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Branfield, Lauren Elizabeth. "Structural and biochemical analysis of protein/RNA interactions during the initiation of dengue virus genome replication." Thesis, University of Leeds, 2018. http://etheses.whiterose.ac.uk/21365/.
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Williams, Kelley J. "Silver Nanoparticles Inhibit the Binding and Replication of Dengue Virus." Wright State University / OhioLINK, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=wright1431880664.
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Trist, Iuni Margaret Laura. "In silico design of novel inhibitors of dengue virus replication." Thesis, Cardiff University, 2014. http://orca.cf.ac.uk/71338/.
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Dengue virus (DENV) is a health burden responsible of 50-100 million new cases and 22,000 deaths per year and its four serotypes are worryingly spreading out of the endemic regions. Current therapy is symptomatic, making antiviral research on DENV an unmet need. Vaccine development is more challenging than expected, so the development of anti-DENV drugs is particularly important for infection management. DENV is a positive sense single stranded RNA virus that replicates within cells exploiting both host and viral enzymes to replicate. Based on the hypothesis that DENV infection can be stopped with the inhibition of one or more of the enzymes that are fundamental for its replication, the aims of the studies reported in this PhD thesis were to: identify novel targets to combat DENV infection, generate new basic knowledge and discover potential novel chemical leads exploiting those targets. Novel approaches combining molecular modelling techniques, classical Medicinal Chemistry approaches, chemical synthesis and in& vitro assays were applied to four essential viral-encoded proteins: the capsid (C), the NS3 NTPase/helicase (NS3hel), the NS5 methyltransferase (NS5 MTase) and the NS5 RNA-dependent RNA polymerase (NS5 RdRp). Novel understanding of the 3'-5' translocation mechanism of NS3hel along the RNA has been hypothesised, increasing awareness about DENV-encoded proteins. Important knowledge on the mode of action of promising antiviral compounds has been acquired, for example that ST-148 stabilises C protein-protein interactions and that published N-sulphonylanthranilic acid RdRp inhibitors bind to a unique allosteric site. Novel promising DENV inhibitor scaffolds have also been developed and the chemical synthesis of one of them has been described, showing that the adopted drug discovery approaches are suitable starting points for the development of anti-DENV medicines. The results obtained represent a significant contribution to DENV research in increasing basic knowledge and in identifying good chemical leads for future work.
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Tran, Tuan Anh. "Screening against the dengue virus polymerase." Thesis, Aix-Marseille, 2016. http://www.theses.fr/2016AIXM4006.
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La dengue, une des maladies les plus largement émergents actuellement, avec 390 millions d'infections chaque année (OMS), est causée par le virus de la dengue contre lequel il n’existe pas de traitements. La protéine NS5 a un rôle important dans le cycle de réplication. Cette protéine se compose d'une méthionine S-transférase d’adénosyl en N-terminal et une ARN polymérase dépendante de l'ARN (RdRp) en C-terminal. Cette NS5 RdRp peut catalyser non seulement la synthèse du brin négatif de l'ARN, utilisé comme matrice pour synthétiser l'ARN brin plus-supplémentaire, mais aussi pour la synthèse d'un ARN complémentaire à partir d'une matrice court e d'ARN sans amorce (de novo). Dans ce travail de thèse, nous présentons la production et le test de l'activité de la protéine NS5, ainsi que du domaine polymérase RdRp pour les quatre sérotypes du virus de la dengue en développant un nouveau test enzymatique, en utilisant comme un réactif fluorescent. L'utilisation de ce réactif fluorescent a également contribué à la détermination des conditions optimisées pour développer un essai de criblage de l'activité polymérase pour identifier des inhibiteurs contre le virus de la dengue. En outre, quatre flavonoïdes, Hinokiflavone, apigénine, la quercétine et Amentoflavone ont montré des valeurs d’IC50 équivalentes contre toutes les constructions NS5 et les domaines polymérase des quatre sérotypesDengue fever, one of the most widely emerging diseases nowadays with 390 million infections each year (WHO), is caused by Dengue virus in which no official antiviral reagent or vaccine is available. The NS5 protein has an important role in the replication cycle. This protein consists of a S-adenosyl methionine transferase at N-terminal and a RNA dependent RNA polymerase (RdRp) at C-terminal. This NS5 RdRp can catalyse for not only synthesis of minus-strand RNA to be used as the template to synthesize additional plus-strand RNA but also synthesizing a complement RNA from a short RNA template without primer (de novo). In this research we present the production and activity test for NS5 protein and N-terminal extended sequence 266-900 from NS5 RdRp of all first four serotypes of Dengue virus and a construct of sequence 273-900 using a new enzymatic assay, using Picogreen as fluorescent reagent. Using this fluorescent reagent also helped determining the optimised conditions to develop a screening assay for inhibitors against dengue polymerase activity. In addition, four flavonoids, Hinokiflavone, Apigenin, Quercetin and Amentoflavone showed approximate IC50 values when testing on all NS5 and polymerase protein constructs of all four serotypes
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Deng, Ruitang. "Molecular cloning, nucleotide sequencing and genome replication of bovine viral diarrhea virus /." The Ohio State University, 1992. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487779914825349.
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Yousuf, Amjad. "High-throughput quantitative proteomic analysis of host proteins interacting with dengue virus replication complex." Thesis, University of Bristol, 2016. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.702423.
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Dengue virus (DENV) is a global health problem with approximately 390 million infections annually. Currently, no completely effective vaccines or medications are available to prevent DENV infection. This study used high-throughput quantitative proteomic analysis to identify cellular proteins that modulate DENV replication and are potential targets for the development of antiviral strategies. DENV is known to replicate and package its genome in association with perinuclear ER membranes. Initially, experimental conditions were optimised to infect human Huh-7 liver cells at high efficiency, with DENV serotypes -2 and -4 and to isolate a subcellular heavy membrane fraction (16K) enriched in the DENV replication complex. Then, stable isotope labelling by amino acids in cell culture (SILAC) coupled with high-throughput mass spectrometry (MS) was used to identify changes in the Huh-7 cell proteome and the subcellular 16K fraction in response to DENV-2 and DENV-4 infection. The analysis led to the identification and quantification of 3650 and 4026 and 3461 and 3668 proteins in the 16K and total cell proteomes from DENV-2 and DENV-4 infected cells respectively. For comparison, the total cell proteomes were also analysed by tandem mass tagging combined with MS. Proteomic bioinformatics was done on the datasets which showed that DENV modulated various cellular pathways including; protein biosynthesis, the secretory pathway, lipid metabolism and the cell cycle. A comparison of changes in the total and 16K proteomes from DENV infected cells identified a number of cellular proteins that selectively increased in the replicase containing fraction. which was validated by Western blotting and immunofluorescence analysis. Seven proteins (APOB, ARFRP1, BAG2, GOLGA1, GOLGB1, GOSRI and TMED9) were further investigated for their role in DENV -2 replication by examining either viral or replicon replication in cells depleted of the proteins by siRNA knockdown which revealed proteins that both positively and negatively modulated DENV replication.
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Beeharry, Yasnee. "Role of RNA Genome Structure and Paraspeckle Proteins In Hepatitis Delta Virus Replication." Thesis, Université d'Ottawa / University of Ottawa, 2016. http://hdl.handle.net/10393/35343.
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The Hepatitis Delta Virus (HDV) is an RNA pathogen that uses the host DNA-dependent RNA polymerase II (RNAP II) to replicate. Previous studies identified the right terminal domain of genomic polarity (R199G) of HDV RNA as an RNAP II promoter, but the features required for HDV RNA to be used as an RNA promoter were unknown. In order to identify the structural features of an HDV RNA promoter, I analyzed 473,139 sequences representing 2,351 new R199G variants generated by high-throughput sequencing of a viral population replicating in 293 cells. To complement this analysis, I also analyzed the same region from HDV sequences isolated from various hosts. Base pair covariation analysis indicates a strong
selection for the rod-like conformation. Several selected RNA motifs were identified,
including a GC-rich stem, a CUC/GAG motif and a uridine at the initiation site of
transcription. In addition, a polarization of purine/pyrimidine content was identified, which might represent a motif favourable for the binding of the host Polypyrimidine tract-binding protein-associated-splicing-factor (PSF), p54 and Paraspeckle Protein 1 (PSP1). Previously, it was shown that R199G binds both RNAP II and PSF, that PSF increased the HDV levels during in vitro transcription and that p54 binds R199G. In the present study, I showed that PSP1 also associates with HDV RNA and I investigated whether these proteins are required for HDV replication. My results show that knockdown of PSF, p54 and PSP1 resulted in a decrease of HDV accumulation. These proteins are highly concentrated in paraspeckles, which are nuclear structures involved in storage of transcripts generated by RNAP II. I found that upon viral replication in 293 cells, PSP1 appeared as bigger foci present outside of the
nucleus, while PSF and p54 foci remained in the nucleus. NEAT1 is a long non-coding RNA essential for the formation of paraspeckles. Upon HDV replication, I found an increase of the intensity and size of NEAT1 foci that correlates with an increase of NEAT1 transcripts. Altogether, these data suggest that HDV replication results in an alteration of the paraspeckles structures, providing foundation for further investigation of the paraspeckles role in HDV cycle. Overall, the present study addresses the importance of the HDV RNA structure and of the host paraspeckle proteins for HDV replication.
