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1
Thomas, C. M. "Cauliflower mosaic virus DNA replication". Thesis, Bucks New University, 1986. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.374828.
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Ekström, Jens-Ola. "Ljungan Virus Replication in Cell Culture". Doctoral thesis, Högskolan i Kalmar, Naturvetenskapliga institutionen, 2007. http://urn.kb.se/resolve?urn=urn:nbn:se:hik:diva-10.
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Ljungan virus (LV) is a recently identified picornavirus of the genus Parechovirus. LV has been isolated from voles trapped in Sweden and also in the United States. LV infected small rodents may suffer from diabetes type 1 and type 2 like symptoms, myocarditis and encephalitis. LV has been proposed as a human pathogen, with indications of causing diabetes type 1, myocarditis and intrauterine fetal deaths. In this thesis, cell culture adapted LV strains were utilised for development and adaptation of several basic methodological protocols to study the LV biology, e.g. real time PCR, highly specific antibodies and a reverse genetics system. These methods allowed detailed studies of this virus and how it interacts with the host cell. The genomic 5'-end was identified and modelling showed unique secondary structure folding of this region. The LV encodes an aphthovirus-like 2A protein with a DvExNPGP motif. This motif was found to mediate primary cleavage of the LV polyprotein in vitro and is proposed to constitute the carboxy terminus of the structural protein VP1 in LV. Rabbit polyclonal antibodies generated against recombinant structural proteins were used to verify that the LV virion is composed of the structural proteins VP0, VP1 and VP3. Cell culture studies showed that LV replicates to low titer with an absent or delayed cell lysis. LV is proposed to be able to spread by a, for picornaviruses, not previously demonstrated direct cell-to-cell transmission. All results taken together suggest a maintenance strategy of LV including low amounts of the LV genome and persistently infected hosts. Stability studies showed that the LV virion not only maintain activity in acidic and alkaline environments but also exhibit resistance to the commonly used disinfectant Virkon®.The results presented in this thesis show that LV has several unique properties, not previously observed for a picornavirus.
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Ekström, Jens-Ola. "Ljungan virus replication in cell culture /". Kalmar : University of Kalmar, 2007. http://urn.kb.se/resolve?urn=urn:nbn:se:hik:diva-10.
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McQuillin, Andrew. "Aspects of cucumber mosaic virus replication". Thesis, Imperial College London, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.321682.
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Evans, Elizabeth Van Amburg. "Molecular genetic analysis of a vaccinia virus gene with an essential role in DNA replication /". Access full-text from WCMC, 1989. http://proquest.umi.com/pqdweb?did=744576211&sid=1&Fmt=2&clientId=8424&RQT=309&VName=PQD.
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Nayak, Arabinda. "Foot and mouth disease virus RNA replication". Thesis, University of Surrey, 2005. http://epubs.surrey.ac.uk/842873/.
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Infection of susceptible cells with foot and mouth disease virus (FMDV) results in multiplication of the RNA genome and assembly of mature virions. The entire process of genome replication is completed in a few hours and encompasses many intracellular events. Like other picornaviruses, FMDV uses a peptide primed RNA replication mechanism. The factors that are required to uridylylate each of the three FMDV VPg peptides and the role of the FMDV cis-acting replication element (cre) or 3B Uridylylation Site (bus) in VPg uridylylation have been determined. The native N-terminus of the FMDV 3Dpol enzyme is a pre-requisite for VPg uridylylation in vitro and the effects of mutations in the RNA template are consistent with a slide-back mechanism. The role of the poly(A) tail in uridylylating VPg was insignificant using full-length FMDV RNA transcripts suggesting the possibility of an alternative mechanism of VPg incorporation into negative strand RNA. The optimal RNA sequences required for VPg uridylylation were found to be within the 5' non-coding region (NCR). Furthermore, the results also showed evidence for RNA-RNA interactions between distinct structures from within the 5' NCR that influence VPg uridylylation. The polymerase precursor 3CDpro is also a prerequisite for uridylylation of each of the FMDV VPg peptides. However BCpro alone can substitute for 3 CD, but is much less efficient. It also appeared that the overall charge of the VPg peptides determines their recognition by the FMDV 3Dpol. The RNA binding activity of the 3C was found to be required for its stimulatory effects on VPg uridylylation. Unlike the poliovirus cloverleaf, the FMDV S-fragment (at the 5' end of the genome) does not interact with the FMDV 3CD precursor protein; however it binds specifically to a cellular factor p48. The crude replication complexes (CRCs) isolated from FMDV-infected cells were found to synthesize viral RNA very efficiently and an in vitro RNA replication system developed using these CRCs can be used to study the complete RNA replication events of FMDV.
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Szemiel, Agnieszka M. "Replication of Bunyamwera virus in mosquito cells". Thesis, University of St Andrews, 2011. http://hdl.handle.net/10023/2570.
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The Bunyaviridae family is one of the largest among RNA viruses, comprising more than 350 serologically distinct viruses. The family is classified into five genera, Orthobunyavirus, Hantavirus, Nairovirus, Phlebovirus, and Tospovirus. Orthobunyaviruses, nairoviruses and phleboviruses are maintained in nature by a propagative cycle involving blood-feeding arthropods and susceptible vertebrate hosts. Like most arthropod-borne viruses, bunyavirus replication causes little damage to the vector, whereas infection of the mammalian host may lead to death. This situation is mimicked in the laboratory: in cultured mosquito cells no cytopathology is observed and a persistent infection is established, whereas in cultured mammalian cells orthobunyavirus infection is lytic and leads to cell death. Bunyaviruses encode four common structural proteins: an RNA-dependent RNA polymerase, two glycoproteins (Gc and Gn), and a nucleoprotein N. Some viruses also code for nonstructural proteins called NSm and NSs. The NSs protein of the prototype bunyavirus, Bunyamwera virus, seems to be one of the factors responsible for the different outcomes of infection in mammalian and mosquito cell lines. However, only limited information is available on the growth of bunyaviruses in cultured mosquito cell lines other than Aedes albopictus C6/36 cells. Here, I compared the replication of Bunyamwera virus in two additional Aedes albopictus cell clones, C7-10 and U4.4, and two Aedes aegypti cell clones, Ae and A20, and investigated the impact of virus replication on cell function. In addition, whereas the vertebrate innate immune response to arbovirus infection is well studied, relatively little is known about mosquitoes’ reaction to these infections. I investigated the immune responses of the different mosquito cells to Bunyamwera virus infection, in particular antimicrobial signaling pathways (Toll and IMD) and RNA interference (RNAi). The data obtained in U4.4 cells suggest that NSs plays an important role in the infection of mosquitoes. Moreover infection of U4.4 cells more closely resembles infection in Ae and A20 cells and live Aedes aegypti mosquitoes. My data showed that the investigated cell lines have various properties, and therefore they can be used to study different aspects of mosquito-virus interactions.
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Napoli, Andrea. "Glycerophospholipid fluorescence imaging during vaccinia virus replication". Thesis, Sorbonne Paris Cité, 2019. https://theses.md.univ-paris-diderot.fr/NAPOLI_Andrea_1_va_20190415.pdf.
