been made any exstensive research of these viruses yet. In case of discover some undiscribed ISFs we wanted to characterize...

This master thesis is dedicated to the preparation of fluorinated derivatives of carbocyclic nucleosides, that may serve as flaviviral replication inhibitors. Preparation of both monofluorinated as well as gem-difluorinated analogs of ribo and 2'-deoxyribonucleoside was attempted. While a suitable and reliable route for the preparation of monofluorinated compounds way found, synthesis of gem-difluorinated turned out to be rather challenging. Although most of the presented work dealt with compounds bearing adenine as a nucleobase, the universal applicability of the developed procedures, demonstrated on the preparation of a guanosine-type molecule, suggests that after slight optimization larger series of this type of compounds could be prepared.

Viral RNA-dependent RNA polymerases (RdRps) are enzymes essential for viral multiplication. The general function of RdRp is universal for all RNA viruses: to recognise viral RNA, bind it and synthesize the complementary RNA strand. This series of steps is absolutely crucial for viral infection. It is important to mention that the non-infected cell is incapable of replicating any RNA. The host cell thus does not naturally express any RdRps. I chose RdRps for my research because these enzymes are key to viral replication and thus an excellent target for antivirals. This study characterises polymerases from ​Picornaviridae and Flaviviridae families, in depth. Picornaviral replication takes place in viral-induced membrane structures called Replication Organelles (ROs), where the polymerase is localised to the membrane. In this study, we investigated the recruitment of picornaviral polymerase membrane. Subsequently, we focused on the activation of picornaviral RdRp induced by the insertion of the very first residue into the protein core. Next, we focused on the flaviviral RdRps specifically from yellow fever virus (YFV) and Zika virus (ZIKV). This study reports the first structure of a full length YFV polymerase and a model of ZIKV polymerase in complex with RNA. The model of ZIKV RdRp in complex with...