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Stirrups, Kathleen Elizabeth. "A defective-RNA expression vector for targeted recombination of the coronavirus infectious bronchitis virus." Thesis, University of Reading, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.285965.
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Hellen, C. U. T. "Studies on the structure, replication and expression of the cherry leaf roll virus genome." Thesis, University of Oxford, 1987. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.305712.
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Mullin, Anne Elizabeth. "Factors affecting the balance between transcription and replication of the influenza A virus genome." Thesis, University of Cambridge, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.615957.
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Turrell, Lauren. "The role of nucleoprotein in transcription and replication of the influenza A virus genome." Thesis, University of Oxford, 2015. https://ora.ox.ac.uk/objects/uuid:abe7310c-9b7e-4023-86a2-bae0182adb2f.
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The eight vRNA genome segments of influenza A virus are bound by the viral RNA polymerase at the 5' and 3' ends, and associated with oligomeric nucleoprotein (NP) to form viral ribonucleoprotein (vRNP) complexes. These vRNP complexes carry out both viral transcription and replication within the nucleus of the host cell. The role of NP in viral transcription and replication, although essential, is not well understood. The findings presented in this thesis support a role for NP in the elongation phase of viral transcription and replication, but suggest that NP does not play a role in the regulation of initiation or termination of replication and transcription by the viral RNA polymerase. For NP to support elongation, it has to be recruited onto the viral RNA and oligomerise, to form a RNP complex. NP is likely to exist in a monomeric state, in the infected cell, prior to being recruited into the vRNP. The data presented here demonstrates that NP recruitment onto the viral RNA is mediated through NP-NP homooligomerisation and that oligomerisation of NP, within the infected cell, is regulated by reversible phosphorylation within the oligomerisation domain. In addition to this, compatibility between NP and the viral polymerase is essential for NP to support RNA polymerase activity. The N-terminus of NP is not required for this compatibility; however, evidence points towards loops within NP, responsible for RNA and polymerase binding, playing a role in the interplay between the RNA polymerase and NP within the RNP complex. Overall, this work provides new mechanistic insights into the assembly and regulation of viral RNP complexes.
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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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Domingues, Patricia da Silva. "Control of hepatitis C virus genome replication by viral factors and mediators of host intrinsic immunity." Thesis, University of Glasgow, 2013. http://theses.gla.ac.uk/4933/.
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Potisopon, Supanee. "Insights into the RNA polymerase activity of the dengue virus NS5." Thesis, Aix-Marseille, 2014. http://www.theses.fr/2014AIXM5019.
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Le virus de la dengue cause une maladie de type grippal qui peut dans certains cas évoluer vers des fièvreshémorragiques mortelles. Mon projet de thèse porte sur la réplication de ce virus. Je focalise sur la compréhension du mécanisme d'action de la protéine NS5 de ce virus. La protéine contient 2 domaines : 1) domaine méthyltransférase, essentiel pour la traduction des protéines virales, 2) domaine polymérase, synthétisant le génome ARN du virus. Premièrement, nous avons démontré que la polymérase joue un rôle principal dans la conservation de l'extrémité 3' et 5' du génome et de l'anti-génome. Puis, j'ai caractérisé l'influence du domaine méthyltransférase sur l'activité polymérase de la protéine NS5. J'ai développé un système d'études mécanistiques en utilisant des techniques biochimiques de cinétique pré-stationnaire pour la protéine NS5, et obtenu des paramètres cinétiques et thermodynamiques de cette protéine envers ses substrats. Avec ce même système, j'ai pu tester des activités de la polymérase NS5 avec des ARN coiffés et triphosphates de différente longueur, mimant les séquences à l'extrémité 5' du génome du virus de la dengue. L'activité polymérase de NS5 est influencée par la présence de la coiffe de l'ARN, ce qui m'a permis de proposer une distance physique correspondant à environ 13 nucléotides entre les sites actifs domaines méthyltransférase et polymérase. Mes travaux ouvrent la voie à la détermination de la structure 3D de NS5 avec ses ARN et des nucléotides 5'-triphosphate.Elucider son mécanisme d'action, c'est être capable d'inhiber son action et donc de pouvoir proposer des molécules capables d'arrêter la prolifération virale lors d'une infectionDengue virus causes dengue fever, which may evolve towards life-threatening hemorrhagic fever. My research projectfocuses on dengue replication, and more precisely on the mechanism of NS5 at the molecular/atomic level. NS5 is a bifunctionalenzyme containing two domains: 1) a methyltransferase domain essential for translation of viral proteins, 2) apolymerase domain synthesizing the viral RNA genome. First, we demonstrated the main role of the polymerase in theconservation of 5' and 3' ends of dengue genome and anti-genome RNAs. Next, I showed the influence of themethyltransferase domain on the activity of the polymerase domain. I also developed a system allowing mechanistic studiesusing pre-steady state kinetics to characterize NS5 in depth. I have made use of this system to determine the catalyticparameters of NS5 towards its substrates. Using the same pre-steady state system, I was able to test the polymerase activityof NS5 with capped and uncapped 5'-triphosphate RNAs of different lengths corresponding to the 5'-end of the dengue RNAgenome. The polymerase activity of NS5 is significantly affected by the presence of the 5'-cap, which allowed me to designan experimental set-up pointing to a minimal physical distance of around 13 nucleotides between the methyltransferase andpolymerase active sites. My work will be useful to characterize the biophysics of NS5 in complex with its RNA and NTPsubstrates, and then to determine the crystal structure of such complex at play during viral RNA synthesis. Knowing thedetailed NS5 mechanism paves the way to inhibit its action and thus design drugs aiming at stopping a viral infection
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Jones, Daniel M. "The contribution of viral and host cell factors to replication of the hepatitis C virus RNA genome." Thesis, University of Glasgow, 2009. http://theses.gla.ac.uk/1056/.
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Studies on the hepatitis C virus (HCV) life cycle have been aided by the development of in vitro systems that permit replication of the viral RNA genome and virus particle production. However, the exact functions of the viral proteins, particularly those engaged in RNA synthesis, are poorly understood. It is thought that NS4B, one of the replicase components, induces the formation of replication complexes (RCs) derived from host cell membranes. These RCs appear as punctate foci at the endoplasmic reticulum (ER) membrane and incorporate the viral and cellular proteins necessary for HCV RNA synthesis. To gain insight into the nature of RCs, green fluorescent protein (GFP) was inserted into the coding region of NS5A, one of the HCV-encoded replicase components. The impact of the GFP insertion was examined in the context of a subgenomic replicon (SGR) based on JFH1, a genotype 2a HCV strain that exhibits efficient RNA replication in cell culture. The resulting construct was capable of robust replication and allowed characterisation of NS5A in live cells that synthesised viral RNA. NS5A displayed a diffuse, ER-like distribution and was also observed in foci. These foci are presumed to represent RCs and NS5A was relatively immobile at these sites. This result was confirmed using SGRs harbouring a photoactivatable derivative of GFP (PAGFP). Utilising plasmid-encoded HCV polyproteins, it was apparent that the targeting of NS5A to these structures was dependent on NS4B. Removal of the NS4B coding region resulted in a diffuse, ER-like distribution of NS5A, with little evidence of the protein within RCs. NS5A was mobile under these conditions, suggesting that the dynamics of NS5A are linked to focus formation by NS4B. To further investigate these findings, a panel of 15 alanine substitutions was constructed in the C-terminal region of NS4B. Transient replication assays revealed that five mutants were incapable of replication, two displayed an attenuated phenotype, and eight exhibited replication levels comparable to the wild-type (wt) genome. Of the five non-replicating mutants, two were defective in their ability to produce foci, while one failed to generate any foci. Thus, the C-terminus of NS4B is important for RC formation. Loss of NS4B foci correlated with decreased NS5A located in these structures. Furthermore, NS5A hyperphosphorylation was reduced for mutants compromised in foci production. This suggests that the membranous changes induced by NS4B provide a favourable environment for post-translational modifications of NS5A. Interestingly, the remaining two non-replicating mutants displayed no impairment in foci production and the characteristics of NS5A were also unaltered. Therefore, in addition to producing the cellular environment for HCV genome synthesis, NS4B is likely to play a more direct role in RNA replication. HCV RCs are believed to be relatively enclosed structures that permit limited exchange of materials with the cytoplasm. In support of this hypothesis, previous reports have shown that NS5A is the only replicase component capable of restoring replication to defective genomes when supplied in trans. In those studies, SGRs harbouring replication-lethal NS4B mutations could not be rescued by trans-complementation. Utilising the five novel non-replicating genomes described above, the potential to trans-complement NS4B in transient replication assays was re-examined. Wt protein produced from a functional HCV replicon could trans-complement defective NS4B expressed from two of the five mutants. Moreover, active replication could be reconstituted from two defective viral RNAs harbouring mutations within NS4B and NS5A. These findings have important implications for our understanding of RC formation. Genome-length JFH1 RNA produces infectious virus particles in Huh-7 cells. Using this system, it has become increasingly apparent that some HCV-encoded replication components are also involved in virus assembly and release. To determine whether NS4B had any influence on these latter stages of the virus life cycle, the NS4B mutations that did not block RNA replication were introduced into the full-length JFH1 genome. While the majority of mutants had no effect on virus production, one mutant consistently enhanced infectious virus titres by up to five-fold compared to wt JFH1. Interestingly, introduction of the same mutation into a chimeric J6-JFH1 genome resulted in repressed virion production. Together, these results suggest that NS4B contributes to virus assembly and release in a genotype-specific manner. In an attempt to identify novel cellular proteins involved in HCV genome replication, a siRNA library targeting 299 nucleotide-binding proteins was screened. For the screen, a robust system was established using two cell lines (derived from Huh-7 and U2OS cells) that replicated tri-cistronic SGRs. While the U2OS cell line supported HCV RNA replication less efficiently compared to Huh-7 cells, this cell type was efficiently transfected with siRNA. Consequently, increased gene-silencing and greater effects on HCV replication were observed in the U2OS cell line. Thus, U2OS cells may be a suitable alternative to Huh-7 cells for HCV-related siRNA studies. For the library screen, all siRNAs were tested in both cell lines, and cell viability measurements allowed specific effects on viral RNA synthesis to be characterised. The screen identified several cellular proteins that enhanced and suppressed HCV RNA replication. This study provides an important framework for more detailed analyses of these proteins in the future.