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Le virus de la vaccine (VACV) est l'organisme modèle pour l'étude des Poxviridae. Son cycle de réplication dans le cytoplasme de la cellule hôte a été largement étudié par microscopie optique et microscopie électronique. Grâce à des études génétiques approfondies, le rôle de certaines des 250 protéines du virus a été élucidé. Cependant, les mécanismes d’acquisition de la membrane du virus, notamment le rôle des lipides cellulaires impliqués, restent mal connus. L’étude de la composition des membranes de VACV purifiés par spectrométrie de masse a montré qu’elles présentent un enrichissement en acide phosphatidique (PA) et en dérivés de phosphatidylinositoles (PIPs). De plus, des études in vitro ont permis d’identifier certaines protéines virales capables de se lier aux PIPs in vitro. Le rôle de ces lipides dans le cycle de vie du virus, en particulier, dans la biogenèse de ses membranes n'a pas été identifié. L'objectif de ce projet de thèse est de déterminer l’implication du PA et des PIPs dans la biogenèse des membranes virales. L’expression transitoire de protéines recombinantes contenant des domaines de liaison à ces lipides a permis de déterminer la localisation du PA et des PIPs au cours de la réplication du virus. Afin de compléter ces résultats nous avons également utilisé des anticorps reconnaissant la PI4K et le PI4P. Enfin, l’utilisation d’inhibiteurs des PI3Ks et des PI4Ks a permis d’étudier le rôle de ces kinases durant l’assemblage de la membrane virale. A l'aide de ces outils, j'ai pu montrer que la localisation de ces lipides, à l'exception du PI3P, n'est pas altérée dans les cellules infectées. De plus, aucune co-localisation n’a été observée entre ces lipides et les sites de réplication du virus. Par ailleurs, nous avons observé une co-localisation entre le PI4P et les virus enveloppés ce qui est en accord avec les études précédentes montrant que les membranes du virus mature seraient dérivées de l'appareil de Golgi. Toutefois, des inhibiteurs de la synthèse du PI3P et du PI4P n'ont pas montré d’effets sur la production des membranes virales observables par microscopie optique. En conclusion, ce travail a permis de mieux définir le rôle des lipides durant la réplication de VACV. Ces résultats mettent en lumière un rôle potentiel du PI4P au cours de l’acquisition de l’enveloppe du virus ainsi qu’un rôle PI3P et de protéines reconnaissant spécifiquement le PI3P au cours des phases tardives de la réplicationVaccinia Virus (VACV) is the model organism for the study of the Poxviridae. Its cytoplasmic life cycle has been studied extensively by light- and electron microscopy. Thanks to a robust genetic system the role of some of its 250 proteins is beginning to be understood. Nevertheless, the acquisition of its membranes is still a matter of debate, in particular the role of cellular lipids. Lipid mass spectrometry of purified VACV previously showed an enrichment of phosphatidic acid (PA) and phosphatidylinositol derivatives (PIPs) in the viral membrane. Although some viral proteins have been shown to bind PIPs in vitro the role of these lipids in the viral life cycle, in particular viral membrane biogenesis, remains elusive.The aim of this work is to determine whether PA and PIPs are relocated in infected cells to the site of viral membrane biogenesis. For both PA and PIPs, I used recombinant proteins containing PA or PIP binding domains fused to eGFP, expressed them by transient transfection to follow their localization during viral replication. In addition, I used antibodies for the recognition of PI4K and PI4P. In order to understand the biochemical role of PIPs, I used pan-PI3K and PI4K inhibitors to study their effect on viral assembly. Using these tools, I could show that the lipids under investigation did not display an altered localization, with the exception of PI3P which showed a different pattern in infected cells. None of the PIPs analyzed co-localized with the sites of primary VACV membrane biogenesis. Consistent with the fact that the mature virus acquires additional membranes derived from the Golgi complex, I could show a co-localization of wrapped virus with PI4P, known to localize to this cellular organelle. However, drugs inhibiting PI3P and PI4P biosynthesis did not show any effect on VACV membrane biogenesis, at least at the light microscopy level. In conclusion, this work sharper defines the role of lipids during VACV replication. In particular, it opens the way to further studies on the putative role of PI4P during wrapping and the fate of PI3P and PI3P binding proteins during late replication
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Lu, Jia. "Norovirus translation and replication". Thesis, University of Cambridge, 2018. https://www.repository.cam.ac.uk/handle/1810/278610.
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Human norovirus (HuNoV) is the leading cause of gastroenteritis worldwide. Despite the significant disease and economic burden, currently there are no licensed vaccines or antivirals. The understanding of norovirus biology has been hampered by the inability to cultivate HuNoV in cell culture. To establish a tissue culture system, infectious HuNoVs were purified from clinical stool samples. HuNoV replication was tested in different cell types. The B-cell and intestinal organoids culture systems were validated. In addition, using organoids culture a DNA-based reverse genetic system was shown to recover infectious HuNoV. Due to the challenges associated with cultivating HuNoV, murine norovirus (MNV) was used as a surrogate system to understand the role of eIF4E phosphorylation in norovirus pathogenesis, and VP1-RdRp interaction in regulating viral genome replication. MNV infection results in the phosphorylation of the translation initiation factor eIF4E, re-programming host-cell translation during infection. Inhibiting eIF4E phosphorylation reduces MNV replication in cell culture suggesting a role in viral replication. A mouse model with eIF4E S209A, a phosphor-ablative mutation, was established to understand the role of eIF4E phosphorylation in MNV pathogenesis. In vitro and in vivo characterisations demonstrated that eIF4E phosphorylation may have multiple roles in norovirus-host interactions, but overall has little impact on MNV pathogenesis. The shell domain (SD) of norovirus major capsid protein VP1 interacts with viral RNA-dependent RNA polymerase (RdRp) in a genogroup-specific manner to enhance de novo initiation of RdRp, and to promote negative-strand RNA synthesis. To understand how VP1 regulates norovirus genome replication, chimeric MNVs with genogroup-specific residues mutagenised were characterised in vitro and in vivo. A single amino acid mutation was shown to destabilise viral capsid. SDs with reduced VP1-RdRp interaction showed less capacity to stimulate RdRp, resulting in delayed virus replication. In vivo, the replication of an MNV-3 with homologous mutations was abolished, highlighting the crucial role of this interaction.
10
Xing, Xuekun. "DNA replication and telomere resolution in vaccinia virus". Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp04/mq23557.pdf.
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Bao, Yiming. "The DNA replication of rice tungro bacilliform virus". Thesis, University of East Anglia, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.386295.
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Frampton, Nicholas Ross. "Impact of hypoxia on hepatitis B virus replication". Thesis, University of Birmingham, 2018. http://etheses.bham.ac.uk//id/eprint/8467/.
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Hepatitis B virus (HBV) is one of the world’s unconquered diseases, with 370 million chronically infected globally. HBV replicates in hepatocytes within the liver that exist under a range of oxygen tensions from 11% in the peri-portal area to 3% in the peri-central lobules. HBV transgenic mice show a zonal pattern of' viral antigen with expression in the peri-central areas supporting a hypothesis that low oxygen regulates HBV replication. We investigated this hypothesis using a recently developed in vitro model system that supports HBV replication. We demonstrate that low oxygen significantly increases covalently closed circular viral DNA (cccDNA), viral promoter activity and pre-genomic RNA (pgRNA) level, consistent with low oxygen boosting viral transcription. Hypoxia inducible factors (HIFs) regulate cellular responses to low oxygen and we investigated a role for HIF-1\(\alpha\) or HIF-2\(\alpha\) on viral transcription. A combination of HIF inhibitors and silencing of HIF-l\(\alpha\) and HIF-2\(\alpha\) ablated the effect of low oxygen on cccDNA and pgRNA, suggesting a role in regulating HBV transcription. This study highlights a new role for hepatic oxygen levels to regulate multiple steps in the HBV life cycle and this may impact on future treatments for viral associated pathologies.
