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Berglund, Peter. "Alphavirus vectors as recombinant vaccines /." Stockholm, 1997. http://diss.kib.ki.se/1997/91-628-2657-3.
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Skoging, Nyberg Ulrica. "Protein interactions involved in alphavirus assembly /." Stockholm, 2000. http://diss.kib.ki.se/2000/91-628-4329-x/.
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Shabman, Reed Solomon Heise Mark T. "Alphavirus evasion of type I interferons." Chapel Hill, N.C. : University of North Carolina at Chapel Hill, 2008. http://dc.lib.unc.edu/u?/etd,1879.
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Thesis (Ph. D.)--University of North Carolina at Chapel Hill, 2008.Title from electronic title page (viewed Dec. 11, 2008). "... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Microbiology and Immunology." Discipline: Microbiology and Immunology; Department/School: Medicine.
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Lim, Elisa X. "Host-pathogen interactions during alphavirus infection." Thesis, Griffith University, 2021. http://hdl.handle.net/10072/410163.
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Arthritogenic alphavirus infection causes debilitating pain in the joints and muscles with many patients experiencing such symptoms chronically. However, there is insufficient evidence to explain the underlying causes behind symptoms of persistent arthralgia and myalgia. Joint-associated tissues are the main site of inflammation during alphavirus infection, and it has been shown that alphaviruses induce damage to the cartilage and synovium. Therefore, the cell types present in these tissues play critical roles in disease pathogenesis. The findings described in this thesis contribute to the general understanding of host-pathogen interactions during alphavirus infection of joint-associated cell types. Here, the analysis of murine joints revealed chondrocytes as a target of RRV infection (Chapter 1). Further evaluation of human primary chondrocytes and skeletal muscle cells through short-term in vitro cell culture showed that these cell types could support productive RRV infection. Our study presents the first evidence of the role of chondrocytes in alphavirus disease pathogenesis. Currently, there are gaps in our understanding of chronic alphavirus disease, especially in the absence of detectable viraemia after recovery from infection. Here, we have investigated the r sponses of several cell types in joint-associated tissues during chronic infection. Human primary cells and their corresponding cell line counterparts for chondrocytes, muscle cells and fibroblast-like synoviocytes (FLS) were infected with four alphaviruses of clinical importance, namely Ross River virus (RRV), Barmah Forest virus (BFV), chikungunya virus (CHIKV) and o’nyong’nyong virus (ONNV). We found that all cell types studied were able to retain residual alphaviral nucleic acids after recovery from infection despite several passages in culture (up to 10 weeks), indicating the potential of these cell types as reservoirs for the virus and/or viral RNA (Chapter 2). Regretfully, we were unable to determine the roles of the lingering viral nucleic acids though we hypothesise that they may play roles in causing chronic inflammation. During this study, we also established persistent alphavirus infection in chondrocyte C28/I2 and muscle RD cell lines (Chapter 2) and hypothesise that these two cell types could act as potential harbours for virus evasion from the immune system. The characterisation of genetic variants present in samples from persistent infections led to the identification of several mutations which could potentially be important for alphavirus persistence. We speculate that C28/I2 and RD cell lines are suitable candidates for exploring alphavirus evolution through selective pressures applied by in vitro serial passaging of infected cells. Our findings indicate that infected chondrocytes, muscle cells and FLS contribute to alphavirus disease pathogenesis through increased expression of pro-inflammatory cytokines associated with clinical disease such as IL-6, MCP-1 and IL-8 (Chapter 1 and 2). However, further studies are required to determine if the presence of residual alphaviral nucleic acids serves as PAMPs that are responsible for eliciting chronic inflammatory responses. While we have shown that RRV-infected chondrocytes play a role in causing alphavirus-induced inflammation, we also observed that these cells cause cartilage damage through disruption of ECM homeostasis. As the main cell type of the cartilage, chondrocytes are responsible for the regulation of ECM synthesis and degradation. During RRV-infection of chondrocytes, we observed reduced gene expression of key ECM constituents COL1A1, COL2A1 and ACAN and elevated gene expression of ECM breakdown enzymes like HPSE, ADAMTS4 and MMP9 (Chapter 1, 2 and 3). We also observed evidence of this through our transcriptomic analysis of RRVinfected and uninfected bystander chondrocytes. This is also the first study that investigates the direct and indirect responses to alphavirus infection of chondrocyte (Chapter 3). As an avascular tissue type, chondrocytes are not easily accessible to virus infection. However, we found evidence of RRV RNA in the chondrocytes of infected mice (Chapter 1) and have shown that these cells are susceptible to alphavirus infection (Chapters 1-4). Therefore, we can only speculate on the possible routes of alphavirus infection of chondrocytes. The use of in vitro chondrocyte models with complex ECM architecture allows for greater physiological relevance in the study of cartilage and their responses to alphavirus infection. The synovium is a neighbouring tissue with access to the blood supply network and provides nutrients to the cartilage. Therefore, it is possible that chondrocytes can acquire alphavirus infection via the synovium. Fibroblast-like synoviocytes (FLS) are the main resident cell type of the synovium and maintains the synovial fluid through the expression of ECM components and breakdown enzymes like MMP3. We found that interactions between chondrocytes and FLS result in increased viral infectivity profiles (Chapter 4). Our study also demonstrates that the ECM surrounding the chondrocytes acts as a physical barrier that prevents access to virus particles. Treatment of cells with MMP3 was able to loosen the interactions of the ECM and expose the chondrocytes to virus infection, resulting in greater virus attachment and infectivity compared to non-treated cells. Taken together, this thesis presents key findings on the possible mechanisms involved in alphavirus disease pathogenesis and the roles of cell types of joint-associated tissues in causing the chronic symptoms of joint and muscle pain felt by the majority of infected patients.Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
Institute for Glycomics
Griffith Health
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Li, Ke-Jun. "Semliki forest virus-derived packaging system for production of retroviral vectors /." Stockholm, 1998. http://diss.kib.ki.se/1998/91-628-3268-9/.
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Gershy-Damet, Guy-Michel. "Etude épidémiologue et virologique des infections à arbovirus en Côte d'Ivoire : aspects ultrastructuraux de l'infection expérimentale du nourrisson par la souche vaccinale antiamarile." Aix-Marseille 2, 1985. http://www.theses.fr/1985AIX21901.
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Nubgan, Amer S. "The role of the deubiquitylase MYSM1 during alphavirus infection." Thesis, University of Liverpool, 2017. http://livrepository.liverpool.ac.uk/3015357/.