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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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Bhrigu, Vipul. "Replication of Adeno-Associated Virus in Murine Fibroblasts with Mouse Adenovirus Provided Helper Functions." University of Toledo Health Science Campus / OhioLINK, 2009. http://rave.ohiolink.edu/etdc/view?acc_num=mco1242316433.
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Hughes, Mair Elisabeth. "Identification of residues in hepatitis C virus NS5A with a critical role in genome replication of particle assembly." Thesis, University of Leeds, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.531528.
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Payan, Christopher. "Developpement d'une pcr competitive et alternative etablissant la replication des genomes des virus d'hepatites b et c in vitro." Paris 11, 1997. http://www.theses.fr/1997PA114811.
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Tran, Anh Thuy. "Genome-wide RNA-interference screen for human host factors vital to influenza A virus-induced cell death and viral replication." Journal of Virology, 2013. http://hdl.handle.net/1993/18323.
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Influenza virus is a globally significant infectious agent with the potential to cause catastrophic pandemic outbreaks. Present treatment of influenza infections is restricted to only four anti-viral drugs, but there are increasing global reports of anti-viral resistance in several seasonal strains and also the 2009 pandemic swine-origin influenza virus H1N1. Possible future pandemic outbreaks, emerging new strains and drug resistance underscore the need to understand this complex virus and its pathogenicity with the goal that novel targets can be uncovered for future therapeutic development.
Extensive lung tissue damage during influenza virus infection is proposed to contribute to the development of aberrant host immune responses. Strong evidence now demonstrates the significance of the cellular death pathway in promoting efficient influenza virus replication and disease progression. Viruses rely heavily on the machinery of their host for productive replication, which is also an Achilles’ heel that could be targeted for treatment. In pursuit of unraveling the complex nature of influenza virus replication, I carried out a global shRNA screen to identify specific host factors and signaling pathways that are involved in influenza-induced cell death and replication. In this study I identified 138 genes required for influenza viruses to induce infected host cell death. These genes were found to be involved in Protein Kinase A, NF-kB and PI3K signaling cascades. These signaling pathways are well known regulators of cell death and survival, which suggests influenza viruses may carefully regulate these pathways to reach a balance that suit their requirements for efficient proliferation, eventually at the cost of the host cell. I chose five candidate genes—BAD, MxB, TNFSF12-13,TNFSF13, and USP47—that were associated with apoptosis and the major signaling pathways determined in my network analysis to further verify the genome-wide screen as well as elucidate the role of these potentially novel host factors in influenza virus replication.
I show in my study that influenza virus-induced cytopathology and cell death are considerably inhibited in BAD knockdown cells and both virus replication and viral protein production also are dramatically reduced. I also report here that MxB depletion protected cells from virus-mediated cytopathology and resulted in significant inhibition of influenza virus replication for H1N1 and H3N2 subtypes. Additionally, I report that TNFSF12-13, TFNSF13, and USP47, similarly, are required for efficient influenza virus replication and induction of cell death. Depletion of these proteins resulted in significant inhibition of viral propagation and conferred protection of host cells to virus killing.
Overall, my study has provided a list of novel host factors that play significant roles during influenza virus infection. Further studies on these potential genes and their encoded protein products may uncover possible new targets for drug development for future therapeutic treatment. In addition to providing greater understanding of influenza virus infection, these studies will also highlight important fundamentals of cellular processes that may be broadly applicable to other fields of research.
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Monteil, Vanessa. "Analyses phénotypiques et génotypiques de différentes souches de dengue : applications en épidémiologie et recherche de facteurs de virulence." Thesis, Aix-Marseille, 2013. http://www.theses.fr/2013AIXM5038.
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De 50 à 100 millions de cas de maladie de dengue sont recensés chaque année dans le monde. Le virus de la dengue présente aujourd’hui un problème de santé publique avec son émergence en Europe et particulièrement en France. L’infection par le virus peut être asymptomatique ou être responsable de trois pathologies: l’une avec des symptômes grippaux (DF), une autre avec des hémorragies modérées (DHF) et une dernière avec des hémorragies sévères entraînant un syndrome de choc (DSS).Les facteurs de l’hôte jouent un rôle important dans le développement de formes sévères mais les facteurs viraux impliqués restent peu décrits. Le but de ce travail de thèse était de mieux comprendre ces facteurs viraux au travers de l’étude des dynamiques de circulation de souches de dengue 3 génotype III en Afrique et de la caractérisation de trois souches de dengue de sérotype I du Cambodge. Ce travail nous a permis de mettre en évidence la circulation de variants pendant les épidémies, permettant de supposer que la présence de variants permet une meilleure dissémination, ainsi que des caractéristiques génotypiques et phénotypiques particulières in vitro aux souches associées aux formes hémorragiques et aux formes avec syndrome de choc chez l’homme. Ces travaux ont été complétés par le développement d’un système original de détection du virus de la dengue et des autres virus du genre Flavivirus. Ce travail de thèse a permis d’identifier de potentiels facteurs de virulence propres au virus, ouvrant la voie à la recherche sur le rôle de certaines protéines virales dans la pathogénicitéFrom 50 to 100 million cases of dengue illness occurred every year in the world. Today, dengue virus is a public health problem with its emergence in Europe, particularly in France. DENV infection can be asymptomatic or be responsible for three distinct pathologies: one with flu-like symptoms (DF), another with moderate hemorrhage (DHF) and the last one with severe bleeding leading to shock syndrome (DSS). Host factors have an important role in the development of severe forms but implicated viral virulence factors stay not well described. The aim of this research work was to better understand these viral factors through study of dengue serotype 3 genotype III dynamics of circulation in Africa and through the characterization of three dengue serotype 1 strains in Cambodia. This work highlighted the circulation of variants during epidemics, allowing us to suppose that the presence of variants permits a better dissemination, as well as specific phenotypic and genotypic characteristics in vitro of strains associated with hemorrhagic forms or forms with shock syndrome in humans. These works were completed by the development of an original system of detection of dengue virus and other viruses of genus Flavivirus. This research work allowed identifying potential virulence factor specific to virus, opening the way for research on the role of certain viral proteins in pathogenicity
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Li, Xing Xing Li. "Viral structural proteins, genome, and in vivo replication : a comparison between nuclear and cytoplasmic types of Adoxophyes orana granulosis virus /." [S.l.] : [s.n.], 1993. http://e-collection.ethbib.ethz.ch/show?type=diss&nr=10326.
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Abrahams, Melissa-Rose Hilda. "The transmitted HIV-1 subtype C: characterization of the transmitted/founder full-length virus genome and the influence of early immune selective pressure on virus replication." Doctoral thesis, University of Cape Town, 2014. http://hdl.handle.net/11427/12712.
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Includes bibliographical references.The identification of targets of early immune responses associated with control of HIV-1 infection will inform immunogen design for vaccine interventions. The early evolution of transmitted/founder subtype C virus sequences was investigated to determine the location and frequency of immune selection, and the impact of early immune escape mutations on viral replicative capacity. Single-genome amplified env sequences from 26 acutely-infected women were evaluated for conformance to a model of random evolution to elucidate multiplicity of infection. Near fulllength genome sequences from the first six months of infection were generated for five women and sites evolving under immune selection were mapped. CD8+ cytotoxic Tlymphocyte escape mutations in HLA-B-restricted epitopes were introduced into infectious molecular clones of cognate transmitted/founder viruses by site-directed mutagenesis and their impact on viral replicative fitness was evaluated using parallel replication assays. In 77% of women (n=20) a single transmitted/founder variant established infection and two to five variants in the remaining 23% (n=6). Near full-length genome sequencing in five women confirmed single variant/low-diversity transmission and identified fifty-five genome regions evolving under immune selection, 40% of which was attributed to CD8+ cytotoxic Tlymphocyte pressure, 35% to antibody-mediated pressure, 16% to reversion and 9% could not be classified. The rate of sequence diversification and number of sites evolving under immune selection was highest in nef. The majority of evolving CD8+ cytotoxic T-lymphocyte epitopes (82%) contained shuffling/toggling mutations. A novel B*15:10-associated mutation, A164T, combined with a V85A Pol mutation reduced viral replication capacity in one individual. In a second individual, the attenuating HLA-B*58:01-associated mutation, T242N, enhanced viral replication capacity due to pre-existing compensatory polymorphisms in the transmitted/founder virus. A third individual, who had extremely rapid disease progression, was infected with the virus with the highest replication capacity. This thesis describes the complex nature of early immune selection and escape in transmitted/founder viruses. Although attenuating escape mutations were identified in viruses from two individuals, this was not associated with clinical benefit. The extensive variability of epitopes evolving under early selection may implicate many early immune targets as poor candidates for vaccine immunogens; however some early targets may be useful if clinical benefit is conferred through attenuating escape mutations.