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Nilsson, Benjamin Erik. "Viral and host factors regulating influenza virus replication". Thesis, University of Oxford, 2017. https://ora.ox.ac.uk/objects/uuid:b8953952-e6d5-4f6d-a7ba-cd55277611d1.
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Avian influenza A viruses typically do not replicate very efficiently when exposed to a mammalian host species. One of the reasons for this is the low activity of the viral RNA-dependent RNA polymerase of avian influenza viruses in mammalian cells. This host restrictive effect can be overcome by adaptive mutations in the avian polymerase, many of which are found in the 627-domain of the PB2 polymerase subunit. Deletion of the 627-domain revealed that this domain is not required for enzymatic functions of the polymerase in vitro, but that it essential for viral replication in a cellular environment in a nucleoprotein-independent manner. While the 627-domain is not necessary for viral RNA synthesis, it was demonstrated that it is involved in mediating encapsidation of nascent replication products. Recently the host factor ANP32A was shown to be the main determinant of host range restriction of the viral polymerase. It was demonstrated that during viral infections ANP32A interacts with KPNA2, a host factor strongly linked to host range restriction of the viral polymerase. It was also revealed that avian polymerases specifically are restricted in vRNA synthesis, a defect that was reversed in the presence of avian ANP32A. ANP32A was shown to be an enhancer of vRNA synthesis in vitro. Viral polymerase-polymerase interactions have been reported previously and presumably fulfil several essential functions during viral replication. Here the potential interaction interfaces of two different polymerase dimers were investigated and a role of polymerase dimers in replication and in trans-activation of cRNA-bound polymerases was found. RNA-binding proteins are essential for RNA metabolism and therefore cell physiology. It has been reported that the RNA-binding proteome responds to biological stimuli. Here the response of the RNA-binding proteome to influenza virus infection was investigated using an in vivo UV crosslinking interactome capture technique. It was demonstrated that the RNA-binding proteome is significantly altered and that this effect is independent of protein abundance. Several host RNA-binding proteins were identified that change their RNA-binding behaviour and that could have pro- or antiviral functions during influenza virus infection.
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Pina, Mery. "Replication of extrachromosomal elements of hyperthermophilic archaea". Paris 6, 2011. http://www.theses.fr/2011PA066557.
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La réplication des éléments extrachromosomales des Crenarchaeota est un sujet qui reste inconnu. Plus de 96% des ORFs virales n’ont pas des homologues dans des basses des données publiques et donc leurs fonctions ne peuvent pas être prédites par similarité de séquences. L’objectif de cet étude a été de comprendre les mécanismes de réplication des éléments extrachromosomales chez les Crenarchaea dans l’exemple du génome linaire du virus filamenteux d’Acidianus 1 (AFV1) qu’infecte Acidianus hospitalis, et celle du plasmide pRN1 de Sulfolobus islandicus. En utilisant l’analyse in silico de la séquence génétique, l’électrophorèse en deux dimensions des gel d’agarose (2DAGE) et la microscopie électronique en champ noir on postule que le mécanisme de réplication du AFV1 c’est un mécanisme dépendante de la recombinassions. On a trouvé une protéine terminal attache aux extrémités 5’ du génome viral. La formation des structures tertiaires sont favorises par l’interaction de cette protéine avec des régions internes du génome. La technique de 2DAGE a été utilisée pour étudier les caractéristiques des intermédiaires de réplication du plasmide pRN1. La présence des intermédiaires du même taille, mais différent structure secondaire support l’hypothèse d’un boucle stem à l’intérieur du réplicon minimal du plasmide. L’origine de réplication par 2DAGE se trouve au même site. La caractérisation biochimique de la protéine AFV1-ORF157 est incluse aussi dans cet manuscrit. Une activité de nucléase en ADN double brin est prouvée d’être présent dans l’ORF-157. Cette propriété a inspiré l’hypothèse que cette protéine peut être implique dans la maturation du génome avant son encapsidationReplication of extrachromosomal elements of the Crenarchaeota remains to be poorly understood. More than 96% of viral ORFs do not have homologs in public databases, and thus their functions cannot be predicted based on sequence similarity. The aim of the present study was to understand the mechanisms of replication of extrachromosomal elements in Crenarchaeota in the frame of the genome of the Acidianus filamentous virus 1 (AFV1) of Acidianus hospitalis, and of the plasmid pRN1 of Sulfolobus islandicus. By applying in silico genome sequence analysis, two dimensional agarose gel electrophoresis (2DAGE) and dark field electron microscopy we postulated that the replication mechanism exploited by AFV1 is recombination-dependent replication. A terminal protein was found to be strongly attached to the 5’- ends of the encapsidated viral genome. Tertiary structures were promoted by the interaction of this protein with internal regions of the genome. Characterization of the replicative intermediates of pRN1 was performed using 2DAGE. The existence of intermediates of the same size, but different secondary structure supported the hypothesis of a stem-loop that was experimentally found to be inside the minimal replicon of the plasmid. A replication origin was found in the 2DAGE at this same site. The biochemical characterization of AFV1-ORF157 is also included in this dissertation. A nuclease activity on linear double-stranded DNA was shown to be present in ORF-157, giving rise to the hypothesis that this protein might be involved in edition of mature genomes for encapsidation
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Kandpal, Manish. "Role of defective hepatitis B virus in wild-type hepatitis B virus replication". Thesis, IIT Delhi, 2017. http://localhost:8080/xmlui/handle/12345678/7247.
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Penzes, Zoltan. "Defective replicating RNAs of coronavirus infectious bronchitis virus : investigation of replication and genome packaging signals". Thesis, University of Hertfordshire, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.283879.
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Rozen-Gagnon, Kathryn. "Chikungunya virus nonstructural proteins regulate replication fidelity and pathogenicity in vivo". Paris 7, 2014. http://www.theses.fr/2014PA077199.