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The members of the genus Alphavirus are positive-sense RNA viruses and it is one of two within the family Togaviridae. Most alphaviruses are predominantly transmitted to susceptible vertebrates by a mosquito vector. Alphavirus disease in humans can be severely debilitating, and depending on the particular viral species, infection may result in encephalitis and possibly life threatening symptoms. Chikungunya virus (CHIKV) is the aetiological agent represents a substantial health burden to affected populations, with clinical symptoms that include severe joint and muscle pain, rashes, and fever, as well as prolonged periods of disability in some patients. In recent years, CHIKV has received significant attention from public health authorities as a consequence of the dramatic emergence infections in the Indian Ocean islands and the Caribbean as well as the recent emergence of CHIKV in the Americas. Infections have also been reported around Europe such as in Italy, France and Greece. Currently, no safe, approved or effective vaccine or treatment exists for CHIKV infection. The ubiquitin-proteasome system (UPS), the major intracellular proteolytic pathway, mediates different kinds of cellular processes, which may be targeted by viruses to aid their replication within cells. In recent years it has been well established that both the forward reaction of ubiquitination, and the reverse reaction of deubiquitination are targeted during virus infection to enhance their replication, either by targeting of cellular proteins or encoding viral homologues of key pathway proteins. The reverse reaction is undertaken by a large family of enzymes termed deubiquitylases or DUBs, and many of these have been shown to play a crucial role, not only in virus replication but also in the regulation of the immune system and vesicle trafficking. The DUBs are attractive drug targets and have increasingly been implicated in cellular processes germane to malignancy which makes the continued characterisation of the role of DUBs during virus infection a worthwhile objective. In on-going experiments in the research group a DUB siRNA pools library screen identified 12 DUBs (USP1, USP4, USP5, USP34, USP45, USP46, OTUD6A, UCHL1, JOSD2, BRCC3 and MYSM1). Depletion of these hits in HeLa cells lead to an increase in cell viability following Semiliki Forest Virus (SFV) infection (and predicted to be pro-viral) and thus could potential be candidate antiviral targets. Inroads into understanding the role of the DUB hits during the alphavirus infection, focusing initial on the BSL2 model virus SFV, and extending this to CHIKV (at BSL3). In the present study, further screening focused on the deconvolution siRNA pools for the DUB hits. Investigation of the subsequent follow up experiments with one strong candidate DUB from this list, MYSM1. Two different approaches were taken. Firstly, the effect of depletion of MYSM1 by siRNA treatment was further investigated in HeLa cells. Secondly, the analysis was extended to investigate the role of MYSM1 in fibroblasts utilising MYSM1 genetic knockout murine embryo fibroblasts. Results from this study indicate that depletion of MYSM1 in HeLa cells by siRNAs resulted in a reduction in both SFV and CHIKV replication, as assayed by measuring RNA levels and plaque formation. It was also found that MYSM1 genetic knockout in MEF cells lead to increase in both SFV and CHIKV replication. In addition, depletion of MYSM1 by siRNAs in MRC-5 cells lead to increase in SFV replication. In conclusion, MYSM1 generated interesting data, implying a role during virus infection that appeared to depend on the cell type being infected. Up to now it is unclear what the effector mechanisms are that contribute to these observations, subject to further mechanistic and functional studies, may increase the options available for targeting this vital DUB during Alphavirus infections.
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Plaskon, Nicole Elyse. "The Development of New Tools to Investigate Alphavirus Replication Kinetics." Thesis, Virginia Tech, 2009. http://hdl.handle.net/10919/34787.
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Members of the alphavirus genus pose a serious or potential threat to public health in many areas of the world. Nearly all alphaviruses are maintained in nature by transmission cycles that involve alternating replication in a susceptible vertebrate and invertebrate host. The maintenance of this transmission cycle depends on the establishment of a life-long persistent infection in the invertebrate vector host. Although alphavirus replication has been extensively studied in vertebrate models, the strand-specific replication kinetics of alphaviruses during persistent infections of the invertebrate host have not been reported. We investigated the strand-specific replication of different alphavirus genotypes in invertebrate cells.
By comparing different detection strategies and chemistries, we identified an optimal ssqPCR assay design for strand-specific quantification of viral RNAs in infected cells and tissues. We found that primer sets incorporating the use of a non-target tag sequence were able to avoid real-time PCR detection of amplicons that were falsely-primed during reverse-transcription. We also determined that DNA hydrolysis probes increased the sensitivity of ssqPCR assays when compared to a double-stranded DNA-specific dye, SYBR Green.
Using this information, we determined the replication kinetics of two different genotypes of o'nyong nyong virus (ONNV) and chikungunya virus (CHIKV) in infected mosquito cells. We found that (-) strand viral RNAs persisted in invertebrate cells for up to 21 days after infection. We also found that significantly less (-) strand RNA was present in cells infected with opal variants of both ONNV and CHIKV than sense variants at several time points post infection, suggesting that the opal codon has a functional role in (-) strand RNA regulation. We also report the development of an ONNV replicon expression system.
In total, the tools we developed for this report will facilitate future replication studies in the mosquito that may shed light on questions regarding the regulatory role of the opal codon and the persistence of (-) strand RNAs during long-term infections. The strand-specific replication kinetics of ONNV and CHIKV genotypes reported here will serve as a foundation for such investigations.
Master of Science in Life Sciences
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Belarbi, Essia. "Etude de la physiopathologie des infections à alphavirus arthritogènes par une approche d’imagerie in vivo." Thesis, Université Paris-Saclay (ComUE), 2017. http://www.theses.fr/2017SACLS073.
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Les alphavirus arthritogènes de la rivière Ross (RRV) et du chikungunya (CHIKV) sont des arbovirus à l’origine de maladies inflammatoires musculosquelettiques chez l'homme. Ils sont largement distribués dans le monde et provoquent périodiquement des épidémies explosives. Les principaux signes cliniques lors d’une infection par un alphavirus arthritogène sont les myalgies, polyarthrites et arthralgies intenses pouvant persister plusieurs mois après l'infection. Les mécanismes de développement de l’infection et des manifestations persistantes sont peu connus. Pour étudier la pathogenèse de l'infection par RRV, nous avons généré un virus recombinant exprimant une nouvelle luciférase brillante et brillante. Nous avons montré que les monocytes humains, malgré une faible susceptibilité à l'infection in vitro par RRV, étaient capables de maintenir une réplication virale jusqu'à 45 jours post infection indiquant leur rôle potentiel dans les formes chroniques. Grâce un modèle expérimental de l’infection par RRV, nous avons suivi les phases aiguë et chronique de la maladie in vivo. Nous avons montré que les cinétiques de réplication du virus recombinant étaient proches de celles du virus parental. Nous avons également observé un tropisme musculaire et articulaire et une corrélation entre le signal bioluminescent et la charge virale confirmant ainsi la relevance de ce modèle. En étudiant la dissémination virale, nous avons montré que le Bindarit, une molécule anti-inflammatoire diminuant le développement de la maladie dans le modèle murin, induit une plus grande réplication dans le tissu cardiaque. Enfin, nous avons pu observer une réplication virale dans les tissus musculaires durant la phase chronique de la maladie et avons montré le rôle de la dose inoculée dans le développement de la persistance virale. Suite à un traitement immunosuppresseur, nous avons observé une légère augmentation du signal bioluminescent indiquant un contrôle de la réplication virale persistante par la réponse immunitaire adaptative. Ce nouveau modèle d’imagerie in vivo permet un suivi en temps réel de la dissémination virale permettant des études de pathogenèse et l'évaluation de stratégies thérapeutiquesRoss River virus (RRV) and chikungunya virus (CHIKV) are mosquito-transmitted viruses that cause musculoskeletal inflammatory diseases in humans. They are widely distributed and periodically cause explosive epidemics. After infection with RRV, patients experience fever, maculopapular rash, myalgia and intense pain in the peripheral joints. Approximately 30% of patients develop a chronic form of the disease with myalgia and poly-arthralgia persisting for months to years after infection. The mechanisms underlying these persistent symptoms remain unclear. To study the dynamics and pathogenesis of RRV infection in vitro and in living animals, we generated a recombinant virus expressing a novel small and bright luciferase. First we showed that human monocytes, despite a low susceptibility to RRV infection, were able to maintain viral replication in vitro up to 45 days post infection. Then, using a murine model of RRV infection, we monitored the acute and chronic phases of the disease. We observed near native replication kinetics and a muscular/articular tropism after infection with our recombinant virus. Moreover, the bioluminescent signal correlated with the viral load further confirming the relevance of this new imaging model. After monitoring of the viral dissemination in live mice, we showed that Bindarit, an anti-inflammatory molecule known to prevent the development of the alphaviral disease in a mouse model, induces a higher replication in the cardiac tissue; thereby indicating that caution must be used before treatment of patients. We were also able to observe viral replication in the muscles during the chronic stage of the disease when using a low inoculation dose. Finally, following an immunosuppressive treatment, we observed a slight increase in the bioluminescent signal indicating a control of remnant viral replication by the adaptive immune response. This new model provides a non-invasive real-time assessment of viral replication and dissemination allowing pathogenesis studies and therapeutic strategies evaluation
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Castro, Ceyla Maria Oeiras de. "Análise metabolômica de alterações induzidas pelo vírus mayaro em células vero." Faculdade de Medicina de São José do Rio Preto, 2015. http://hdl.handle.net/tede/373.