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Germi, Raphaële. "Étude virologique de la fixation et de la replication du virus de l'hépatite C sur des cellules permissives : analogie avec deux autres Flaviviridae, les virus de la dengue et de la fièvre jaune." Université Joseph Fourier (Grenoble), 2001. http://www.theses.fr/2001GRE18008.
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L'étude du cycle du virus de l'hépatite C ( VHC) et la découverte de nouveaux traitements antiviraux ont été entravés par l'absence de systèmes cellulaires permettant la multiplication efficace du virus. Ce constat a motivé la première partie de nos travaux. Nous avons évalué l'adsorption et la multiplication du VHC dans cinq lignées cellulaires. Les cellules Vero (rein de singe) et AP61 (moustique) ont permis la détection de l'ARN viral pendant 28 jours et la réalisation de 4 passages viraux successifs. Le système cellulaire Vero , associé à une technique originale de quantification des ARN viraux par RT-PCR en temps réel a permis l'étude de l'adsorption de CHC. Ce travail a révélé le rôle des glycosaminoglycanes cellulaires dans l'adsorption du VHC mais aussi de deux autres Flaviviridae, les virus de la dengue et de la fièvre jaune utilisés, en parallèle, comme modèle et étudiés à l'aide d'une techique de titrage viral en culture de cellules. Le rôle important du récepteur des lipoproéines de faible densité (LDL) dans l'adsorption du VHC a été confirmé (inhibition de la fixation virale par les LDL et des anticorps anti-recepteurs des LDL). Les techniques développées dans cette étude pourraient être utilisées pour le criblage de molécules inhibant l'adsorption cellulaire des FlaviviridaeThe study of Hepatitis C virus ( HCV) cycle and the discovery of new therapy have been hampered by the lack efficient virus culture systems. We assessed adsorption and replication of HCV on five cell lines. Monkey Vero cells and mosquito AP61 cells were selected for their ability to blind and replicate HCV and to support 4 virus passages. HCV adsorption was studies using Vero cells and quantification of virus RNA by a real time RT-PCR method. This work showed that cellular glycosaminoglycans were involved in HCV adsorption. These molecules were shown to have an important role in the binding of tow other Flaviviridea , Dengue virus and Yellow fever virus which were studies in parralel, as models, by virus titration in cell culture. The important role of low density lipoprotein (LDL) receptor in HCV adsporption was confirmed ( the viral binding was inhibited by LDL and anti-LDL receptor antibody). The methods descripted in this study might be screening of molecules inhibiting Flaviviridae cell-adsorption
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Shen, Ni 1969. "Role of the leader sequence of human immunodeficiency virus type 1 in viral replication, genome dimerization, encapsidation, and proviral DNA synthesis." Thesis, McGill University, 2002. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=84434.
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Human immunodeficiency virus type 1 (HIV-1) genome consists of two identical RNAs that appear noncovalently linked near their 5' ends. The 5' untranslated region is called leader region. The 3' part of leader, i.e. nucleotides U200 to G335 in HIV-1 genomic RNA, between the primer binding site and the gag gene, can fold into 3 stem-loops: the kissing-loop domain (KLD) or stem-loop 1 (SL1), the 5' splicing junction hairpin (SD) or SL2 and SL3. The KLD, from nucleotide (nt) 243 to 277, forms a stem-loop (kissing loop hairpin) seated on top of a small stem bulge (stem B and loop B). The kissing-loop hairpin, or dimerization initiation site (DIS) hairpin consists of stem C and loop C. Loop C contains the autocomplementary sequence (ACS) GCGCGC262 or GUGCAC262, also called DIS.In the kissing-loop model of HIV-1 genome dimerization, HIV-1 RNA dimerization is initiated by base pairing between the ACS of one RNA monomer and that of an adjacent monomer.
To understand the role of the ACS in HIV-1 replication and HIV-1 genomic RNA dimerization, we replaced the central CGCG261 (or tetramer) of the HIV-1 Lai ACS by other tetramers. Genomic RNAs containing the UUAA tetramer (non-HIV-1 tetramer) were half dimeric, but UUAA genome packaging was unaffected. This was the first evidence that genomic RNA dimerization and packaging can be dissociated (Chapter 2). Destroying stem-loop C reduced genomic RNA dimerzation by ~50%, proviral DNA synthesis by ~85%, and reduced viral infectivity by ~3 logarithmic units. Destroying stem-loop B had similar effects on genome dimerization, reverse transcription, and viral infectivity. We also observed that mutations in stem-loop B and in the DIS hairpin were "non additive" (Chapter 2).
The existence of stem-loop C is supported by phylogenetic evidence, while that of stem-loop B is not, namely, its sequence is completely conserved. We investigated the role of stem B and loop B nucleotides in viral replication, and genomic RNA dimerization. The putative CUCG246/CGAG277 duplex was replaced by 9 alternative complementary sequences, 4 likely to base pair in long (~500 nts) RNAs, as assessed by the algorithm mfold. Among the 4 sequences, 3 preserved genome dimerzation, 1 did not significantly inhibit it, and 2 preserved viral replication. We also asked if 9 deletions or nucleotide substitutions within nucleotide 200 to 242 and/or 282 to 335 could influence genome dimerization. Delta200--226 and Delta236--242 genomic RNAs dimerized relatively poorly despite having neutral or positive influences on stem B, loop B and klh folding (Chapter 3).
Mutations within the Matrix, Capsid, p2 and nucleocapsid genes suppress several functional defects caused by KLD destruction. We tested the effect of these suppressor mutations on genome dimerization and infectiousness of viruses bearing moderate to crippling KLD mutations. Our conclusion is that these suppressor mutations can restore genomic RNA dimerization when DIS is weakened, but not when DIS is denatured or the KLD is destroyed (Chapter 4).
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Steil, Benjamin Peter. "Protein primers and a telomerase-like mechanism of poliovirus RNA replication maintain the 3' end of the RNA genome /." Connect to abstract via ProQuest. Full text is not available online, 2008.
Find full textAbstract:
Thesis (Ph.D. in Microbiology) -- University of Colorado Denver, 2008.Typescript. Includes bibliographical references (leaves 198-225). Online version available via ProQuest Digital Dissertations.
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Genevois, Marion. "Comparaison de la pathogenèse hépatique des virus fièvre jaune et dengue dans un modèle d’hépatocytes humains dérivés de cellules souches." Thesis, Lyon, 2019. http://www.theses.fr/2019LYSE1088/document.
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Les formes sévères de l’infection par les virus de la fièvre jaune (YFV) et de la dengue (DENV) sont caractérisées par une atteinte du foie, plus sévère lors d’une infection YFV. L’objectif de cette thèse est de comparer les infections de YFV et DENV dans un modèle d’hépatocytes humains dérivés de cellules souches (iHeps) afin d’identifier des facteurs à l’origine de cette différence de pathogenèse. Dans un premier temps, nous avons comparé le tropisme de YFV aux 4 sérotypes de DENV dans notre modèle hépatique établi en monocouche cellulaire. Nous avons observé une faible propagation de DENV dans les iHeps par rapport YFV. Les mêmes observations ont été faites dans des hépatocytes primaires. L’utilisation de souches chimériques 17D/DENV a permis de mettre en évidence que cette faible propagation serait liée à une faible efficacité d’entrée de DENV dans les hépatocytes. Nous avons également étudié l’infection dans des sphéroïdes iHeps, métaboliquement plus actifs que les iHeps 2D. Une infection productive a été observée uniquement avec YFV. Ce résultat pourrait s’expliquer par la faible accessibilité des cellules à l’intérieur des sphéroïdes. Dans un 2ème temps, nous avons étudié les réponses cellulaires induites dans les iHeps 2D après infection par les différents virus en utilisant une approche RNAseq. Les résultats préliminaires suggèrent un lien entre le taux de réplication et le nombre de gènes activés. La réponse interféron est plus précocement détectée dans le cas de YFV, mais l’infection par DENV induit un plus grand nombre de gènes. De plus, DENV-1 et DENV-4 induisent une augmentation d’expression de certains gènes impliqués dans la présentation d’antigène comme HLA-E et TAP-2, alors que YFV diminue l’expression de certains gènes de chimiokines et molécules d’adhésion. L’analyse préliminaire des voies liées au métabolisme hépatique révèle une inhibition de la voie de la coagulation dans le cas de l’infection par YFV, qui n’est pas observée lors de l’infection par DENV. Des observations similaires ont été décrites in vivo, au niveau protéique, confirmant la pertinence du modèle iHepsSevere forms of infection with yellow fever virus (YFV) and dengue virus (DENV) are characterized by liver damage, with more severe symptoms observed during YFV infection. The aim of this thesis is to compare YFV and DENV infections in a model of human hepatocytes derived from stem cells (iHeps) in order to identify factors that could explain their difference in pathogenesis.First, we compared YFV tropism to the four DENV serotypes in 2D iHeps. We observed a low spread of DENV compared to YFV in both iHeps and primary hepatocytes. By using chimeric 17D/DENV strains, we demonstrate that this low propagation is linked to a low DENV entry efficiency in hepatocytes. We also studied infection in iHeps spheroids, metabolically closer to primary cells than 2D iHeps. A productive infection was observed with YFV only. The low accessibility of cells inside the spheroids could explained this result. Second, we studied cellular responses induced following infection by different viruses in 2D iHeps using an RNAseq approach. Preliminary results suggest a link between replication rate and the number of activated genes. The interferon response is detected earlier following YFV infection, but DENV induces a greater number of genes implicated in this pathway. Moreover, DENV-1 and DENV-4 up-regulate some genes involved in antigen presentation such as HLA-E and TAP-2, while YFV down-regulates genes encoding chemokines and adhesion molecules. Preliminary analysis of hepatic metabolism pathways reveals inhibition of the coagulation pathway induced by YFV infection, which is not observed during DENV infection. Similar observations have been described in vivo, at the protein level, confirming the relevance of the iHeps model
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Jaubert, Chloe. "Extrémités 3’ de l’ARN du Virus de l’Hépatite C : structures et Rôles dans la réplication du génome." Thesis, Bordeaux, 2016. http://www.theses.fr/2016BORD0219/document.