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Le virus Chikungunya (CHIKV) est un arbovirus ré-émergent mais aussi l'alphavirus le plus responsable de maladie humaine. La réplication de CHIKV est assujettie d'erreurs ; c'est une caractéristique de tous les virus à ARN, qui présentent les taux de mutation les plus élevés de la nature. Par conséquent, un seul virus génère des millions de descendance virale qui portent de mutations distinctes dans leurs génomes. Ces populations peuvent être décrites comme une quasi-espèce, car la population agira comme (quasi) une unité d'évolution unique (espèces). La diversité de la population virale a des implications importantes dans la biologie du virus à ARN et son évolution, et il a été prouvé que les taux de mutation virale ont été optimisées au fil du temps. Les taux de mutation doivent être suffisamment élevés pour générer de nouveaux variants, ce qui permet une adaptabilité rapide dans des environnements changeants. Toutefois, si les taux de mutation sont trop élevés, l'intégrité du génome est perdue. La découverte récente de variants de fidélité a souligné l'importance du maintien d'un taux de mutation idéal. Les variants de fidélité sont des virus qui présentent des taux de mutation modifiés, entraînant une restriction (antimutateur) ou une expansion (mutateur) des quasi-espèces virales. En général, ces variants contiennent des mutations dans l'ARN polymérase ARN-dépendante (RdRp) qui modifient la fidélité intrinsèque. Mais, on ne sait pas comment la diversité affecte le fitness et le phénotype des arbovirus. Nous avons retrouvé des variants mutateurs de CHIKV avec des fréquences de mutation plus élevés que le virus sauvage et les caractérises in vitro et in vivoArboviruses cycle through both vertebrates and invertebrates, which requires them to adapt to disparate hosts while maintaining genetic integrity during genome replication. To study the genetic mechanisms and determinants of these processes, we use chikungunya virus (CHIKV), a re-emerging human pathogen transmitted by the Aedes mosquito. We isolated novel mutators with decreased replication fidelity and higher mutation frequencies, allowing us to examine the fitness of error-prone arbovirus variants. Although CHIKV mutators displayed no major replication defects in mammalian cell culture, they were attenuated in vivo. Unexpectedly, mutator phenotypes were suppressed in mosquito cells and the variants exhibited significant defects in RNA synthesis. Consequently, these replication defects resulted in strong selection for reversion during inection of mosquitoes. Since residue 483 is conserved among alphaviruses, we examined the analogous mutations in Sindbis virus (SINV), which also reduced polymerase fidelity and generated replication defects in mosquito cells. However, replication defects were mosquito cell-specific and were not observed in Drosophila S2 cells, allowing us to evaluate the potential attenuation of mutators in insect models where pressure for reversion was absent. Indeed, the SINV mutator variant was attenuated in fruit flies. These findings confirm that residue 483 is a determinant regulating alphavirus polymerase fidelity and demonstrate proof of principle that arboviruses can be attenuated in mammalian and insect hosts by reducing fidelity
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Fodor, Ervin. "Studies on the vRNA promoter of influenza A virus". Thesis, University of Oxford, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.294232.
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Kabdulov, Timur O. "Mechanisms of retroviral replication". Morgantown, W. Va. : [West Virginia University Libraries], 2001. https://etd.wvu.edu/etd/controller.jsp?moduleName=documentdata&jsp%5FetdId=2256.
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Molin, Ylva. "Arsenic Influences Virus Replication in Experimental Coxsackievirus B3 Infection". Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis : Univ.-bibl. [distributör], 2010. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-112049.
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Basak, Sanjukta. "Studies of Hepatitis C virus immunology : translation and replication". Thesis, McGill University, 2005. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=97903.
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Hepatitis C virus (HCV) has become a worldwide problem. Roughly 3% of world population are estimated to be infected with the virus, producing high rates of progressive liver disease, leading to cirrhosis and hepatocellular carcinoma. The present therapy, a combined administration of pegylated interferon-alpha (IFN) and ribavirin is costly and only successful in 50% of patients infected with HCV. It is also associated with serious side effects. Thus, there is an urgent need for better tolerated and more effective treatment modalities. A therapeutic vaccine may be the solution.Recent efforts to produce efficient vaccines require not only the identification of potential viral antigens but also vaccine adjuvants or enhancers of immunity. Dendritic cells (DC) are being considered one such adjuvant for the activation of CD4+ and CD8+ T-cells. As potent antigen presenting cells, they are capable of capturing antigens, processing them into peptides, and presenting them on products of the MHC to T cells. For such reasons, peptide loading of antigens onto DCs to enhance T cell responses is becoming of increasing interest. Using cell penetrating peptides, or motifs capable of transporting cargo freely across cell membranes, we have developed a peptide based delivery system suitable for the transport of all HCV proteins into immature DCs. In our studies we demonstrated that 3.1% of immature DCs internalized the reporter cargo, eGFP. This system was then optimized to 53.81 % in target HeLa cells.
Another area of recent focus is the regulation of HCV translation and replication. Positive stranded viruses such as HCV use the genomic RNA as a common template for translation as well as for RNA replication, both proceeding in inverse directions. Thus, specific regulatory mechanisms must be in place in order to coordinate these two antagonistic processes. In this study, we investigated the role of HCV Core protein as a translational inhibitor and enhancer of replication. Using several transient and stable in vivo reporter assays, we showed that Core expression inhibited HCV IRES-mediated translation in trans, in a dose-dependent manner. Furthermore, HCV Core protein is able to dramatically inhibit HCV translation in the Huh7 replicon system, more so than the bicistronic reporter systems tested and subsequently increase total levels of replicon RNA by 1.5 log fold and thus, affect replication. We believe that Core may indeed be the sought regulator of translation and replication.
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Cotton, Sophie. "Characterization and movement of turnip mosaic virus replication complexes". Thesis, McGill University, 2010. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=86558.
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Viruses are intracellular pathogens that use the host cell to produce new infectious progeny. For all positive-strand RNA viruses that have been investigated so far, viral replication takes place in cytoplasmic virus-induced membrane structures. For turnip mosaic virus (TuMV), the generation of vesicles likely associated with the replication complex depends on the synthesis of the viral protein 6K. To further characterize the vesicles formed during TuMV infection, Nicotiana benthamiana plants were agroinfiltrated with a TuMV infectious clone expressing 6K protein fused to GFP. Using confocal microscopy, cytoplasmic aggregates were observed, corresponding to the 6KGFP-induced vesicles which house the viral replication complexes (VRCs). Intracellular movement of these vesicles was visualized by time-lapse imaging. Vesicle trafficking was inhibited when plants were infiltrated with latrunculin B, an inhibitor of microfilament polymerization. The absence of movement had severe effects on viral accumulation. Viral vesicles also aligned with actin filaments. These results indicate that microfilaments are necessary for VRC trafficking which is important for virus infectivity.The biogenesis of viral vesicles was investigated by infecting cells with two recombinant TuMVs producing 6KGFP or 6KmCherry-labelled vesicles. Individual vesicle within a cell contained unique protein products derived from each recombinant demonstrating the origin of a vesicle from a single viral genome. Green and red sectoring was also observed, meaning that vesicles could fuse together. The presence of the eukaryotic translation factors eIF(iso)4E, PABP and eEF1A enclosed in VRC was demonstrated previously by our group. These data combined to a single-genome origin suggest that viral translation occur within these structures. The same host factors were also found to co-localize with the active replicating sites along with viral proteins VPg-Pro, RdRp and CI using immunofluorescence labelling in infected protoplasts. These data bring accumulating evidence for the possible coupling of viral translation and replication.
Les virus sont des parasites intracellulaires qui utilisent la cellule hôte pour produire une nouvelle descendance infectieuse. Pour tous les virus à ARN positif étudiés jusqu'à maintenant, la réplication virale prend place dans des structures membranaires induites par le virus. Chez le virus de la mosaïque du navet (TuMV), la formation de vésicules probablement associées au complexe de réplication dépend de la synthèse de la protéine virale 6K. Afin de caractériser les vésicules formées durant l'infection de TuMV, des plants de Nicotiana benthamiana ont été agroinfiltrés avec un clone infectieux de TuMV exprimant la protéine 6K fusionnée à GFP. Des agrégats cytoplasmiques ont été observés par microscopie confocale, correspondant aux vésicules induites par la protéine 6KGFP et abritant le complexe de réplication virale (VRC). Le mouvement intracellulaire de ces vésicules a été visualisé par imagerie time-lapse. Le trafic des vésicules a été inhibé lorsque les plantes étaient infiltrées avec de la latrunculin B, un inhibiteur de polymérisation des microfilaments. L'absence de mouvement a également conduit à une sévère diminution de l'accumulation virale. Les vésicules colocalisent avec les filaments d'actine. Ces résultats indiquent que les microfilaments sont nécessaires au mouvement des vésicules lequel est important pour l'infection virale.