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This study aimed at assessing the extracellular metabolic profile of Vero cells infected by Mayaro virus. In this metabolomic study, the use of nuclear magnetic resonance associated to multivariate analytical methods, devices of standard recognition, showed metabolic variations which can be attributed to the effect of Mayaro virus infection. Vero cells were infected and incubated for 2, 6 and 12 hour periods. Differentiated variations in the levels of several metabolites such as amino acids, organic acids, guanidine compound, monoamine, carbohydrates and fatty acids have occurred in each period. These organic compounds are metabolites involved in the glycolysis pathway, tricarboxylic acid cycle, pentose phosphate pathway, and the oxidation pathway of fatty acids (via the β-oxidation). This study demonstrates footprinting analysis representing the effect of the virus action on the Vero cell metabolism, furthermore, these analyzes point out the intracellular metabolic state, improving the knowledge of the microorganism influence on cellular metabolism.
O presente estudo tem como objetivo avaliar o perfil metabólico extracelular de células Vero infectadas pelo vírus Mayaro. Neste estudo metabolômico o uso da ressonância magnética nuclear combinado a métodos analíticos multivariados, ferramentas de reconhecimento padrão que demonstraram variações metabólicas que podem ser atribuídas ao efeito da infecção do vírus Mayaro. As células Vero foram infectadas e incubadas em períodos de 2, 6 e 12 horas. Em cada período ocorrem variações diferenciadas nos níveis de vários metabólitos, como aminoácidos, ácidos orgânicos, composto de guanidina, monoamina, carboidratos e ácidos graxos. Esses compostos orgânicos são metabólitos envolvidos na via da glicólise, ciclo do ácido tricarboxílico, via das pentoses-fosfato, e via da oxidação dos ácidos graxos (via da β-oxidação). Este estudo demonstra footprinting analysis que representa o efeito da ação do vírus no metabolismo da célula Vero, além disso, essas análises indicaram o estado metabólico intracelular, e contribuem para o conhecimento da influência do microorganismo no metabolismo celular.
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Mostafavi, Helen. "Adaptive Immunity in Alphavirus-Induced Disease – A Focus on Interleukin-17." Thesis, Griffith University, 2022. http://hdl.handle.net/10072/417290.
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Mosquito-borne arthritogenic alphaviruses such as Ross River virus (RRV) are responsible for outbreaks of arthritic disease in Australia and worldwide. RRV induces the development of painful, incapacitating musculoskeletal inflammation which can last from months to years after initial acute disease, and is mediated by host cellular immune responses. While previous studies have described the role of innate immune responses in RRV infection, the role of adaptive immune responses still remain poorly understood. In addition, no current vaccine or specific-treatment exists, with patients relying on analgesia, palliative care or disease modifying anti-rheumatic drugs. RRV shares similar characteristics with rheumatoid arthritis (RA), such as the development of arthritis, arthralgia, and the influx of proinflammatory mediators. The IL-17 signaling pathway, specifically the role of IL-17‒producing T cells, has been widely described to contribute to inflammatory responses and bone/cartilage damage in RA. Further, inhibiting IL-17 has been shown to be an effective treatment strategy in alleviating RA-associated pathology and disease severity. Therefore, in this thesis we aimed to investigate if the IL-17 pathway contributed to RRV disease by tracking it throughout the course of infection, and if inhibiting IL-17 would alleviate disease and dampen inflammatory responses in RRV-infected mice. Mice were infected with RRV, and feet and muscle tissues collected at the pre-onset phase of disease, disease onset, peak disease, and disease recovery for various analyses. Confocal microscopy and flow cytometry analysis revealed the presence of IL-17A+ and IL-17F+ cell subsets produced by CD4+ and CD8+ T cells in the feet and muscle of RRV-infected mice. These cells followed the course of disease, in that they peaked during severe disease before contracting while mice started to recover. However, we found T cells and IL-17+ T cell persisted at the recovery phase when disease had fully resolved. Interestingly, we also found that IL-17‒producing neutrophils are important contributors of IL-17 in the cellular infiltrate during peak disease. In addition, treating RRV-infected mice with an anti-IL-17A/F monoclonal antibody significantly reduced disease severity and led to decreased proinflammatory proteins, reduced cellular infiltration in synovial tissues, decreased cartilage damage at peak disease, and reduced viral titres during early infection. Further, we demonstrated that anti-IL-17A/F treatment triggered a shift in the transcriptional profile of both leukocyte infiltrates and synovial stromal cells by downregulating proinflammatory genes, and upregulating genes associated with tissue repair and cell homeostasis.
In the final component of this thesis, we investigated the role of the stromal compartment and how it interplays with the leukocyte compartment during alphavirus infection, which remains poorly understood. We therefore developed a joint and muscle organotypic cell culture model which was susceptible to RRV infection and reflected the immune responses in the leukocyte and stromal compartment observed in in vivo RRV infection. To our knowledge, this is the first organotypic cell culture model for not just RRV-induced infection, but also the first organotypic model for alphavirus-induced infection, as previous studies have relied on either the use of animal models, or monocultures of human/murine primary cells or commercial cell lines.
Altogether, this thesis highlights a previously uncharacterised role of the IL-17 signaling pathway in an animal model of acute RRV disease. We also highlight that inhibiting the IL-17A/F heterodimer using a monoclonal antibody reduced disease severity and inflammatory responses, indicating that both isoforms of IL-17 may play a role in RRV disease pathogenesis, and targeting IL-17 is a potential effective therapeutic strategy in RRV patients. We also highlight that the stromal compartment plays a major role in alphavirus infection and identify multiple stromal populations in the joint and muscle using a novel organotypic cell culture model which can also be used in future high-throughput studies to test potential treatment strategies.Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Pharmacy & Med Sci
Griffith Health
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Wimmer, Wolfgang. "Cytokines in alphavirus induced arthritis as possible targets for novel treatments." Thesis, Curtin University, 2015. http://hdl.handle.net/20.500.11937/48524.