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Le génome du virus de l’Hépatite C est constitué d’un ARN monocaténaire linéaire de polarité positive (+). Les interactions ARN-ARN prennent une place essentielle dans la régulation du cycle viral.La synthèse de l’ARN est réalisée par l’ARN-polymérase ARN-dépendante (RdRp) codée par le virus. Elle serait initiée à l’extrémité 3’ des molécules à répliquer. Un ARN génomique complémentaire de polarité négative (-) est d’abord synthétisé. Il sert ensuite de matrice pour la production des brins génomiques. Les mécanismes qui président au recrutement de la polymérase et à l’initiation de la synthèse d’ARN restent, aujourd’hui, mal connus.Les structures ARN présentes aux extrémités 3’ et leurs rôles ont donc étés étudiés au cours des travaux de cette thèse. Au niveau de l’extrémité 3’ de l’ARN (+), la dimérisation a été montrée indispensable à la réplication du virus in cellulo. Ces travaux ont par la suite permis de caractériser par gel retard et cryo-microscopie la dimérisation des ARN génomiques en solution. Au niveau de l’extrémité 3’ de l’ARN (-), la dimérisation de deux molécules a également pu être caractérisée par des approches biochimiques et biophysiques. Par ailleurs la présence d’un G-quadruplex introduit un remaniement conformationnel qui se révèle indispensable à une synthèse performante de l’ARN. De manière similaire au brin génomique, la dynamique structurale résultante de ces interactions semble donc nécessaire à une réplication efficace de l’ARN par la RdRp.Les résultats obtenus soulignent l’importance de la dimérisation et des variations conformationnelles prisent aux extrémités 3’ pour la réplication de l’ARN. Ces données ouvrent alors la voie vers de nouvelles perspectives quant à la compréhension des mécanismes qui président au fonctionnement de la polymérase du VHCThe hepatitis C virus genome consists of a linear positive sens (+) single-stranded RNA. RNA-RNA interactions play an essential role in the regulation of the viral cycle.RNA synthesis is performed by the RNA-dependent RNA-polymerase (RdRp) encoded by the virus. It would be initiated at the 3 'end of the molecule to be replicated. A complementary genomic RNA of negative polarity (-) is first synthesized. It then serves as a matrix for the production of genomic strands. The mechanisms that govern the recruitment of the polymerase and the initiation of RNA synthesis remain poorly understood today.The RNA structures found at the 3 'ends and their roles have therefore been studied during the work of this thesis. At the 3 'end of the (+) RNA, dimerization was shown to be essential for replication of the virus in cellulo. This work made it possible to characterize by gel shift assay and cryo-microscopy the dimerization of the genomic RNAs in solution. At the 3 'end of (-) RNA, the dimerization of two molecules could also be characterized by biochemical and biophysical approaches. Moreover, the presence of a G-quadruplex introduces a conformational reshuffle which proves to be indispensable for an efficient RNA synthesis. Similarly to the genomic strand, the resulting structural dynamics of these interactions appear to be necessary for efficient RNA replication by the RdRp.The results obtained here underline the importance of dimerization and conformational variations at the 3 'ends for RNA replication. These data then open the way to new perspectives on understanding the mechanisms that govern the functioning of HCV polymerase
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Weiss, Etienne. "Caracterisation structurale et fonctionnelle de sous-populations de minichromosomes du virus sv40." Université Louis Pasteur (Strasbourg) (1971-2008), 1987. http://www.theses.fr/1987STR13019.
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HEHN, ALAIN. "Les genes 3' proximaux du rna 2 du virus des nervures jaunes et necrotiques de la betterave (ou bnyvv) : leur role dans la replication et la traduction du genome viral ainsi que dans le mouvement du virus de cellule a cellule." Université Louis Pasteur (Strasbourg) (1971-2008), 1995. http://www.theses.fr/1995STR13092.
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Le virus des nervures jaunes et necrotiques de la betterave est le phytovirus responsable de la rhizomanie chez la betterave a sucre, maladie caracterisee par un rabougrissement du pivot et une proliferation anarchique du chevelu racinaire. Mon travail de these peut etre subdivise en trois parties: (1) etude du role des proteines codees par les 4 genes 3' proximaux du rna 2. Nous avons montre que les produits des orf iii, iv et v etaient directement impliques dans le mouvement du virus de cellule a cellule. La proteine p14 synthetisee a partir de l'orf vi, quant a elle, agit en cis sur la replication du rna 2 et en trans sur la traduction de la proteine de capside. (2) dans le but de developper une strategie de lutte contre la multiplication du bnyvv, nous avons construit des rna defectifs interferant (di) artificiels en introduisant des grandes deletions dans le rna 2 du bnyvv. Ces rna di sont capables d'inhiber efficacement la replication des rna 1 et 2 lorsqu'ils sont coinocules a des protoplastes de chenopodium quinoa. Enfin (3), nous avons mis en evidence la maturation de la proteine p237 codee par le rna 1, confirmant ainsi les predictions faites par des comparaisons de sequences avec d'autres virus
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Simões, Margarida Pires. "Spatiotemporal study of morpho-functional modifications on cell nucleus during African swine fever virus infection." Doctoral thesis, Universidade de Lisboa. Faculdade de Medicina Veterinária, 2015. http://hdl.handle.net/10400.5/9844.
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Tese de Doutoramento em Ciências Veterinárias. Especialidade de Ciências Biológicas e BiomédicasStudies on virus-host interactions are decisive to enhance our understanding on how African swine fever virus (ASFV) subverts cellular mechanisms, and also to better characterize host nucleus changes enabling this infection. Immunofluorescence studies and immunoblotting analysis of ASFV-infected cells, allowed us to identify the Ataxia telangiectasia mutated and Rad3-related (ATR) pathway as the specific DNA damage response (DDR) mechanism activated by ASFV infection. Additionally, the use of ATR kinase-dead cells confirmed that ATR has an essential role for the infection success. The viral intranuclear replication was then pursued using BrdU-pulse experiments, supported on previous reports about ASFV genome presence inside the host nucleus and the proven ATR activation. BrdU-labelled DNA molecules confirmed the active viral replication at early infection times, exclusively within the cell nucleus. Related spatial and morphological nuclear changes during ASFV infection were further addressed, particularly on subnuclear domains and host chromatin epigenetic signatures. Promyelocytic leukaemia nuclear bodies (PML-NBs), nuclear speckles and Cajal bodies displayed major alterations, accompanied by a repressive nuclear environment. PML knockdown revealed an essential proviral activity for ASFV successful infection. Herein, suggestions on how this work may help in the development of therapeutic strategies against ASFV infections can be found.
ABSTRACT - Estudo espacio-temporal das alterações morfo-funcionais do núcleo celular durante a infeção pelo vírus da peste suína africana - Estudar as interacções vírus-núcleo da célula hospedeira é fundamental para melhor compreendermos a subversão dos mecanismos nucleares pelo Vírus da peste suína africana (VPSA), e como estas facilitam a infecção. Ensaios de imunofluorescência e expressão proteica (imunodetecção) permitiram a identificação da via ATR (Ataxia telangiectasia mutated and Rad3-related) como mecanismo de reparação de ADN especificamente activado pelo VPSA. Seguidamente, através de células modificadas na expressão de ATR, confirmou-se o papel essencial do ATR para esta infecção. Atendendo às evidências de presença do vírus no núcleo celular e à activação da via ATR, quisemos desvendar a replicação intranuclear do VPSA pelo meio de ensaios pulso-caça de BrdU. Moléculas de ADN marcadas com BrdU confirmaram a replicação viral, exclusivamente no núcleo, na fase precoce da infecção. Seguiu-se o estudo das alterações espaciais e morfológicas do núcleo induzidas pelo VPSA, particularmente as modificações dos sub-domínios nucleares e das assinaturas epigenéticas da cromatina da célula. Domínios PML (Promyelocytic leukaemia), speckles nucleares e corpos de Cajal mostram-se alterados, associando-se a um ambiente nuclear repressivo. Estudos funcionais da proteína PML revelaram o seu papel proviral desta infecção. Nesta tese apresentam-se ainda sugestões acerca do potencial destes estudos para o desenvolvimento de estratégias terapêuticas no combate anti-viral.