La biogenèse des vésicules virales a été investiguée en infectant les cellules avec deux clones infectieux de TuMV produisant des vésicules étiquetées par 6KGFP ou 6KmCherry. Des vésicules individuelles contenant des protéines uniques dérivant d'un seul clone recombinant a démontré que l'origine des vésicules provient d'un seul génome. Des vésicules ayant des secteurs vert et rouge ont aussi été observé, indiquant que la fusion de vésicules est possible. La présence des facteurs eucaryotes de traduction eIF(iso)4E, PABP et eEF1A à l'intérieur des vésicules a été démontré par notre groupe. Ces données combinées à l'origine unique des vésicules suggèrent que la traduction virale se produit à l'intérieur de ces vésicules. Les mêmes facteurs de traduction ainsi que les protéines virales VPg-Pro, RdRp et CI colocalisent avec les sites de réplication active dans des protoplastes infectés. Ces données apportent des indices supplémentaires sur la possibilité de couplage entre la traduction et la réplication virale chez TuMV.
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Stirling, Julie M. "Studies on the replication and assembly of bluetongue virus". Thesis, University of Reading, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.362312.
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Mioulet, Valerie. "Analysis of transcription and replication signals in rinderpest virus". Thesis, University of Hertfordshire, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.323458.
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Patel, Hershna. "Evolutionary targeted discovery of influenza A virus replication inhibitors". Thesis, University of Hertfordshire, 2017. http://hdl.handle.net/2299/19623.
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Influenza A is one of the most prevalent and significant viral infections worldwide, resulting in annual epidemics and occasional pandemics. Upon infection, antiviral drugs targeting the neuraminidase protein and M2 protein are the only treatment options available. However, the emergence of antiviral drug resistance is concerning, therefore the aim of this work was to identify inhibitor molecules that may bind to highly conserved regions of selected internal influenza A proteins. Sequences of the non-structural protein 1 (NS1), nuclear export protein (NEP) and polymerase basic protein 2 (PB2) from all hosts and subtypes were aligned and the degree of amino acid conservation was calculated based on Valdar's scoring method. Missing parts of the experimental structures were predicted using the I-TASSER server and ligand binding hot spots were identified with computational solvent mapping. Selected binding sites in conserved regions were subjected to virtual screening against two compound libraries using AutoDock Vina and AutoDock 4. Two out of twelve top hit compounds predicted to target the NS1 protein showed capability of reducing influenza A H1N1 replication in plaque reduction assays at concentrations below 100 μM, although the target protein and mechanism of action could not be confirmed. For the NEP, conservation analysis was based on 3000 sequences and binding hot spots were located in common areas amongst three structures. Docking results revealed predicted binding affinities of up to -8.95 kcal/mol, and conserved amino acid residues interacting with top compounds include Arg42, Asp43, Lys39, Ile80, Gln101, Leu105, and Val109. For the PB2 protein, conservation analysis was based on ~12,000 sequences and fifteen potential binding hot spots were identified. Docking results revealed predicted binding affinities of up to -10.3 kcal/mol, with top molecules interacting with the highly conserved residues Gln138, Gly222, Ile539, Asn540, Gly541, Tyr531 and Thr530. The findings from this research could provide starting points for in vitro experiments, as well as the development of antiviral drugs that function to inhibit influenza A replication without leading to resistance.
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Berthold, François. "Grapevine fanleaf virus replication : viral proteins and host factors". Strasbourg, 2015. http://www.theses.fr/2015STRAJ086.
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Stoner, Terri Dorene. "Indole-3-Carbinol Inhibition of Herpes Simplex Virus Replication". Kent State University / OhioLINK, 2008. http://rave.ohiolink.edu/etdc/view?acc_num=kent1228328838.
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Baillie, Andrew James. "Cellular and viral determinants for hepatitis C virus replication". Thesis, Imperial College London, 2010. http://hdl.handle.net/10044/1/6865.
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The recent discovery of an HCV isolate which replicates in cell culture has opened up opportunities to study the full viral life cycle in vitro. This genotype 2a isolate (JFH-1) and its derivatives are the only ones known to replicate efficiently in cell culture, and recent work has indicated that viral determinants for efficient replication may lie in the non-structural protein coding region of the genome. In this thesis chimaeric JFH-1 virus containing full length NS3, NS3 helicase and NS3 protease sequences from genotype 1a and 1b were constructed. The replication efficiencies of chimaeric viruses were tested in cell culture, and were shown not to replicate, indicating that vital viral determinants for JFH-1 replication exist in NS3. The JFH-1 model also provides the opportunity to study the effect of the full viral life cycle on the host cell. Microarray analyses were performed to identify gene expression changes in Huh7 and Huh7.5 cells that had been infected with JFH-1 for 6, 12, 18, 24 and 48 hours. A large number of host genes were found to be regulated during JFH-1 infection, including those involved in lipid metabolism, oxidative stress, apoptosis and intracellular transport. The microarray data were validated by quantitative PCR analyses of separate infection experiments. A selection of the most highly regulated genes was assessed for their necessity to HCV replication by RNA interference studies. The knockdown by siRNA of genes ABLIM3, SPTLC3 and CYP1A1 resulted in significant impairment of HCV replication. The knockdown by siRNA of gene TXNIP (thioredoxin interacting protein) resulted in up to 90% reduction in HCV replication. This is a novel finding which may be of importance to the study of HCV as TXNIP plays roles in oxidative stress, lipid metabolism and glucose metabolism, all of which have potential to influence the HCV lifecycle. Magnetic resonance spectroscopy indicated a change in levels of choline metabolites in JFH-1 infected cells, which has implications for the aspects of the HCV lifecycle associated with lipid membranes and other lipid structures.
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Rosskopf, John J. "CIS-acting signals for replication of Nodamura virus RNA1". To access this resource online via ProQuest Dissertations and Theses @ UTEP, 2009. http://0-proquest.umi.com.lib.utep.edu/login?COPT=REJTPTU0YmImSU5UPTAmVkVSPTI=&clientId=2515.
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Clark, Hayley. "The effect of iron on hepatitis C virus replication". Thesis, Clark, Hayley (2011) The effect of iron on hepatitis C virus replication. Honours thesis, Murdoch University, 2011. https://researchrepository.murdoch.edu.au/id/eprint/7179/.
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Hepatitis C virus (HCV) is a small RNA encoded virus that causes acute and chronic infections of the liver and is associated with fibrosis, cirrhosis, hepatocellular carcinoma and end-stage liver disease. The infection is linked with elevated serum ferritin levels and iron deposits within the liver which are associated with an increased risk of progression of HCV disease. It is hypothesised firstly that the presence of excess iron affects HCV replication and secondly that iron homeostasis is altered by HCV replication. The main aim of this study was to investigate the influence of iron on HCV replication by analysing the spread of infection and the titre of infectious HCV produced in the presence of excess iron. The second aim was to determine if HCV infection itself alters the level of iron stored as ferritin in liver cells.