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This study highlighted the involvement of macrophages, muscle cells and adipocytes in the pathogenesis of RRV disease and investigated the release of various cytokines from these cells during infection, such as TNFα, MIF, NO, HMGB proteins, IL-6, IL-18, IL-33, IL-10 and others. Several known cytokine inhibitors were tested for their anti-inflammatory properties in RRV infected cell lines as possible future treatment options. These inhibitors included erythromycin, clarithromycin, roxithromycin, ethyl pyruvate, pentoxifylline and resveratrol.
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Moriette, Coralie. "Le virus de la maladie du sommeil des Salmonidés : mise au point d'un ADNc infectieux et obtention d'anticorps monoclonaux." Paris 11, 2005. http://www.theses.fr/2005PA112140.
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Le Virus de la Maladie du Sommeil (VMS) a été caractérisé au laboratoire comme étant le premier alphavirus aquatique appartenant donc à la famille des Togaviridae. Au cours de ce travail de thèse nous avons poursuivi 3 objectifs : (i) élaboration d'un ADNc infectieux, (ii) génération d'un éventail d'anticorps monoclonaux contre les protéines non structurales et structurales du VMS, et cartographie de certains de ces anticorps, (iii) développement de méthodes diagnostiques pour le VMS. (i) Le génome du VMS est un ARN positif de 12 kb environ, entièrement séquencé. Il a été converti en ADNc et cloné dans un vecteur d'expression eucaryote sous le contrôle de différentes séquences promotrices : soit SP6 (dérivée du phage SP6), soit T7 (dérivée du phage T7), soit du CMV (promoteur précoce du cytomégalovirus). En premier lieu, des minigénomes dans lesquels un gène rapporteur (luciférase, GFP) remplace la région codante pour les protéines structurales ont été construits. Leur expression a été testée et mise au point en transfectant à des cellules de poisson, soit l'ARN synthétisé in vitro à partir du promoteur SP6, soit l'ADN plasmidique porteur des promoteurs CMV ou T7. De cette façon, nous avons déterminé les conditions nécessaires à la réplication du génome viral et à sa détection. Le VMS ne peut être exprimé lorsqu'il est fusionné à la séquence promotrice qui le précède, ce qui constitue une autre particularité de cet alphavirus atypique. Dans le système d'expression CMV/T7, le remplacement du gène rapporteur par la région codant pour les protéines structurales du VMS conduit à la production de particules virales réplicatives identiques au virus sauvage lorsque l'ADNc est transfecté à des cellules de poissonSleeping Disease Virus (SDV) has been characterised in our laboratory as being the first aquatic alphavirus belonging to the Togaviridae family. During this work, we pursue three objectives : (i) elaboration of an infectious cDNA, (ii) generation of a panel of monoclonal antibodies directed against structural and non structural viral proteins, (iii) development of an RT-PCR diagnostic method. (i) The SDV genome is a positive single strand of RNA of approximately 12 kb which sequence is determined. It has been converted in a full length cDNA and cloned in an eukaryotic expression vector under the control of several different promoters : either the SP6 promoter (derived from SP6 phage), or the T7 promoter (derived from T7 phage), or the CMV promoter (early promoter of cytomegalovirus). First, replicons expressing a reporter gene (luciferase, GFP) instead of the structural genes have been constructed. Expression of these replicons has been tested through transfection of fish cells using either RNA transcribed from SP6 promoter, or plasmid DNA carrying CMV or T7 promoters. It was not possible to express SDV genome when it is directly fused to the promoter. In the CMV/T7 expression system, replacement of reporter gene by the structural genes allowed the recovery of infectious viral particles when cDNA was transfected into fish cells. The virulence of this recombinant virus has been studied in vivo on juvenile rainbow trouts. We show that this virus is greatly attenuated and induce a long lasting protection. Otherwise, this system has been tested for development of a gene vector : a second transcriptional unit coding to the GFP protein has been inserted either upstream, or downstream of the structural genes
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Li, Changqing. "Insights into mRNA capping enzyme and Macro domain of alpha-like viruses." Thesis, Aix-Marseille, 2015. http://www.theses.fr/2015AIXM4082.
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Alphavirus et virus de l'hépatite E, appartiennent à l'alpha-like supergroupe de virus à ARN simple brin positif. Dans cette thèse, la caractérisation fonctionnelle de l'ARNm plafonnement enzyme et le domaine macro sont abordées, afin d'élucider leur rôle dans la réplication virale et de les évaluer en tant que cibles antivirales possibles.Les alphavirus possèdent un mécanisme unique de coiffe de l'ARNm viral impliquant la protéine non structurale nsP1. Nous présentons ici la caractérisation biochimique de nsP1d'alphavirus et son potentiel comme cible antivirale. Pour cela, différents tests enzymatiques ont été développés afin de mieux comprendre et de découpler les différentes étapes de la réaction catalysée par nsP1. Nous avons pu montrer pour la première fois chez les alphaviurs le guanylyltransfert de m7GMP sur l'extrémité 5'-diphosphate d'un ARN. Les techniques développées mises au point ont été mises à profit pour élucider le mode d'action d'une nouvelle classe d'antiviraux d'alphavirus.Le Macro domaine est un domaine protéique ancien et conservé et largement distribué dans tout le règne vivant. Nous déclarons que le domaine Macro de virus de l'hépatite E sert une protéine hydrolase ADP-ribose pour inverser la protéine ADP-ribosylation. L'abolition de l'activité diminue considérablement la réplication d'un réplicon sub-génomique du VHE. L'activité est également présente dans les macro domaines du SRAS-CoV et VEEV. Nos résultats montrent que les macro domaines viraux servent ADP-ribose protéine hydrolase et jouent un rôle important dans la réplication virale, peut-être grâce à la modulation de la réponse antivirale de l'hôteAlphavirus and Hepatitis E virus, belong to alpha-like supergroup of positive single stranded RNA viruses. In this thesis, the functional characterization of mRNA capping enzyme and Macro domain are addressed, in order to elucidate their role in the viral replication and to evaluate them as possible antiviral targets. Part I: Alphaviruses are known to possess a unique viral mRNA capping mechanism involving the viral non-structural protein nsP1. Here we report the biochemical characterization and antiviral investigation of alphavirus nsP1. Different enzymatic assays were developed to further understand and uncouple the different reaction steps catalyzed by nsP1. The final guanylyltransfer of m7GMP onto a 5'-diphosphate RNA oligonucleotide was observed for the first time in vitro with an alphavirus nsP1. Taking advantage of the developed techniques, the mode of action of a novel class of alphavirus antivirals, [1,2,3]triazolo[4,5-d]pyrimidin-7(6H)-ones, was deciphered. Part II: Macro domain is an ancient and highly evolutionarily conserved protein domain widely distributed throughout all kingdoms of life. Here, we report that the Macro domain from hepatitis E virus serves as an ADP-ribose protein hydrolase to reverse protein ADP-ribosylation. Abbrogation of this activity dramatically decreases replication of a HEV sub-genomic replicon. This activity is also present in Macro domains from SARS-CoV and VEEV virus. Collectively, our results show that viral Macro domains serve as ADP-ribose protein hydrolase and play important roles in viral replication, possibly through modulating the antiviral host response
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Serra, Otacília Pereira. "Arbovírus dos gêneros Flavivirus e Alphavirus em culicídeos capturados em Cuiabá, Mato Grosso." Universidade Federal de Mato Grosso, 2015. http://ri.ufmt.br/handle/1/694.