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Morch, Marie-Dominique. "Organisation et expression genetiques du virus de la mosaique jaune du navet : contribution a l'etude des strategies d'expression des virus a arn, a la discussion de leur variabilite et a l'elaboration de strategies de protection." Paris 7, 1988. http://www.theses.fr/1988PA077124.
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On procede au sequencage du genome complet du virus constitue d'un arn de polarite positive a 6318 nucleotides et contenant 3 cistrons. Parmi les strategies mises en oeuvre pour synthetiser ses proteines, on presente la maturation proteolytique de la proteine 206 k codee par ce virus. Une approche "arn-sens" pour la lutte anti-virale est testee experimentalement: des arn "pseudogenomiques" rentrent en competition avec l'arn du virus et sont capables d'inhiber sa replication in vitro
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Muszynski, Bartosz. "Structure Function Studies of Rotavirus NSP5." Doctoral thesis, Scuola Normale Superiore, 2008. http://hdl.handle.net/11384/85974.
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Rotaviruses, causative agents of gastroenteritis in young animals and humans, are
large icosahedral viruses with a complex architecture. The double-stranded RNA (dsRNA)
genome composed of 11 segments, that codes for 6 structural and 6 non-structural proteins, is
enclosed within three concentric capsid layers.
NSP5, a non structural protein, is encoded by segment 11. It is produced early in
infection and localizes in ‘viroplasms’, cytoplasmic inclusion bodies in which viral RNA
replication and packaging take place. NSP5 is essential for the replicative cycle of the virus
since, in its absence, viroplasms are not formed and viral RNA replication and transcription
do not occur.
NSP5 is known to undergo two different types of posttranslational modifications, a
cytoplasmic O-glycosylation and phosphorylation, which lead to the formation of proteins
differing in electrophoretic mobility. Although the hyperphosphorylation process of NSP5
seems to be very complex, its role in the replicative cycle of rotavirus is unknown.
We demonstrated that NSP5 operates as an auto-regulator of its own phosphorylation
as a consequence of two distinct activities of the protein: substrate and activator. In the first
part of the thesis we have shown, that phosphorylation of Ser-67 within the SDSAS motif
(amino acids 63-67) was required to trigger hyperphosphorylation by promoting the activation
function. The evidence coming from iv vitro experiments, including kinase assay with
recombinant casein kinase 1α from zebrafish, proved that this enzyme is responsible for a key
phosphorylation step that initiates the whole hyperphosphorylation cascade of NSP5.
In the second part of the dissertation, using MALDI TOF/TOF spectroscopy, we added
new data to the information about the posttranslational modifications of NSP5. We confirmed
that the region of the protein encompassing Ser-67 is phosphorylated in vivo. Additionally we
managed to map which parts of NSP5 sequence carries N-acetyloglucosamine and which
regions bear phosphorylated serines or threonines.
There is no evidence about structure of NSP5 so far. In the last chapter we focused on
investigating the structural organization of this crucial viral protein. To achieve this, in
addition to the full length protein, one point mutation and two different truncation mutants
were constructed, expressed, purified and refolded. The secondary structure of the different
proteins was analyzed by circular dichroism spectroscopy and general information about
protein conformation was provided. Our findings, together with an analysis of NSP5 sequence
indicate that NSP5 can be an intrinsically unfolded/disordered protein.
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Delmas, Véronique. "Structure et proprietes biologiques du papovavirus de hamster." Paris 6, 1986. http://www.theses.fr/1986PA066550.
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Le papovavirus de hamster (hapv) possede un tropisme restreint in vivo vis a vis des keratinocytes et des lymphocytes. Il se replique dans les tumeurs cutanes qui apparaissent chez des hamsters syriens, et induit egalement des lymphomes chez le hamster. L'organisation genetique du hapv deduite de sa sequence a montre qu'il appartient a la famille des polyomavirus. Le hapv est present dans les lymphomes sous forme de multiples copies libres possedant toujours une deletion localisee dans la meme region du genome. Les signaux de transcription precoce du hapv semblent etre actives par la region precoce de ce virus
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Srinivasa, Ramya. "Flavivirus RNA replication: Probe development, structural dynamics, and role of zinc ions." Thesis, 2023. https://etd.iisc.ac.in/handle/2005/6085.
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Flaviviruses share a typical genome architecture where the highly conserved UTRs flank a central coding region. They also share a common genome replication strategy called genome cyclization where the UTRs interact to form a long-range, dynamic, 3D RNA interactome. Recent studies focused on the involvement of the capsid coding region in modulating these UTR interactions. Along similar lines, we have investigated the effect of CCR on the UTRs interactions in Dengue using ensemble FRET with cyanine dye-labeled Dengue UTRs. As a part of this study, we have developed a novel, generic chemoenzymatic, bead-based method for base-specific modification of RNA called “T7 RNA polymerase extension-based RNA modification (TERM)”. Here, we describe TERM development, demonstrate the synthesis of single, site-specific cyanine dye-labeled Dengue UTRs and discuss the UTR interaction studies performed using the TERM-synthesized cyanine dye-labeled UTRs. Our results bridge the knowledge gap in understanding the role of the capsid coding region in Flavivirus UTRs.
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ChiChiu and 邱琦. "Study on the mechanism of dengue virus replication: the role of proteases during dengue virus replication." Thesis, 2013. http://ndltd.ncl.edu.tw/handle/x63pgv.
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碩士國立成功大學
醫學檢驗生物技術學系碩博士班
101
Dengue virus (DENV) infection can causes dengue fever and potentially lethal diseases such as dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Until now, there is no effective vaccine or therapeutic drugs that can be used specifically to block DENV replication in clinic. One of the difficulties of developing anti-DENV drugs is lacking of a suitable platform for compound selection. Thus, we constructed a sequence which contains secreted alkaline phosphatase (SEAP) and dengue NS2B-NS3 cutting site, followed by ligation with vector pEGFP-C1. After transient transfection of pEG(Δ2B3)SEAP into HuH7 and HT29 cells, results showed that the EGFP signals were degraded after few passages of cell culture. According to previous studies, the midgut trypsin activity can affect DENV-2 infection and dissemination in Aedes aegypti. In order to investigate the role of proteases in DENV replication, we used inhibitors that can inhibit trypsin, chymotrypsin or both of them to understand the effect of cellular enzymes on DENV replication. Supplementation of pEG(Δ2B3)SEAP transfected cells with trypsin inhibitor revealed that the dregradation of EGFP signals was inhibited. Furthermore, tosyl phenylalanyl chloromethyl keton (TPCK), which is a chymotrypsin-like protease and NF-κB inhibitor, demonstrated the most significant inhibitory ability. We then used several NF-κB inhibitors to investigate the role of NF-κB during DENV infection. The results revealed that NF-κB not only inhibit DENV-induced autoaphgy, which is acquired for DENV replication, but also decrease the level of DENV-induced cell apoptosis, and the production of macrophage migration inhibitory factor (MIF) were also decreased after inhibition of NF-κB. Taken together, these results suggested that both trypsin-like enzymes and NF-κB activation play important roles in DENV replication, DENV-induced autophagy and apoptosis, and MIF secretion. Combination of protease and NFκB inhibitors may provide an alternative therapeutic strategy for dengue patients.
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Hsin-HuaLu and 盧信樺. "Dengue virus requires glucose metabolism for replication." Thesis, 2018. http://ndltd.ncl.edu.tw/handle/2kg7sz.
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Chen, Chun Yu, and 陳俊宇. "Regulation of Hepatitis Delta Virus Genome Replication/Transcription." Thesis, 2009. http://ndltd.ncl.edu.tw/handle/64556828029638168263.
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碩士長庚大學
生物醫學研究所
97
The unbranched rod-like structure of hepatitis delta virus (HDV) is dependent on host RNA polymerase for its replication and transcription. The HDAg mRNA transcribed from HDV genome has a 5’ cap and 3’ poly(A) tail consistent with its role as mRNA for translation of the HDAg which is involved in HDV replication and assembly. Although it is agreed that RNA pol II is involved in the replication to produce HDV genomic RNA, the RNA polymerase involved in the replication to produce HDV antigenomic RNA is not sure. Furthermore, the HDV RNA promoter regions required for the transcription and replication are not clear. In order to identify the promoter region in HDV RNA replication and transcription, first, I constructed HDV mini-genome, and the cis-elements localized in the end of rod-like structure of HDV are important to HDV replication. Second, I constructed RNA recombinant, and the rod-like structure of HDV is required for the genome replication, but not for HDAg-mRNA transcription. According to the results, I constructed the plasmids to produce different HDV RNA fragments that contain HDAg ORF and the potential promoter region. This data showed that the potential RNA promoter region for HDV transcription is located on the top of rod-like structure of HDV. In these results, two experimental systems which were designed to investigate the potential promoter region for HDV replication and transcription were established, and the specific region is remained to be identified.
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Lin, Yu-Wen, and 林育雯. "Virus replication and cytokine production of dengue virus-infected human B lymphocytes." Thesis, 2002. http://ndltd.ncl.edu.tw/handle/kg6378.