Os Flavivirus são transmitidos por mosquitos (Diptera: Culicidae) que se refugiam em remanesctentes de Mata Atlântica. Essas áreas verdes correspondem às Unidades de Conservação e parques urbanos, que estão espalhados pela região metropolitana de São Paulo. Este estudo foi realizado com o intuito de conhecer as espécies de culicídeos que circulam na Área de Proteção Ambiental (APA) Capivari-Monos, na zona Sul do município de São Paulo, e no Parque Estadual da Cantareira (PEC), na zona Norte do mesmo município, e de investigar infecção natural por Flavivirus na fauna de culicídeos amostrada. Também foi proposto relacionar a variedade, a quantidade e identidade dos Flavivirus detectados com os padrões de riqueza, abundância e diversidade das assembleias de mosquitos. Foram realizadas 14 coletas, mensalmente, em quatro pontos de coleta na APA e três no PEC, todos com diferentes níveis de intervenção antrópica, no período de março de 2016 a abril de 2017. Armadilhas automáticas luminosastipo CDC (com atração de CO2 e ácido lático) foram instaladas na copa das árvores e no nível do solo. O esforço amostral foi equivalente para os todos os pontos, sendo que foram instaladas duas armadilhas em cada ponto (uma na copa e outra no solo), com 18 horas de coleta, permitindo amostragem de culicídeos de hábitos diurnos, crepusculares e noturnos. Os espécimes foram transportados com vida para o Laboratório de Saúde Pública da Faculdade de Saúde Pública da Universidade de São Paulo, criopreservados a temperatura -70ºC, identificados morfologicamente e agrupados em pools (com até 10 indivíduos). Os pools foram submetidos à técnica de isolamento viral em cultura de células (C6/36), seguida do teste de imunofluorescência indireta. Os pools positivos foram submetidos à reação de RT-qPCR e, posteriormente, sequenciados. Duas árvores de similaridade foram construídas para confirmação dos Flavivirus. No total, 1216 exemplares de culicídeos foram amostrados (13 gêneros), cuja riqueza foi de 42 táxons. A APA registrou a maior abundância (878 espécimes) e maior riqueza (37 táxons). A Cachoeira foi o ponto de coleta na APA que amostrou a maior riqueza e abundância, contudo, com a mais baixa diversidade. Entretanto, a Borracharia obteve alta riqueza, baixa abundância e a maior diversidade. O PEC amostrou 338 indivíduos e a riqueza foi de 23 táxons. Dentre os pontos do PEC, a Trilha do Pinheirinho amostrou a maior riqueza e abundância. An. (Ker.) cruzii, Cx. (Cux.) sp, Cx. (Mel.) vaxus, Li. durhami, Wy. (Prl.) confusa e Wy. (Pho.) theobaldi foram detectadas com infecção natural por Flavivirus. O sequenciamento revelou infecção por ZIKV em An. (Ker.) cruzii, Li. durhami e Wy. (Prl.) confusa, e infecção por DENV-2 em Cx. (Cux.) sp e Cx. (Mel.) vaxus. Concluiu-se que a riqueza, abundância e diversidade estão relacionadas entre si e, juntas, influenciaram na detecção de espécies de culicídeos naturalmente infectadas por Flavivirus, sendo que estes foram detectados em espécies provenientes de pontos de coleta cuja riqueza e abundância foram altas, e a diversidade baixa. A quantidade e a variedade dos Flavivirus também foram influenciadas por esses três fatores, para ocorrer na natureza. Não foi possível correlacionar a identidade dos Flavivirus com os três fatores uma vez que as espécies detectadas com infecção natural por esses vírus não são apontadas como potenciais vetoras. Além disso, a abundância e a diversidade pareceram ter uma relação inversa entre si. / Flaviviruses are transmitted by mosquitoes (Diptera: Culicidae) that take refuge in remnants of the Atlantic Forest. These green areas correspond to Conservation Units and urban parks which are spread throughout the metropolitan region of São Paulo. This study was carried out in order to identify the Culicidae fauna that circulate in Capivari-Monos Environmental Protection Area (APA), located in the South area of the city of São Paulo, and in Cantareira State Park (PEC), North area of the same municipality and to investigate natural Flaviviruses infection in this sampled Culicidae fauna. It was also proposed to relate the variety, quantity and identity of the Flaviviruses detected with patterns of richness, abundance and diversity of mosquito assemblages. Fourteen collections were carried out monthly at four collection sites in the APA and three in the PEC, all sites with different levels of anthropogenic intervention, during March 2016 to April 2017. CDC automatic traps (with attraction of CO2 and lactic acid) were installed in the canopy and on ground. The sampling effort was equivalent for all the points, and two traps were installed at each point (one in the canopy and the other on ground), with 18 hours of sampling, allowing sampling culicidae of daytime, morning and evening twilight, and nightlyl habits. The specimens were carried alive to the Public Health Laboratory of the School of Public Health of the University of São Paulo, were cryopreserved at a -70ºC temperature, identified morphologically and grouped in pools (with up to 10 individuals). The pools were submitted to the virus isolation technique in cell culture tissue (C6 / 36), followed by the indirect immunofluorescence test. The positive pools were submitted to the RT-qPCR reaction and, subsequently, sequenced. Two similarity trees were made only to confirm Flaviviruses infection. In total, 1216 specimens of culicidae were sampled (13 genera), and the richness was 42 taxa. In addition to APA recorded the highest abundance (878 specimens) and also highest richness (37 taxa). Cachoeira was the collection site in APA that showed the greatest richness and abundance as well, however, with the lowest diversity. In addition, Borracharia obtained high richness, low abundance and highest diversity. PEC sampled 338 specimens and the richness was 23 taxa. Among the collection sites of the PEC, Pinheirinho Trail showed the highest richness and also abundance. An. (Ker.) cruzii, Cx. (Cux.) sp, Cx. (Mel.) vaxus, Li. durhami, Wy. (Prl.) confusa and Wy. (Pho.) theobaldi were detected with natural Flaviviruses infection. The sequencing analyzes revealed ZIKV infection in An. (Ker.) cruzii, Li. durhami and Wy. (Prl.) confusa, and DENV-2 infection in Cx. (Cux.) sp and Cx. (Mel.) vaxus. It has concluded that the richness, abundance and also diversity are related to each other and, together, influenced the detection of species of culicidae naturally infected by Flaviviruses, which were detected in species from collection sites whose richness and abundance were high. About quantity and variety of Flaviviruses, these were also influenced by the three factors on nature. It was not possible to correlate the identity of the Flaviviruses with the three factors since the species detected with natural infection by these viruses are not indicated as potential vectors. Moreover, abundance and diversity appeared to have an inverse relation.

CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico / No Brasil, a maioria das viroses transmitidas por artrópodes (arboviroses) pertence ao gênero Flavivirus, no entanto, aqueles transmitidos por carrapatos são menos estudados do que os transmitidos por insetos. Além dos vírus, outros microrganismos possuem grande relevância em saúde pública. Riquétsias são os principais agentes de zoonoses transmitidas por carrapatos no Brasil, especialmente as do Grupo da Febre Maculosa (GFM), que inclui diversas espécies patogênicas e que são transmitidas por carrapatos. Dentre os diversos biomas, dois foram particularmente degradados no Brasil, a Mata Atlântica e o Cerrado. Diante destes motivos, esta tese compõe-se de três capítulos, que tiveram como objetivo identificar carrapatos vetores em áreas do Cerrado e Mata Atlântica adjacentes a áreas com atividade humana, pesquisando por patógenos causadores de zoonoses, Rickettsia spp. e Flavivirus. No primeiro capítulo, a presença de riquétsias foi averiguada em carrapatos de cães e carnívoros da região de Cumari-GO. Pesquisou-se por esse microrganismo em oito espécies de carnívoros silvestres e em cães domésticos. Duas amostras amplificaram fragmentos do gene ompB, presente em riquétsias do GFM, e ao mesmo tempo amplificaram fragmentos específicos para Rickettsia bellii, que não pertence a esse grupo. Isso demonstra uma possível infecção cruzada, podendo o carrapato manter concomitantemente estas duas espécies divergentes. O segundo capítulo, dados de carrapatos de tamanduás (Myrmecophaga tridactyla e Tamandua tetradactyla) referentes a um período de 18 anos foram analisados, 169 amostras foram avaliadas quanto à presença de riquétsias. Quatro destas amostras exibiram sequência de ompA com 100% de identidade com outras sequências de carrapato Amblyomma nodosum, indicando que Rickettsia spp. do grupo da febre maculosa (SFG) em Amblyomma nodosum estava circulando no entorno da cidade de Uberlândia e estado de São Paulo, podendo outros carrapatos, tais como A. sculptum serem infectados, gerando risco para animais domésticos e seres humanos. No último capítulo, carrapatos de seis áreas, uma pertencente à Mata Atlântica e as outras do bioma Cerrado, foram avaliados quanto à presença de Flavivirus. Nestes locais foram pesquisados carrapatos de doze espécies diferentes, sendo onze da família Ixodidae e um da família Argasidae, estes pertenciam às espécies: Amblyomma sculptum, Rhipicephalus sanguineus, Rhipicephalus microplus, Dermacentor nitens, Amblyomma ovale, Amblyomma dubitatum, Amblyomma parvum, Amblyomma rotundatum, Amblyomma incisum, Amblyomma brasiliense, Amblyomma naponense e Ornithodoros sp. Detectou-se a presença de um flavivírus em carrapatos R. microplus coletados na região de Uberlândia. Estas amostras exibiram aproximadamente 98% de identidade com o Vírus do Carrapato Mogiana (MGTV). Todos os carrapatos que exibiram resultados positivos para flavivírus ou riquétsias, foram coletados em áreas antropizadas, confirmando a importância do estudo de vetores hematófagos na epidemiologia de agentes virais e bacterianos de animais domésticos e selvagens. / In Brazil, most arthropod-transmitted viruses (arboviruses) belong to Flavivirus genus, being tick-transmitted less studied than insect-transmitted ones. Other microorganisms have great public health relevance, such as Rickettsia, the main agent of tick-borne diseases in Brazil, specially the Macular Fever (MF) group, which includes several pathogenic species that are transmitted by ticks. Within different biomes, both the Atlantic Forest and the Cerrado were severely degraded in Brazil. This thesis consisted of three