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Arbovírus são transmitidos por artrópodes hematófagos, representando um problema de saúde pública em áreas tropicais. O objetivo deste estudo foi investigar a diversidade de espécies de culicídeos e sua frequência de infecção por Alphavirus e Flavivirus em Cuiabá, MT. Foram realizadas capturas com aspirador de Nasci e puçá entre janeiro e abril de 2013 em três locais de 200 setores censitários, definidos aleatoriamente. Os culicídeos foram identificados com chave dicotômica de Forattini, alocados em pools (1-20 mosquitos) segundo sexo, espécie, data, local de coleta e armazenados a -80˚C. Pools de fêmeas foram submetidos à extração de RNA e DNA total, à multiplex semi-nested-RT-PCR para cinco alphavírus e 11 flavivírus e Nested- PCR para identificação de Culex (Cx.) quinquefasciatus. Amostras positivas para SLEV, DENV-1, -4 e MAYV foram submetidas a single RT-PCR e sequenciamento nucleotídico. Pools positivos para o MAYV foram inoculados em células Vero e submetidos a RT-PCR para o gene de envelope E1 dos alphavirus. Pools positivos para flavivirus foram inoculados em células C6/36 (Flavivirus). Calculou-se a taxa de infecção mínima (MIR). Foram capturados 11.090 mosquitos, 4.556 fêmeas de 14 espécies, perfazendo 610 pools. Foram analisados 171 pools de Aedes (Ae.) aegypti; 1 Ae. albopictus; 1 Aedes sp.; 5 Cx. bidens/interfor; 1 Cx. spinosus; 403 Cx. quinquefasciatus; 1 Galindomyia sp; 6 Limatus sp; 2 Mansonia wilsoni; 5 Psorophora (Ps.) sp; 1 Ps. ciliata; 11 Ps. varipes/albigenu; 1 Sabethes chloropterus e 1 Uranotaenia sp. Encontrou-se 1/171 (MIR=0,92) pool de Ae. aegypti positivo para DENV-1; 1/403 (MIR= 0,2) Cx. quinquefasciatus para SLEV genótipo V-A, 12/403 (MIR=3,5) de Cx. quinquefasciatus, 4/171 (MIR=3,67) de Ae. aegypti para MAYV; destes, cinco pools apresentaram co-infecção com DENV-4. Um produto de 1,3 kb obtido com o protocolo para o gene de envelope de três pools positivos para o MAYV resultou em sequências nucleotidicas inespecíficas. O MAYV foi isolado de dois pools contendo duas fêmeas não ingurgitadas de Ae. aegypti (#958) e duas de Cx. quinquefasciatus (#489). Positividade para DENV-4 foi identificada em 58/171 (MIR=53,35) Ae. aegytpi, 105/403 (MIR=30,65) Cx. quinquefasciatus, 2/5 (MIR=400) Psorophora sp, 2/11 (MIR=142,85) Ps. varipes/albigenu, 1/1 (MIR= 1000) Sabethes chloropterus, 2/5 (MIR=285.7) Cx. bidens/interfor e 1/1 (MIR=1000) Aedes sp. O DENV-4 foi isolado de dois pools contendo três (#329) e 16 (#806) fêmeas de Cx. quinquefasciatus não ingurgitadas. O SLEV, MAYV e os sorotipos do DENV foram identificados em pacientes com suspeita de dengue na cidade de Cuiabá em estudos prévios do Laboratório de Virologia. Experimentalmente, Culex e Aedes spp. são vetores competentes do MAYV. A identificação do vírus em fêmeas não ingurgitadas sugere que estas espécies podem estar envolvidas no ciclo urbano do MAYV em Cuiabá. Dentre os sorotipos do DENV, somente o DENV-1 e o DENV-4 foram identificados em culicídeos. O DENV-4 tem produzido importantes epidemias em MT desde 2012. A positividade para DENV-4 em diferentes espécies de culicídeos pode ser decorrente de infecção natural ou da hematofagia em humanos, pois muitos destes pools apresentavam fêmeas ingurgitadas. Cuiabá possui ecossistema favorável para a ocorrência de arboviroses e proliferação de vetores. Estudos envolvendo vigilância entomológica e virológica são importantes para estimar a situação epidemiológica dos arbovírus no estado.
Arbovirus are transmitted by hematophagous arthropods, posing a public health issue in tropical areas. The aim of this study was to investigate the diversity of culicidae and their frequency of infection by arboviruses from Alphavirus and Flavivirus genus in Cuiabá, MT. To achieve that, culicids were captured with Nasci aspirators and hand net between January and April 2013 in three locations of 200 censitary sectors randomly defined. The specimens were identified using the Forattini dichotomy key, allocated in pools (1-20 mosquitoes), according to sex, species, day and place of capture, and stored at -80˚C. Female pools were subjected to total RNA and DNA extraction and to multiplex semi-nested-RT-PCR for five alphaviruses and 11 flaviviruses and Nested-PCR for Culex (Cx.) quinquefasciatus identification. The pools positive for SLEV, DENV-1, -4 and MAYV were subjected to single RT-PCR and nucleotide sequencing. MAYVpositive pools were inoculated in Vero cells and subjected to RT-PCR for the E1 envelope gene of alphaviruses. Pools positive for flaviviruses were inoculated in C6/36 cells. The minimum infection rate (MIR) was calculated. 11,090 mosquitoes were captured, 4,556 females belonging to 14 species, comprising 610 pools, 171 pools of Aedes (Ae.) aegypti specimens; 1 Ae. albopictus; 1 Aedes sp.; 5 Cx. bidens/interfor; 1 Cx. spinosus; 403 Cx. quinquefasciatus; 1 Galindomyia sp.; 6 Limatus sp.; 2 Mansonia wilsoni; 5 Psorophora sp.; 1 Ps. ciliata; 11 Ps. varipes/albigenu; 1 Sabethes chloropterus e 1 Uranotaenia sp. Among them, 1/171 (MIR=0.92) Ae. aegypti pool was positive for DENV-1 and 1/403 (MIR=0.3) Cx. quinquefasciatus for SLEV genotype V-A. For MAYV, 12/403 (MIR=3.5) Cx. quinquefasciatus, 4/171 (MIR=3.67) Ae. aegypti were positive, five of them were also infected by DENV-4. A DNA product with 1.3 kb obtained from three pools positive for MAYV with the protocol for the envelope gene resulted in unspecific nucleotide sequences. MAYV was isolated from two pools, both containing two non-engorged females of Ae. aegypti (#958) and Cx. quinquefasciatus (#489). DENV-4 was detected in 58/171 (MIR=53.35) Ae. aegytpi, 105/403 (MIR=30,65) Cx. quinquefasciatus, 2/5 (MIR=400) Psorophora sp., 2/11 (MIR=142.85) Ps. varipes/albigenu, 1/1 (MIR= 1000) Sabethes chloropterus, 2/5 (MIR=285.7) Cx. bidens/interfor and 1/1 (MIR=1000) Aedes sp. DENV-4 was isolated from two pools containing three (#329) and 16 (#806) non-engorged females of Cx. quinquefasciatus. The SLEV, MAYV and the four DENV serotypes were identified in patients suspected of harboring dengue infection in Cuiabá in previous studies of the Virology Laboratory. Experimentally, Culex and Aedes spp. are competent vectors for MAYV. The identification of the virus in nonengorged females suggests these species may be involved in the urban cycle of MAYV in Cuiabá. Among the DENV serotypes, only DENV-1 and DENV-4 were identified in culicids captured in the city. DENV-4 has been responsible for major outbreaks in MT since 2012. The identification of DENV-4 in several mosquito species might be resultant either from natural infection or hematophagy in humans, since several of these pools presented engorged females. Cuiabá presents a favorable ecosystem for the occurrence of arboviruses and vector proliferation. Studies involving entomological and virological surveillance are important to estimate the epidemiological situation of arboviruses in the state.