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碩士國立成功大學
微生物及免疫學研究所
90
ABSTRACT Dengue virus (DV) infection is a major problem in public health because it can cause fatal diseases such as dengue hemorrhagic fever and dengue shock syndrome. Unfortunately, there is no specific antiviral treatment or prophylaxis vaccine because the lack of understanding of pathogenesis. Even the most important question, which cells are infected by virus in humans remains to be determined. Monocytes have long been assumed to be the major virus producer, however, recent reports demonstrated that most DV was found in B cells but not in monocytes of peripheral blood mononuclear cells of infected patients. Using B cell line, my research established that DV2 (PL 046) replicated in Raji cells from 4 to 120 hours post infection at a multiplicity of infection (MOI) of 10 and that a 1:60,000 dilution of human DV3 immune serum (5 fold beyond the neutralizing titer) yielded antibody dependent enhancement effect. Meanwhile, I investigated DV infection, antibody-enhanced virus infection, and cytokines responses of human primary B cells and compared them with those of monocytes. The presence of replication template (negative strand RNA intermediate), virus antigens (envelope, core and nonstructural proteins), and increasing amount of viruses in infected B cells indicated the DV actively replicated in B cells. Additionally, the levels of virus replication, antibody-enhanced virus replication, and cytokine response observed in B cells are comparable to those in monocytes. The results indicated cells support DV growth and may play an important role in the pathogenesis of DV-induced disease.
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Heckmann, Anna Louise. "Targeting the Dengue genome with a Hepatitis Delta Virus ribozyme." 2010. http://etd.nd.edu/ETD-db/theses/available/etd-04162010-160534/.
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Wu, Yi-Shan, and 吳依珊. "Dengue virus NS5 protein nuclear localization is crucial for virus replication in Aedes aegypti." Thesis, 2019. http://ndltd.ncl.edu.tw/handle/mpdr2e.
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碩士國立臺灣大學
微生物學研究所
107
Mosquitoes are one of the fatal animals in the world and they act as vectors for several diseases, including malaria, dengue fever, Zika, japanese encephalitis, West Nile fever, and chikungunya. Among these diseases, dengue fever is currently one of the world’s most important tropical diseases. Previous studies have indicated that the non-structural protein 5 (NS5) was able to translocate into the nucleus and influence the dengue virus serotype 2 (DENV2) infectivity in mammalian cell lines. However, the detail mechanisms of NS5-mediated infectivity remain largely unknown. Therefore, the aim of this study is to investigate the role of the dengue virus NS5 nuclear localization on DENV replication in the mosquito Aedes aegypti. We showed that dengue virus NS5 was localized in the nucleus only in the case of DENV2 (16681 strain) infection in Aedes aegypti. Next, we investigated the effect of NS5 nuclear localization in viral replication by application of Ivermectin, which was demonstrated to be an inhibitor for NS5 protein nuclear localization. We showed that treatment of Ivermectin resulted in the significant reduction of DENV2 replication and infectivity. We also showed that Caspar splicing was inhibited in the case of NS5 nuclear localization, suggesting the important role of NS5 nuclear localization on host mRNA splicing. In the future, we will further investigate the detail mechanisms underlying the regulation of NS5 nuclear localization on DENV2 replication and immune response modulation.
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Tsai, Yi-Ting. "Human TLR3 Recognizes Dengue Virus and Modulates Viral Replication In Vitro." 2008. http://www.cetd.com.tw/ec/thesisdetail.aspx?etdun=U0001-2801200816391800.
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Yen-Chang, Lu, and 盧彥彰. "Effects of autophagy on dengue 2 virus replication in Aedes aegypti." Thesis, 2012. http://ndltd.ncl.edu.tw/handle/68367854696020216477.
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碩士國立陽明大學
微生物及免疫學研究所
100
Dengue virus (DENV) infection has been a global public health challenge for the past 50 years. DENV is transmitted mainly by mosquitoes Aedes aegypti and Aedes albopictus. Autophagy was initially described as a key physiological process of cell survival where recently it has been demonstrated that autophagy plays an important role in the innate immunity against the microbes. However, autophagy as an innate immune mechanism has not been described in Ae. aegypti. In this study the autophagy biomarker:, LC3-II, was used to monitor the autophagic flux in Ae. aegypti. This study found that DENV2 infection can persistently induce autophagic flux in mosquitoes. The induction of autophagy by feeding mosquitoes with rapamycin; an autophagy inducer, can enhance DENV2 replication. A significant decrease of DENV2 replication was observed in Ae. aegypti fed with autophagy inhibitor, 3-methyladenine and L-asparagine. Results from this study indicated that DENV 2 infection could induce autophagy machinery in Ae. aegypti which leads to enhanced viral replication in mosquitoes.
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Hung, Chiao-Ling, and 洪巧玲. "Study of the role of dengue virus NS4B in virus replication by reverse genetics approach." Thesis, 2011. http://ndltd.ncl.edu.tw/handle/11142196988704771492.
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碩士國立陽明大學
生命科學暨基因體科學研究所
99
Dengue virus (DENV) encodes three structural proteins and seven nonstructural (NS) proteins. Most, if not all, DENV NS proteins are involved in viral replication. NS4B, the largest membrane associated NS protein, contains two membrane-associated domains and three trans-membrane domains (TMD3~TMD5). The role of NS4B in virus life cycle is less known compared to the cytoplasm located NS3 and NS5 proteins, each of which has multiple enzymatic activities. In this thesis, a reverse genetics approach was taken to assess the role of NS4B in DENV life cycle. Mutagenesis analysis was carried out on five conserved proline residues and the Lp45 region (the region between TMD 4 and TMD 5) of NS4B in the context of DENV replicons. The P104 to E, K, or R mutation (in TMD3) as well as the P200 to L mutation (in Lp45) of NS4B was deleterious to viral replication. Alanine scanning of the Lp45 showed that this region of NS4B needed to keep the native sequence for viral replication. Interestingly, some replication competent revertants of P104 and P200 mutants were identified, and these revertants had the #104 or #200 residue substituted to an accessible amino acid(s). Several revertants of the P200L mutant contained an intragenic second site mutation in TMD3, TMD4, or Lp45 of NS4B, and each second site mutation by itself was replication competent and they all had a rescuing effect on P200L mutation. Besides, an NS4A intergenic second site mutation was found to rescue the replication deficiency of NS4B P104E mutation. Some replication competent NS4B mutations were found to have deficiency in infectious virus production. Overall, the reverse genetic studies using the replicon system indicate that NS4B is essential to DENV life cycle and NS4B is likely to have interaction with other NS protein such as NS4A to exert its biological function.
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Lee, Tai-Chi, and 李岱冀. "Utilizing Liver-specific microRNA-122 to Modulate Replication of Dengue Virus Replicon." Thesis, 2010. http://ndltd.ncl.edu.tw/handle/65261131739777588538.
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博士國防醫學院
生命科學研究所
98
MicroRNAs (miRNAs) are endogenous non-coding RNAs that spatiotemporally target and modulate mRNAs in a post-transcriptional manner. Exploiting reverse genetics approach that engineering viruses by insertion of a tissue-specific miRNA recognition element (MRE) into viral mRNA can restrict viral tissue tropism during infection. It is intriguing to ask whether this miRNA targeting approach can also be applied to flavivirses with positive-sense single-stranded RNA viral genome lacking poly(A) tail. In this study we employed dengue virus (DEN) replicon as a model to investigate whether miRNAs are able to suppress flavivirus replication through the targeting of nonpolyadenylated and highly structured viral mRNA. Because liver infection by DEN may contribute to the virus pathogenesis, we inserted an MRE of hepatic-specific and liver-enriched microRNA-122 (miR-122) into its 3′-untranslated region (3′-UTR) to test the feasibility of creating a liver-restricted DEN replicon. Our results demonstrate that incorporation of miR-122 target sequence appears to confer upon the DEN replicon an inhibitory susceptibility to endogenous miR-122 or exogenous miR-122-like siRNA, suggesting that DEN can be engineered to exert the desired replication restriction effect to avoid infection of vital tissues/organs and may allow to further successfully construct an engineered DEN vector that was shown to be highly-susceptible to miRNA-mediated gene silencing. This approach provides an additional layer of biosafety and thus has a great potential for use in the rational development of safer flavivirus vaccines
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Hsu, Chia-Wei, and 徐嘉偉. "MicroRNA-based antiviral transgenic mosquitoes Aedes aegypti to suppress dengue virus replication." Thesis, 2016. http://ndltd.ncl.edu.tw/handle/22771471066131273554.
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碩士國立臺灣大學
分子與細胞生物學研究所
104
Dengue is an arthropod-borne viral disease prevalent in tropical and subtropical regions, caused by the four dengue virus serotypes (DENV 1–4), which are transmitted by Aedes mosquitoes. Until now, effective vaccine and positive treatment are not available, and the use of insecticides also leads to resistance in mosquito vectors. Therefore, the alternative strategies for mosquito vector control are needed. Genetic manipulation via RNA interference (RNAi) in the mosquito can permanently reduce vector competence and subsequent transmission of DENV to the human. Recent studies have found that expression of virus-specific double stranded RNAs is a potential way to block the DENV replication and vector competence to virus. In this study, using the mariner (Mos1) transposase and genomic integration system, we generated serial transgenic mosquito lines which express anti-viral microRNAs under the control of A. aegypti poly-ubiquitin (Pub), serine-type enodpeptidase (AAEL001703) and carboxypeptidase A (CPA) promoters. We verified the integration of the transgene in the mosquitoes by qPCR and Southern blot. By using virus challenge with viremic bloodmeal, the viral titer and infection rate was significantly suppressed in the transgenic mosquitoes of CPA>8-miRNA, Pub>8-miRNA and AAEL001703>4-miRNA at day-7 post bloodmeal, compared to those of wild type mosquitoes. Repeat bloodmeal on CPA>8-miRNA provided effective antiviral efficiency at day-14 post infection. Transmission in vitro of virus from CPA>8-miRNA line was significantly diminished when compared to Pub>8-miRNA and control mosquitoes at day-14 post bloodmeal. Our study provides an evidence for DENV control strategy which suppress viral replication via genetic manipulation of miRNA in Aedes aegypti.