HCV Japanese fulminant hepatitis (JFH-1) infection of the human hepatoma cell line, (Huh7), was used as the model for this study. The culture conditions were first optimised to allow detection of ferritin by western blot. Huh7 cells were then treated with iron provided by 5, 10 or 20 μg/ml ferric ammonium citrate (FAC) prior to HCV infection (0.001 MOI). No evidence of inhibition of HCV replication was found on day 7 post infection (PI) by analysis of the level of viral protein detected by western blot, the proportion of infected cells determined by immunohistochemistry and flow cytometry or the titre of infectious HCV produced. A comparison of iron donors was then undertaken and conditions were changed to add iron donors 1 day after HCV. Viral RNA levels were not significantly reduced on treatment with 20 μg/ml FAC and 25 μM holo-transferrin and assays for viral protein and production of infectious virus again showed no evidence of inhibition. Fe-PIH, a lipophilic iron donor, had the greatest effect, reducing both the proportion of infected cells and the titre of infectious virus produced. When the reduction in the number of cells present in cultures treated with Fe-PIH was taken into account, the decrease in virus titre was explained.
HCV infection was found to alter iron storage in Huh7 cells. A significantly lower level of ferritin was detected (p < 0.05) by western blot in HCV infected compared to uninfected Huh7 cells whereas in other situations highly infected cells were found to have increased iron storage. While it had been intended to analyse iron metabolism by examining gene expression during iron deficiency and iron storage, the conditions which give rise to these outcomes will need to be elucidated first.
This study has shown that iron has the potential to inhibit HCV replication although in vitro it depends on the type and concentration of iron donor used and the time during infection that excess iron is present in the cell. This study has confirmed that HCV infection alters the iron status of the liver cell. The outcome of these interactions between iron and HCV is expected to influence the course of HCV infection and the development of liver disease.
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Piccininni, Sabina. "Structure and function of Hepatitis C virus replication complex". Thesis, University of Manchester, 2004. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.488274.
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Hafner, Gregory. "Replication of banana bunchy top virus : mechanisms and interference". Thesis, Queensland University of Technology, 1998.
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Dirks, Clarissa A. "The role of cellular factors in retrovirus replication /". Thesis, Connect to this title online; UW restricted, 2001. http://hdl.handle.net/1773/5072.
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駱淑芳 i Suk-fong Anna Lok. "Replication of hepatitis B virus in Chinese patients with chronic hepatitis B virus infection". Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 1990. http://hub.hku.hk/bib/B31981392.
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Brett, Katharina. "Molecular requirements of influenza virus hemagglutinin for site-specific S-acylation and virus replication". Doctoral thesis, Humboldt-Universität zu Berlin, Lebenswissenschaftliche Fakultät, 2015. http://dx.doi.org/10.18452/17274.
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Das Hämagglutinin (HA) des Influenzavirus ist post-translational durch S-Acylierung von drei Cysteinen modifiziert. Zwei davon befinden sich in seiner zytoplasmatischen Domäne (CD) und enthalten Palmitat und eines am Cytosol-zugewandten Ende der Transmembranregion (TMR) wird bevorzugt mit Stearat acyliert. Es wird vermutet, dass entweder die Aminosäureumgebung der Acylierungsstelle oder dessen Lage relativ zur Membran bestimmt welcher Fettsäuretyp angeheftet wird. Diese Acylierungstellen sind zudem essentiell für die Virusreplikation. Ob auch andere Aminosäuren der CD essentiell sind, ist nicht bekannt. Nach einem umfangreichen Sequenzvergleich zur Identifikation konservierter Aminosäuren wurden rekombinante Viren mit Aminosäureaustauschen in der Nähe der drei Acylierungstellen hergestellt. Diese Austausche enthielten Punktmutationen, Verschieben des TMR Cysteins in die CD sowie die Deletion der gesamten CD. Viren ohne CD und ein Austausch neben einem acylierten Cystein verhinderten die Virusreplikation. Eine konservative Substitution derselben Position, andere Austausche in TMR und CD sowie das Schieben des TMR-Cysteins in die CD dagegen beeinflussten das Viruswachstum nur schwach. Einige der mutierten Codons revertierten zur ursprünglichen oder einer neuen Aminosäure. Rekombinante Viren wurden in MDCK-Zellen und embryonierten Hühnereiern vermehrt und mittels Massenspektrometrie analysiert. Es wurden keine unteracylierten Peptide detektiert, und selbst die zwei Letalmutationen behielten die Acylierung. Punktmutationen beeinträchtigten nur mäßig den Stearat-Gehalt, wogegen die Verlagerung des TMR-Cysteins in die CD die Stearylierung praktisch eliminierte. Mehr Stearat wurde angeheftet, wenn humane Viren in Säugerzellen im Vergleich zu aviären Zellen angezüchtet wurden. Die Position einer Acylierungsstelle repräsentiert relativ zur TMR-Spanne das Hauptsignal der Stearylierung während der Sequenzkontext und der Zelltyp das Fettsäuremuster modulieren.Influenza virus’s hemagglutinin (HA) is post-translationally modified by S-acylation of three cysteines. Two are located in its cytoplasmic tail (CT) and contain palmitate and one at the end of the transmembrane region (TMR) is acylated primarily with stearate. It is hypothesized that either the acylation site’s amino acid environment or its location relative to the membrane determines which type of fatty acid is attached. Additionally, these acylation sites are essential for virus replication. Whether other amino acids in the CT are required for virus replication, is not known. Based on a comprehensive sequence comparison to identify conserved amino acids, recombinant viruses with amino acid substitutions in the vicinity of HA’s acylation sites were created. These substitutions included point mutations, shifting of a TMR cysteine to the CT and the deletion of the entire tail. The truncated tail mutation and a substitution adjacent to an acylated cysteine disabled virus replication. In contrast, a conservative substitution at this position, other exchanges in TMR and CT and moving the TMR cysteine to the CT had only subtle effects on virus growth. Yet, some of the mutated codons reverted to the original or other amino acids. Recombinant viruses were propagated in MDCK cells and embryonated chicken eggs and analyzed by mass spectrometry. No under-acylated peptides were detected, even the two lethal mutations did not abolish acylation. Point mutations only moderately affected the stearate content, while relocating the TMR cysteine to the CT virtually eliminated attachment of stearate. More stearate was attached if human viruses were grown in mammalian compared to avian cells. Hence, the location of an acylation site relative to the TMR represents the principal signal for stearate attachment, while the sequence context and the cell type modulate the fatty acid pattern.
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Najmabadi, Hossein. "Characterization of the Self-Replicating Kirsten Murine Leukemia Viral DNA: Replication and Tetracycline Resistance". Thesis, University of North Texas, 1989. https://digital.library.unt.edu/ark:/67531/metadc798479/.
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This research project deals with the characterization of self-replicating Kirsten murine viral DNA. The replication of this viral DNA and tetracycline resistance conferred to bacteria by this viral DNA will be studied. The restriction endonuclease and Southern blot analysis revealed a fragment of pBR322 from the Hind III and Pst I site that is located in the 3' end of the MLV-K:E molecule. Single stranded sequencing of the two terminal ends of this fragment verified that the 3' end of MLV-K:E contains identical sequence homology to pBR322. The presence of this pBR322 fragment explains the unusual properties of the MLV-K:E molecule. However, tetracycline resistance is less in E. Coli containing MLV-K:E than E. coli containing pBR322 as determined by zone of inhibition assay. This may be due to alteration in the promoter region of the tetracycline gene.
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Roy, Dominic. "Improving the Delivery and Replication of Oncolytic Viruses". Thesis, Université d'Ottawa / University of Ottawa, 2016. http://hdl.handle.net/10393/35227.