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Fleeton, Marina N. "Genetic vaccination against acute viral disease /." Stockholm, 1999. http://diss.kib.ki.se/1999/91-628-3811-3/.
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Duggan, Jacqueline Marie. "Production and evaluation of recombinant single-chain antibodies for the detection of Venezuelan equine encephalomyelitis virus." Thesis, Oxford Brookes University, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.323908.
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Pastorino, Boris. "Etude du complexe protéasique des flavivirus et des alphavirus : caractérisations enzymatiques et recherche de cibles cellulaires." Aix-Marseille 2, 2008. http://www.theses.fr/2008AIX20671.
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Cresson, Marie. "Study of chikungunya virus entry and host response to infection." Thesis, Lyon, 2019. http://www.theses.fr/2019LYSE1050.
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Les alphavirus sont un groupe de virus enveloppés à ARN simple brin positif retrouvés sur la totalité du globe et responsables de nombreuses maladies humaines et animales. Durant la dernière décennie, une réémergence du virus du chikungunya (CHIKV) a été observée causant de nombreuses épidémies sur tous les continents. Malgré les nombreuses études, les mécanismes moléculaires de réplication du CHIKV et les interactions hôte-virus restent peu caractérisées. L’objectif de mon travail était de mieux comprendre et caractériser l’entrée du virus du chikungunya et les facteurs de l’hôte impliqués dans la réplication chez les mammifères. Plusieurs approches distinctes ont été utilisées dans ce projet. Dans un premier temps, nous avons mis en avant une diminution de l’infection du CHIKV après un traitement avec du fer sous forme de citrate d’ammonium ferrique et nous avons étudié le rôle potentiel dans l’entrée virale de NRAMP2 et TFRC, deux protéines impliquées dans le transport cellulaire du fer et connus comme récepteurs d’entrée de plusieurs virus. D’autre part, nous nous sommes intéressés à deux autres protéines, CD46 et TM9SF2, identifiés à travers un criblage par ARNi réalisé en collaboration, dans le but de déterminer si elles sont utilisées comme facteurs d’entrée par le virus du chikungunya. Dans un dernier axe, nous avons mis en place et réaliser un criblage perte de fonction sur le génome entier en utilisant la technologie CRISPR/Cas9 afin d’identifier des facteurs de l’hôte importants pour l’entrée du CHIKV, sa réplication ou la mort viro-induite. Bien qu’il soit apparu que l’approche utilisée pour le criblage devrait être optimisée, nous avons pu identifier des candidats potentiellement nécessaires pour l’infection par le CHIKV. Ces candidats sont testés individuellement afin de confirmer leur implication dans la biologie du virusAlphaviruses are a group of enveloped, positive-sense RNA viruses which are distributed almost worldwide and are responsible for a considerable number of human and animal diseases. Among these viruses, the Chikungunya virus (CHIKV) has recently re-emerged and caused several outbreaks on all continents in the past decade. Despite many studies, molecular mechanisms of chikungunya virus replication and virus-host interactions remain poorly understood. The aim of my project was to better understand and characterize the CHIKV entry and the host factors involved during replication steps in mammals. Several different approaches have been used in this work. As a first step, we have demonstrated a decrease of CHIKV infection after iron treatment in form of ferric ammonium citrate and we have studied the potential role in viral entry of NRAMP2 and TFRC, two proteins involved in iron transport and known receptors for other viruses. On the other hand, we have also focused on two proteins, CD46 and TM9SF2, identified through an RNAi screen in collaboration, in order to determine if they are required as entry factors for chikungunya virus. In a last axis, we have set up and carried out a genome-wide loss of function screen with the CRISPR/Cas9 technology in order to identify host factors important for chikungunya virus entry, replication or virus-induced cell death. Although it appears that screen conditions should be optimized, we have identified potential candidates required for CHIKV infection and we are currently testing them
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Storm, Nadia. "Epidemiology of Sindbis fever in South Africa and development of a real-time pan-alphavirus PCR assay for the detection of Sindbis and other medically important alphaviruses." Diss., University of Pretoria, 2013. http://hdl.handle.net/2263/30942.
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The genus Alphavirus in the family Togaviridae consists of approximately 30 single
stranded, positive-sense RNA viruses which cause diseases ranging from mild, febrile
illness with rash and arthritis to life-threatening encephalitis. Sindbis virus is the prototype virus for this genus and is the most widely distributed alphavirus globally.
Little epidemiological data exists for this virus due to inadequate surveillance efforts
in most countries, and it is suspected that Sindbis and other alphavirus infections are
largely underreported. Additionally, changing patterns of alphavirus infections are
challenging conventional knowledge of these diseases and have created the need for
improved diagnostic methods and increased vigilance and disease surveillance around
the globe.
To date, no real-time PCR assay exists that is able to detect all of the medically
important (and other) alphaviruses. This study was therefore aimed at developing and
evaluating such an assay in order to improve the diagnostic and surveillance activities
of these viruses in South Africa. Additionally, this study was aimed at providing
novel information on the molecular and epidemiological characteristics of Sindbis
virus in South Africa, and to provide insights in the diversity of this virus compared to
elsewhere in the world.
This study has revealed that an outbreak of Sindbis fever occurred during 2010, with
the Free State and Northern Cape Provinces being most affected. The outbreak
coincided with an outbreak of Rift Valley fever. The risk for acquiring a Sindbis
virus infection was found to be highest during late summer/early autumn, and was
slightly higher among males of increasing age. The phylogenetic results obtained in
this study have demonstrated that Sindbis isolates form five separate groups with a
considerable amount of genetic variation among these groups. The grouping of the
isolates corresponded to the major migratory patterns of birds, suggesting that birds
may play a large role in the dispersal of Sindbis viruses between continents. In
addition, a real-time reverse transcription PCR assay has been developed which is
able to detect Sindbis virus and other medically important alphaviruses with varying
sensitivities. This project contributes to the update of epidemiological and
phylogenetic data on Sindbis virus in South Africa and delivers potential tools for
improved surveillance activities of alphaviruses in future.Dissertation (MSc)--University of Pretoria, 2013.