chapters, aiming to identify ticks in areas of Cerrado and Atlantic Forest with surrounding human activity, looking for both Rickettsia and Flavivirus infected ticks. In the first chapter, the presence of Rickettsia was investigated in ticks from dogs and other carnivores, being eight wild carnivores species and domestic dogs in Cumari, Goiás state, Brazil. Two samples were positive for ompB gene, common in the MF group, being also positive for specific genes found in Rickettsia bellii, which does not belong to the MF group. This result shows a possible cross-infection, being tick able to maintain these two different species simultaneously. The second chapter, an 18- year-study-data from anteater ticks (Myrmecophaga tridactyla and Tamandua tetradactyla) was analyzed, accounting 169 samples evaluated for the presence of Rickettsia. Four samples were positive for ompA gene, being 100% identical to other tick sequences present in Amblyomma nodosum, indicating that Rickettsia from the MF group in A. nodosum was circulating around the city of Uberlândia and state of São Paulo. Other ticks, such as A. sculptum, could become infected by the Rickettsia presence, generating risk for domestic animals and humans. In the last chapter, ticks from six areas, being one from the Atlantic Forest and the others from the Cerrado biome, were evaluated for the presence of Flavivirus. 12 different species were accounted, in which eleven were from the Ixodidae family and one was from the Argasidae family, having the following species identified: Amblyomma sculptum, Rhipicephalus sanguineus, Rhipicephalus microplus, Dermacentor nitens, Amblyomma ovale, Amblyomma dubitatum, Amblyomma parvum, Amblyomma rotundatum, Amblyomma incisum, Amblyomma brasiliense, Amblyomma naponense and Ornithodoros sp. The presence of a Flavivirus in R. microplus ticks collected in the Uberlândia region was detected. These samples exhibited approximately 98% identity with the Mogiana Tick Virus (MGTV). All ticks that were positive for Flavivirus or Rickettsia were collected in areas with human activities, stating the importance of studies on hematophagous vectors in the epidemiology of both viral and bacterial agents in domestic and wild animals. / Tese (Doutorado)

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.

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.

Recently, non-cellular viral agents became the focus of a large number of scientific groups. A prominent and widespread group of these viruses are flaviviruses, which include, for example, Zika virus, Dengue fever virus, tick-borne encephalitis virus and West Nile virus. There is a considerable diversity among these viruses, however, highly conserved proteins can be found throughout this viral genus. The largest and most conserved protein encoded by flaviviruses is the nonstructural NS5 protein. Its N-terminal domain bears the methyltransferase (MTase) activity. Thanks to the methylation of its genome, it allows the virus to initiate translation and at the same time mask it from the host's immune system. By blocking the active site of this enzyme with a small molecule, viral infection could be stopped not only in one flavivirus, but, due to the high conservation of MTases, in all other flaviviruses. This diploma thesis deals with the aforementioned MTase domain of the NS5 protein, specifically of the West Nile virus (WNV). After designing an insert encoding the WNV MTase domain, amplifying it and ligating it into the vector, the MTase domain was prepared by a recombinant expression, followed by purification. Subsequently, complexes of the protein with small molecules (MTase ligands) were formed, in...