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The optimal route for clinical delivery of oncolytic viruses (OVs) is thought to be intravenous (IV) injection; however, the immune system is armed with several highly efficient mechanisms to remove pathogens from the circulatory system. To overcome the challenges in trying to deliver OVs IV, cell carriers have been investigated to determine their suitability as delivery vehicles for systemic administration of OVs. Here we demonstrate the utility of a Drosophila melanogaster cell platform for the production and in vivo delivery of multi-gene biotherapeutic systems. We show that cultured Drosophila S2 cell carriers can stably propagate OV therapeutics that are highly cytotoxic for mammalian cancer cells without adverse effects on insect cell viability or cellular gene expression. Drosophila cell carriers administered systemically to immunocompetent animals trafficked to tumours to deliver multiple biotherapeutics with little apparent off-target tissue homing or toxicity, resulting in a therapeutic effect. S2 cells provide a genetically tractable platform supporting the integration of complex, multi-gene biotherapies while avoiding many of the barriers to systemic administration of mammalian cell carriers.
Once OVs are delivered to tumour beds, they initiate replication in tumour cells, which often possess defects in antiviral pathways and are thus susceptible to infection. However, not all tumours have defects in their antiviral defenses and thus virus replication in these tumours is rather limited. Identifying and modulating host factors that regulate virus replication in OV-resistant cancer cells, but not normal cells, could lead to increased replication in these tumours and potentially improve therapeutic outcomes. We therefore conducted an RNA interference screen using Sindbis virus (SINV) in order to identify host factors that modulate OV replication in tumour cells. Specifically, serial passage of a SINV- artificial microRNA (amiRNA) library in a tumour cell line followed by deep sequencing of
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the selected virus populations led to the identification of several amiRNA sequences that were enriched. Furthermore, the identified amiRNA sequences increased the replication of various OVs both in vitro and in vivo, ultimately resulting in an enhanced therapeutic effect.
Overall, the work presented here highlights strategies in which both the systemic delivery and tumour-specific replication of OVs can be improved.
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Morgan, Rachel E. "Is the Cytoskeleton Necessary for Viral Replication?" Digital Archive @ GSU, 2012. http://digitalarchive.gsu.edu/biology_theses/38.
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The cytoskeleton plays an important role in trafficking proteins and other macromolecular moieties throughout the cell. Viruses have been thought to depend heavily on the cytoskeleton for their replication cycles. However, studies, including one in our lab, found that some viruses are not inhibited by anti-microtubule drugs. This study was undertaken to evaluate the replication of viruses from several families in the presence of cytoskeleton-inhibiting drugs and to examine the intracellular localization of the proteins of one of these viruses, Sindbis virus, to test the hypothesis that alternate pathways are used if the cytoskeleton is inhibited. We found that Sindbis virus (Togaviridae, positive-strand RNA), vesicular stomatitis virus (Rhabdoviridae, negative-strand RNA), and Herpes simplex virus 1 (Herpesviridae, DNA virus) were not inhibited by these drugs, contrary to expectation. Differences in the localization of the Sindbis virus were observed, suggesting the existence of alternate pathways for intracellular transport.
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MAENO, KOICHIRO. "Replication of Influenza B Virus: Biological Functions of Viral Neuraminidase". Nagoya University School of Medicine, 1994. http://hdl.handle.net/2237/15935.
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au, M. Stewart@murdoch edu, i Meredith Stewart. "An investigation into aspects of the replication of Jembrana disease virus". Murdoch University, 2005. http://wwwlib.murdoch.edu.au/adt/browse/view/adt-MU20051222.104106.
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Jembrana disease virus (JDV) is an acutely pathogenic lentivirus affecting Bali cattle in
Indonesia. The inability to propagate the virus in vitro has made it difficult to quantitate
JDV and determine the kinetics of virus replication during the acute disease process.
Reported for the first time are 2 techniques that enable quantification of the virus, and
the use of these techniques to quantify the virus load in plasma of cattle during the
acute disease process. The 2 techniques were a qualitative one-step JDV real-time
reverse-transcription polymerase chain reaction (qRT-PCR) assay for the detection
and quantification of JDV RNA, and a JDV p26 capture ELISA for the detection and
quantification of JDV capsid protein. The limit of detection of the qRT-PCR was 9.8 x
102 JDV viral RNA copies over 35 cycles, equivalent to 4.2 x 104 JDV genome
copies/ml, and a peak virus load of 1.6 x 1012 JDV genome copies /ml during the acute
febrile period. Viral RNA and JDV p26 levels were correlated in 48 plasma samples
obtained from experimentally infected cattle. A significant positive correlation (R =
0.835 and r2 = 0.697) was observed between the 2 techniques within the range of their
detection limits, providing a solid basis for the use of the economical capture ELISA to
quantify JDV load when real-time PCR capability is not available. The detection of
JDV p26 by capture ELISA was, however, much less sensitive than the real-time RTPCR
with a detection limit equating to approximately 1 x 108 JDV genome copies/ml.
The transcriptional pattern of JDV during the acute phase of infection was studied by
RT-PCR, sequencing and northern blot analysis. Analysis revealed a complex pattern
of transcription with the identification of 14 transcripts, which confirmed 6 predicted
splice sites and the identified 7 splice sites not reported previously. A small 78 bp
putative non-coding exon was identified that shared the same splice acceptor as vif
and was associated with the alternative transcripts of tat, rev and env. Four tat, 3 rev
and 2 env transcripts were identified. The rev and env transcripts were demonstrated
to use the same splice site. The study confirmed that the production of a tmx
transcript, a unique gene identified in the two bovine lentiviruses JDV and Bovine
immunodeficiency virus (BIV). Northern blot analysis identified 11 of the 14 transcripts
identified by RT-PCR, including a 7.8 kb gag/pol primary transcript and singly spliced
transcripts. The complexity of the transcript map produced suggested that JDV
replication is a highly regulated process.
One of the aims of this thesis was to determine the functional role of the Tmx and Vif
accessory proteins of the bovine lentiviruses. Although this aim was not achieved,
molecular reagents were produced that will allow these investigations to proceed. The
Vif and Tmx proteins of both JDV and BIV were successfully expressed as C-terminal
fusions with glutathione S-transferase (GST) using the pGEX-6P-1 bacterial
expression system. The recombinant proteins were purified and were recognised by
both BIV and JDV antisera from Bos taurus and Bos javanicus respectively, and by
antibody in sera from cattle that had been vaccinated with a tissue-derived JDV
vaccine and also those that had been naturally infected with JDV. The Vif, Tmx and
Rev proteins of JDV and vif BIV were successfully expressed in a Rev-independent
manner in COS7 and bovine macrophage cells using a pcDNA3.1® mammalian
expression system. Cellular localisation of the recombinant viral proteins varied in the
2 cell types: in COS7 cells, both JDV and BIV Vif were detected predominantly in the
nucleus, whereas in bovine macrophage cells BIV Vif localised in the cytoplasm and
JDV Vif localised in the cytoplasm and nuclear membrane. JDV Tmx localised in the
cytoplasm of COS7 cells but the nuclear membrane of bovine macrophage cells, and
BIV Tmx localised in the nucleus and nuclear membrane in both cell types and
appeared to affect the morphology of the nucleus. Mutations of vif and tmx were also
successfully engineered into an infectious clone of BIV and these mutated clones will
provide a valuable resource for further investigation of the role of Vif and Tmx in
replication of the bovine lentiviruses.
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Huang, Jianhe. "Molecular dissection of the Spodoptera littoralis nucleopolyhedrovirus : virus-host cell interaction and virus DNA replication". Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape2/PQDD_0008/NQ52762.pdf.