Microbiology and Plant Pathology
MSc
Unrestricted
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Zweerink, Susanne [Verfasser]. "Investigations into Interferon Response of Novel Bat Cell Cultures upon Alphavirus Infection / Susanne Zweerink." Bonn : Universitäts- und Landesbibliothek Bonn, 2013. http://d-nb.info/1044970987/34.
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Herath, Tharangani K. "Cellular and molecular pathogenesis of Salmonid alphavirus 1 in Atlantic salmon Salmo salar L." Thesis, University of Stirling, 2010. http://hdl.handle.net/1893/2325.
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Salmonid alphaviruses (SAV) are a group of viruses that have recently emerged as a serious threat to the salmonid aquaculture industry in Europe. Over recent years, diseases caused by SAV have severely hampered the Scottish, Irish and Norwegian Atlantic salmon industry, and are considered to be among the major economically important viral diseases affecting the industry at present. Amongst the six subtypes characterised so far, Salmonid alphavirus 1 (SAV1) causes severe pathology in the heart, pancreas and the skeletal muscle of Atlantic salmon leading to death and growth retardation in the affected fish. The biochemical characteristics of the virus and the sequential pathology of the diseases caused by SAV have been described; however the mechanisms responsible for causing the disease and the host defence mechanisms against the virus are poorly defined. This thesis therefore examined the pathogenesis of SAV infection at the cellular and molecular level in vivo in salmon and in vitro in salmonid cells, with a special emphasis on host immune defence mechanisms against the virus. SAV was first isolated from Chinook salmon embryo-214 (CHSE-214) cells in 1995 in Ireland. Several cell lines have since been used to grow the virus. In the present study, three established salmonid cell lines, Chum salmon heart -1 (CHH-1), CHSE-214 and Salmon head kidney -1 (SHK-1) were evaluated for their ability to support the isolation of SAV-1 from infected fish tissue, with CHH-1 cells giving the fastest cytopathic effect (CPE) during primary isolation. The CPE appeared as localised cell-rounding on CHH-1 and CHSE-214 cells, although in SHK-1 cells, the cells were seen to slough off the monolayer relatively later than with the other two cell lines during the infection. The host response to SAV infection was evaluated by experimentally infecting Atlantic salmon parr using a cell culture-adapted virus isolate. A quantitative reverse transcription polymerase chain reaction (qRT-PCR) was developed to examine the virus load in the fish, from which it was found that the highest viral RNA copy number was detected at 5 day post infection (d.p.i), of the 90 day experimental infection period. Characteristic pathological lesions were only seen in the pancreas and the heart but not in the skeletal muscles of the infected fish. A gene expression study using qRT-PCR revealed the rapid induction of interferon (INF) and INF-associated genes in the head kidney of the infected fish compared to the control fish. The Mx protein was found to be highly expressed in the heart and the mucous membranes of infected fish by immunohistochemistry. Interestingly, the pathological changes that were seen occurred some time after the peak expression of genes associated with the INF-1-pathway. When the host-virus interaction of Atlantic salmon infected with SAV was examined using a microarray, a potent first line defence response was observed, together with the signatures of early activation of the adaptive immune response during the initial stages of the infection. Genes associated with transcription, translation and lipid metabolism were significantly differentially expressed in virus infected fish compared to control fish. A large array of antiviral genes was significantly expressed, amongst which were some of the genes also described in mammalian alphavirus infections. Genes associated with apoptosis and anti-apoptosis were also seen to be differentially regulated showing the complexity of the host-virus interaction. Collectively, all of these findings suggest that a non-specific antiviral immune response takes place providing rapid immune protection during the early stages of SAV infection in salmon. In the study on morphogenesis of SAV in salmonid cells using electron microscopy (EM), a rapid internalization of virus into the cells and generation of replication complexes using the secretory pathway of the cell, similar to mammalian alphavirus replication was observed. The mature viruses were released through surface projections, acquiring envelopes from the host cell membrane. From the ultrastructural studies of the salmonid cells infected with SAV, a progressive chromatin marginalisation and condensation could be seen, leading to cellular fragmentation, forming membrane bound apoptotic bodies, characteristic of progressive apoptosis. The activation of caspase-3 in the cytoplasm and genomic DNA damage were also seen in the infected fish cells, indicating that apoptosis is the main cause of cell death during SAV infection. The results of this study have increased our knowledge and understanding of the cellular and molecular mechanisms involved in the pathogenesis of SAV infection, emphasising the importance of the first line defence mechanisms against SAV infection in salmon. This has given an interesting insight into the host mechanisms used to combat the virus during infection, and will undoubtedly be useful for designing new vaccines and management strategies for prevention and control of this important disease
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Ferguson, Mhairi Catriona. "Mammalian cell stress responses during Semliki Forest virus infection." Thesis, University of Edinburgh, 2013. http://hdl.handle.net/1842/8188.
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Virus infection of mammalian cells induces several stress mechanisms, including autophagy and type-I interferon (IFN). Autophagy, a cellular homeostatic mechanism in which intracellular materials are sequestered into double-membrane vesicles and targeted to lysosomes for degradation, is also activated in response to virus infection. Most positive single-stranded RNA viruses studied to date utilise autophagy to increase virus replication. IFN is a potent anti-viral mechanism, which can be divided into two parts: (i) induction and secretion of IFN and (ii) IFN signalling and priming of uninfected cells for a rapid response upon infection and induction of an anti-viral state in infected cells. Alphaviruses are medically important RNA viruses. Semliki Forest virus (SFV) provides a well-characterised model for studying alphavirus infection. A number of strains have been identified, which differ in virulence in adult mice. In this thesis three hypotheses were investigated: (i) that SFV infection induces autophagy in cell culture and utilises this response to enhance virus replication, (ii) that the quality, quantity and/or protective efficacy of the IFN response differ between virus strains and between human and murine cells and (iii) that non-structural protein (nsP)-2 and/or nsP3 antagonise the IFN response. SFV4, SFV L10 and SFV A7(74) infection induced autophagy in Huh7 cells as early as one hour post-infection. Pharmacological induction or inhibition of autophagy had no affect on SFV4 replication, except at a very low multiplicity of infection. NsP3, capsid and dsRNA rarely colocalised with the autophagosome marker LC3. Taken together these results indicate that SFV does not use autophagosomes for replication and autophagy is not important in controlling SFV4 infection at a high MOI, at least in Huh7 cells. However, autophagy may be important in controlling SFV4 spread at a low MOI. An IFN bioassay was established. In fibroblasts, SFV4, SFV L10 and SFV A7(74) induced relatively little IFN in comparison to that induced by Sendai virus. In human fibroblasts, similar levels of IFN were induced by all three virus strains. In mouse fibroblasts, SFV4 induced more IFN than SFV L10. Treatment of fibroblasts with IFN prior to infection greatly reduced, but did not abolish, the replication and spread of all three strains. Therefore, SFV is sensitive to IFN. Analysis of IFN signalling demonstrated that all three strains of SFV inhibited STAT1 phosphorylation during infection of fibroblasts. The growth and viability of SFV infected cells varied between human and mouse cells. The complete genetic sequences of SFV L10 and SFV A7(74) were determined using Solexa (Illumina) sequencing and compared to the sequence of SFV4. The sequences of SFV L10 and SFV4 were extremely similar; only seven differences were identified. Multiple amino acid substitutions were identified in SFV A7(74) compared to SFV4, these mostly mapped to nsP3. To investigate the hypothesis that nsP2 and or nsP3 antagonise the IFN response, two virus mutants were studied: SFV4nsP2RDR and SFV4nsP3Δ50. SFV4nsP2RDR encodes a point mutation in the nuclear localisation signal of nsP2, which largely restricts nsP2 to the cell cytoplasm. SFV4nsP3Δ50 contains a deletion of 50 amino acids in the C-terminus hyperphosphorylated region of nsP3. Neither mutant inhibited STAT1 phosphorylation as efficiently as WT SFV4; SFV4nsP2RDR was particularly poor at inhibiting STAT1 phosphorylation. Both mutants induced more IFN in fibroblasts than SFV4. In summary, autophagy had a limited affect on SFV replication. In contrast, strains of SFV were highly sensitive to IFN, but antagonised this response through the nsP2 protein inhibiting STAT1 phosphorylation.