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Hsu, Hui-Chun, and 徐惠純. "Therapeutic Inhibition of Dengue Virus Replication and Gene Expression by RNA Interference." Thesis, 2004. http://ndltd.ncl.edu.tw/handle/91591581572494144324.
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碩士國立成功大學
生物化學研究所
92
Abstract The Dengue virus is a member of the virus family Flaviviridae and is transmitted to people through the bite of the mosquitos Aedes aegypti and Aedes albopictus. It can cause dengue fever (DF), dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Dengue vaccine is difficult to produce, because there are four serotypes of dengue virus, and the cross-infection of different serotype will have a high risk at the DHF/DSS development. So far, the most important aspect in treatment of DHF is to prevent further fluid loss. RNA interference (RNAi) is a key mechanism of post-transcriptional gene silencing (PTGS) in which double-stranded, small interfering RNAs trigger a sequence-specific gene-silencing process. Dengue virus is a positive-strand RNA virus, and contains a single RNA genome and translates only a single polyprotein precursor. In this study, we introduce RNAi system against the polyprotein precursor, which can sufficiently inhibit dengue virus replication in mammalian cells. We constructed enhanced green fluorescence protein (EGFP) gene on pSUPER vector. EGFP signal indicates that the cells were transfected with siRNA vectors successfully and pSUPER vector directs siRNAs synthesis in mammalian cells. The designed siRNAs were derived from the genome of dengue virus type 2 (DENV-2) membrane glycoprotein (MG), envelope protein (E), and non-structural protein 1 (NS1) regions. First, we used the MG, E or NS1-firefly luciferase fusion to screen effective siRNA targeting sequences separately. Subsequently, BHK-21 (baby hamster kidney-21) cells were transfected with effective siRNA vectors and challenged with DENV-2 for 24 hours. Then we assayed NS1 antigen content of the EGFP-expressing cells by flow cytometry and observed that the percentage of positive cells was significant reduction. Moreover, we fixed the DENV-2-infected BHK-21 cells on the slides for immunofluorescence staining and saw that EGFP-expressing cells had little virus NS1 and E antigens. We also collected the supernatants separately for plague-forming unit (PFU) assay and calculated that the virus titers decreased. Besides, transfecting effective siRNA vectors into BHK-21 cells could reduce the virus-induced cytopathic effect (CPE) and cell apoptosis. These results suggest that RNAi can block dengue virus replication in mammalian cells and offer a hope for the treatment of dengue virus infection on the therapeutic promise of RNAi
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Chao-HoLi and 李兆和. "The facilitating roles of p62/sequestosome-1 protein in dengue virus replication." Thesis, 2013. http://ndltd.ncl.edu.tw/handle/47833561120072189123.
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碩士國立成功大學
微生物及免疫學研究所
101
Dengue is a re-emerging disease which is endemic in more than 100 countries throughout Africa, South-East Asia, America, and the Western Pacific. Due to global warming effects, the dengue virus (DV) threat is broadening in the world. The symptoms are febrile illness called dengue fever to dengue hemorrhagic fever and dengue shock syndrome. Autophagy, a cellular catabolic system responsible for damaged organelles and misfolded proteins degradation, is reported to facilitate DV replication. A recent study showed that sindbis virus is associated with an autophagy adaptor protein p62 that leads to elimination of virus by autophagy. However, our data here indicate that p62 seems to be required for DV replication. Compared to WT MEF cells, less virus proteins, released DV particles and DV RNA were detected in DV-infected p62-deficient MEF cells. Significant decreased DV protein translation was also observed in p62 knockdown DV-replicon stable expressing cells. This facilitating role of p62 to DV replication is not limited in MEF cells but also in hepatocyes and monocytes. There were no differences in virus cell binding, endocytosis, interferon activation and autophagy induction between DV-infected WT and p62-deficient cells. Lipid accumulation by p62 deficiency also showed no contribution to DV replication. However, DV triggered high NF-κB phosphorylation and nuclear translocation in p62-deficient cells. The reduced DV replication can be rescued by inactivation of NF-κB with inhibitor BAY 11-7082 in p62-deficient cells. Moreover, we found that NF-κB responsible cytokine IL-6 was highly produced in DV-infected p62-deficient cells. The co-localization of p62 with DV prM, E, NS2A and NS3 proteins from confocal microscopy observation indicates that p62 might interact with DV proteins to facilitate virus replication. Our findings uncover a new role of p62 in facilitating DV replication and also provide a potential therapeutic target for dengue infection.
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Tsai, Yan-Yi, and 蔡燕儀. "Effects of mosquito signal peptidases on dengue virus replication in Aedes aegypti." Thesis, 2019. http://ndltd.ncl.edu.tw/handle/v9m69z.
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碩士國立臺灣大學
微生物學研究所
107
Mosquitoes act as vectors to carry and spread disease to humans, including malaria, chikungunya, West Nile fever, dengue fever, and Zika. Among these diseases, dengue fever is currently one of the world’s most important tropical diseases. According to the statistics from World Health Organization, 390 million individuals were infected each year, with up to 96 million symptomatic infections. Taiwan is also one of the prevalent areas of dengue fever, and a serious outbreak occurred in 2014 and 2015 with more than 50,000 cases. Although dengue vaccines have been developed recently, their effectiveness and safety remain to be further elucidated. Hence, vector control becomes an alternative strategy for dengue control. Signal peptidases, which have been shown to be required for flavivirus replication that convert secretory and some membrane proteins to their mature forms by cleaving their signal peptides. Absence of signal peptides resulted in reduced yield of Flaviviridae family members. In the dengue mosquito vector Aedes aegypti, several signal peptidases have been identified but their functions were largely unknown. To investigate the role of signal peptidases on dengue virus reproduction in Aedes aegypti, two signal peptidases gene, SPCS2 and SEC11, were silenced by reverse genetic approach. Silencing of SPCS2 and SEC11 resulted in significant reduction of dengue virus replication. Interestingly, we showed that two isoforms of dengue virus envelope protein were exhibited in the mosquito midgut, salivary gland and fat body whereas only one isoform of E protein was detected in the saliva. We speculate that two isoforms may be generated by an additional cleavage of E protein, and this cleavage is important for dengue virus replication or transmission. We therefore generated a point mutation on the predicted cleavage site of the E protein indicating that K36 may be the additional cleavage site of E protein. In the future, we will investigate the effects of signal peptidases in the cleavage of E protein.
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Lo, Yin Ping, and 羅尹萍. "Ultrastructural studies on the replication cycle of dengue virus in C6/36 cells." Thesis, 2008. http://ndltd.ncl.edu.tw/handle/22310960307911348830.
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碩士長庚大學
基礎醫學研究所
96
Dengue virus is a member of the Genus flavivirus belonging to the family Flaviviridae which comprises a single-strained RNA genome of positive polarity. The morphology of virus is spherical, enveloped, and 50 nm (mature virion) or 60 nm (immature virion) in diameter. Dengue virus usually infects host cells via receptor-mediated endocytosis followed by the release of genomic RNA after uncoating of the nucleocapsid. A single polyprotein is translated, which is cleaved into 10 proteins in consequence, within the endoplasmic reticulum during the cycle of viral replication. It has been reported that flavivirus replication occurs in a virus-induced membrane structure. In addition, the replication was initiated by endocytosis of viral particles which were fused with endosomal membranes in the cytoplasm. However, its detail has not been observed and clearly interpreted thus far. In this study, the replication process of dengue virus was investigated in C6/36 mosquito cells by using transmission electron microscopy (TEM) as well as confocal laser microscopy. The results showed that dsRNAs appeared around perinuclear regions as early as 6 h post- infection. Subsequently, numerous viral particles formed as a crystalline array in and/or adjacent to a unique structure at 6, 12, or 24 h post-infection. Presumably, the structure containing newly-formed viral particles serves as a “viral factory” in which protein translation, RNA replication, and the early stage of virion assembly may occur. Virions containing nucleocapsid were usually seen at 24 h post- infection. It was likely that virions have appropriately modified during dispensing into vacuoles or vesicle packets. A mature virion is measured 45 nm in diameter, smaller than that (60 nm) from the crystalline array. The mature virions were then transported to the cell surface, i.e., intercellular space, probably via the host secretion system. It suggested that the virus could infect neighboring cells via cell-to-cell transmission which is supposed to be efficient for virus infection between cells. The increase of tetraspanin C189 in C6/36 cells with dengue 2 virus infection was previously identified in our laboratory. In this study, using the immunofluorescen staining, C189 and den-2 virus was further demonstrated colocalizing in the cytoplasm of infected C6/36 cells at 24 h post- infection. It is speculated that C189 participate in regulating assembly, maturation, and/or releasing during infection of the virus in mosquito cells.