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Horscroft, Nigel John. "Orbivirus non-structural protein NS2 : its role in virus replication". Thesis, University of Oxford, 1997. http://ora.ox.ac.uk/objects/uuid:9b550db6-dd9d-4127-941f-93eab2b6e038.
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Gowans, E. J. "Studies on markers of hepatitis B virus replication in man /". Title page, contents and summary only, 1986. http://web4.library.adelaide.edu.au/theses/09PH/09phg722.pdf.
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Shaw, Andrew Edward. "The Role of Non-Structural Proteins in Bluetongue Virus Replication". Thesis, University College London (University of London), 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.519459.
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Escaler, Margarita. "Changes in host gene expression associated with plant virus replication". Thesis, University of East Anglia, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.302215.
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Hofer, Julie M. I. "Regulation of gene expression and replication in wheat dwarf virus". Thesis, University of East Anglia, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.334331.
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Desmond, Elizabeth. "The interference of influenza virus replication by subgenomic ribonucleic acid". Thesis, University of Reading, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.339508.
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Abdulsattar, Ban Oday. "Coronavirus proteins and their directed evolution to inhibit virus replication". Thesis, University of Reading, 2017. http://centaur.reading.ac.uk/73736/.
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Coronaviruses are enveloped, positive sense, RNA viruses that infect many species of animals, including humans. Of the six coronaviruses that can infect humans, SARS-CoV and MERS-CoV are the etiological agents of most concern currently. Coronaviruses possess the most complex and largest RNA genomes among all RNA viruses. The genome contains up to 15 genes with multiple open reading frames (ORFs) encoding both structural and non-structural proteins. Coronaviruses encode about 30 proteins that play specific, and often essential, roles in viral replication and assembly. This thesis presents work done to express Murine hepatitis virus strain A59 (MHVA59) proteins such as Nucleoprotein and membrane genes, non-structural proteins (5,6,7,8,9,10,16) from gene1, part of non-structural proteins (Plpro, Y-domain from nsp3 and N-terminal from nsp12) and RdRp from C-terminal part of nsp12 in E. coli BL21 cells and mammalian 17clone-1 cells, the latter of which are permissive for MHV-A59. The efficiency of transfection and expression of the proteins in mammalian cells was evaluated. SUMOStar (small ubiquitin-like modifier) fusion technology was used to enhance protein expression in the eukaryotic system. Expressed proteins were detected by Western blot with an anti-His tag antibody. The ability of virus-expressed proteins to interfere with virus infection was tested and an inhibitory effect was detected by plaque assay. The coronavirus nucleoprotein (N) is an important component for both viral replication and transcription. Error-prone PCR (ep-PCR) was used with the N protein as template to introduce random error and the number of mutations introduced was calculated after 100 random colonies were sequenced to validate the mutagenesis. Transient expression of N protein was shown to increase the efficiency of infection and virus yield. The function of N was investigated by screening for dominant-negative N mutants, using a library of N variants constructed using ep-PCR. The cytotoxicity of N variants was tested by MTT assay. Expressed N variants showed a range of effects ranging from a 10-fold increase in virus yield associated with the wild type N to 10-fold inhibition of virus growth. One particular N variant, mut38, was non-toxic, but reproducibly inhibited virus growth. The potential to screen for dominant-negative N variants using cell survival was also assessed using different N libraries. The thesis also investigated different strategies aimed at purification of non-structural protein 16 (nsp16). The overall findings suggest an ability of virus-expressed proteins in eukaryotic cells to interfere with virus infection and demonstrate that such antiviral activity can be generated by mutating an important viral protein.
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Dewar, Rebecca Amy. "Targeting cellular nuclear export to inhibit influenza A virus replication". Thesis, University of Edinburgh, 2018. http://hdl.handle.net/1842/31349.
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Influenza A virus (IAV) is a global health threat, causing seasonal epidemics and potential pandemics leading to morbidity, death and economic losses. Currently, there are two main classes of licensed antivirals against IAV available in the US and Europe; adamantanes and neuraminidase inhibitors, both of which are hindered by the generation of resistant virus variants. The viral polymerase has a high error rate leading to mutations that allow the virus to overcome selection pressures directed at its own genome from conventional antivirals. The prospect of inhibiting host proteins that the virus exploits to facilitate its replication is of increasing interest as an antiviral strategy as the emergence of resistance has been predicted to be slower when targeting a host cellular factor. IAV utilizes the host nuclear export protein CRM1 to transport viral ribonucleoproteins (vRNPs) from the nucleus to the cytoplasm of an infected cell, a critical late stage of the influenza lifecycle. Leptomycin B (LMB), a Streptomyces metabolite, has been previously shown to target this pathway, resulting in reduced viral propagation; however, LMB's potent cytotoxicity has limited its use as a therapeutic agent. This thesis examined two novel selective inhibitors of nuclear export (SINE), KPT-335 and KPT-185, with less cytotoxicity. In vitro, KPT-335 inhibited replication of human and animal IAV strains in a dose-dependent manner with minimal cytotoxicity. To assess the resistance potential of KPT-335, IAV viruses were serially passaged in the presence of a sub-optimal concentration of the compound and assayed for the development of resistance. Resistance to KPT-335 became evident at 8-10 rounds of passage. Sequencing analysis of independently derived resistant virus clones identified 4 single amino acid changes on a surface exposed patch of the viral nucleoprotein (NP). Introduction of these amino acid changes, into otherwise wild type viruses by reverse genetics, confirmed that changes Q311R and N309T conferred a drug-resistant phenotype. However, these substitutions came at a fitness cost to virus replication. The molecular basis for resistance was unclear but Q311R and N309T NP-mutant viruses produced increased levels of M1 during infection as well as producing virus particles with increased M1:NP ratios. Furthermore, the KPT-335-resistance mutations were surprisingly similar to NP sequence polymorphisms previously associated with susceptibility to the innate defence protein MxA. Consistent with this, viruses harbouring the Q311R mutation displayed increased susceptibility to MxA inhibition compared to wild-type virus. Altogether this study confirms that SINEs have the potential to be successful therapeutic agents against IAV replication and that although resistance could be generated, it may be difficult for the virus to overcome both drug selection pressures and the human innate immune response restrictions by escape mutations.
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Willment, Janet Anne. "Investigations of the molecular determinants of maize streak virus replication". Doctoral thesis, University of Cape Town, 1999. http://hdl.handle.net/11427/9869.
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Includes bibliographical references.Geminiviruses replicate via a rolling circle mechanism, which initiates at the origin of replication located within the long intergenic region (LIR). The viral replication associated-protein (Rep) in conjunction with the host's DNA replication machinery is responsible for the initiation and termination of the replication cycle from a stem-loop structure, located within the LIR and conserved throughout the three genera of Geminiviridae. The specific interaction between the Rep protein with sequences within the intergenic region has been well characterised for the begomoviruses and to some extent the curtoviruses; however, this interaction in the mastreviruses, and in particular maize streak virus (MSV), has yet to be fully explored. A theoretical model has been proposed based on sequence data and informed by the current understanding of replication specificity in begomoviruses. Due to the lack of conservation of the stem sequence of the stem-loop structure amongst mastreviruses, the model implicates a pair of nucleotide sequence repeats called iterons. These are located within the stem structure, and on the complementary sense side of the LIR. The former is the putative site of Rep interaction with the LIR. These iterons would therefore potentially act as the determinants of replication specificity amongst mastreviruses.