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Rückert, Claudia. "Alphavirus and flavivirus infection of Ixodes tick cell lines : an insight into tick antiviral immunity." Thesis, University of Edinburgh, 2014. http://hdl.handle.net/1842/10063.
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Arthropod-borne viruses, arboviruses, have the ability to replicate in both vertebrates and invertebrates and are transmitted to susceptible vertebrate hosts by vectors such as mosquitoes and ticks. Ticks are important vectors of many highly pathogenic arboviruses, including the flavivirus tick-borne encephalitis virus (TBEV) and the nairovirus Crimean-Congo haemorrhagic fever virus. In contrast, alphaviruses are principally mosquito-borne and have been isolated only rarely from ticks; ticks have not been implicated as their vectors. Nevertheless, the alphavirus Semliki Forest virus (SFV) replicates in cell lines derived from many different tick species, including those of the genus Ixodes, which includes vectors of TBEV and its lesspathogenic relative Langat virus (LGTV). In vertebrate cells, arboviruses generally cause cytopathic effects; however, arbovirus infection of arthropod cells usually results in a persistent low-level infection without cell death. While little is known about antiviral immunity in tick cells, the immune system of other arbovirus vectors such as mosquitoes has been studied extensively over the last decade. In insects, pathways such as RNA interference (RNAi), JAK/STAT, Toll, Imd and melanisation have been implicated in controlling arbovirus infection, with RNAi being considered the most important antiviral mechanism. In tick cells, RNAi has been shown to have an antiviral effect, but current knowledge of other immunity pathways is limited and none have been implicated in the antiviral response. In the present study, SFV and LGTV replication in selected Ixodes spp. tick cell lines was characterised and the Ixodes scapularis-derived cell line IDE8 was identified as a suitable cell line for this project. Potential antiviral innate immunity pathways were investigated; putative components of the tick JAK/STAT, Toll and Imd pathways were identified by BLAST search using available sequences from well-studied arthropods including the fruit fly Drosophila melanogaster. Using gene silencing, an attempt was made to determine whether these pathways play a role in controlling SFV and LGTV infection in tick cell lines. Selected genes were silenced in IDE8 cells using long target-specific dsRNA and cells were subsequently infected with either SFV or LGTV. Effects of gene silencing on virus replication were assessed by quantitative real time PCR (qPCR) or luciferase reporter assay. Effects on infectious virus production were measured by plaque assay. Replication of the orbivirus St Croix River virus (SCRV), which chronically infects IDE8 cells, was also quantified by qPCR after silencing of selected genes. Interestingly, SFV or LGTV infection of IDE8 cells resulted in a significant increase in SCRV replication, possibly as a result of interference with antiviral pathways by SFV and LGTV or possibly due to diversion of cellular responses from sole control of SCRV. No evidence for an antiviral role for the JAK/STAT or Toll pathways was found in IDE8 cells. However, an antiviral effect was observed for protein orthologues putatively involved in the RNAi response. Argonaute proteins play an important role in translation inhibition and target degradation mediated by RNAi, and silencing of selected Argonaute proteins resulted in a significant increase in SFV and SCRV replication. The carboxypeptidase CG4572 is essential for an efficient antiviral response in D. melanogaster, and supposedly involved in the systemic RNAi response. A putative tick orthologue of CG4572 was identified and this appeared to be involved in the antiviral response in IDE8 tick cells. When expression of CG4572 was silenced and cells subsequently infected with SFV or LGTV, replication of both viruses was significantly increased. In addition, it was shown that three mosquito orthologues of CG4572 also had an antiviral role against SFV in Aedes mosquito cells. In conclusion, of the tick cell lines investigated, IDE8 provided a suitable model system for investigating tick cell responses against arboviruses and new insight into the nature of the tick cell antiviral response was gained.
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Zuchi, Nayara. "Investigação molecular de alphavirus em pacientes febris durante epidemia de dengue em Mato Grosso, Brasil." Universidade Federal de Mato Grosso, 2014. http://ri.ufmt.br/handle/1/493.
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Introdução: O gênero Alphavirus, família Togaviridae, alberga arbovírus de importância médica relatados em áreas tropicais mundialmente. Nas Américas, os alfavírus de maior importância compreendem os das encefalites equinas e o vírus Mayaro (MAYV). No Brasil, o MAYV tem sido relatado em epidemias de doença febril principalmente no norte do país. O principal objetivo deste estudo foi investigar a situação epidemiológica de alfavírus em pacientes febris durante epidemia de dengue em Mato Grosso (MT). Material e Métodos: Entre 2011 e 2012, 604 amostras de soro de pacientes com doença febril aguda suspeita de dengue durante epidemia em MT foram submetidas a Duplex-RT-PCR seguida de Multiplex-semi-nested-PCR para pesquisa dos alfavírus MAYV, vírus Aura e os vírus das encefalites equinas do Leste, Oeste e Venezuelana. Amostras positivas foram confirmadas em dois testes independentes e os produtos de PCR submetidos a sequenciamento nucleotídico. Amostras positivas foram submetidas a RT-PCR em tempo real (RT-qPCR) e isolamento viral em cultura de células. Todas as amostras foram também investigadas para flavivirus em um estudo paralelo. Resultados: Foram encontrados 15/604 (2,5 %) pacientes positivos para o MAYV em Cuiabá (9), Várzea Grande (3), Nossa Senhora do Livramento (1) e Sorriso (2). Destes, 12 (80,0 %) apresentaram co-infecções com DENV-4 e 3 (20,0 %) infecções únicas pelo MAYV. Dentre 13 amostras submetidas a RT-qPCR, 10 (76,9 %) apresentaram carga viral entre log 0,965-3,321 cópias/μL. Discussão: Casos esporádicos de infecção pelo MAYV foram identificados durante uma grande epidemia de dengue no MT em residentes de áreas urbanas, sem histórico recente de viagem ou visita a áreas rurais e/ou silvestres. A ocorrência do MAYV em estados adjacentes, em cidades afetadas pela rodovia Cuiabá-Santarém e soroprevalência em índios Xavantes no estado corroboram a evidência da circulação de MAYV no MT. Apesar do MAYV ser transmitido principalmente por Haemagogus janthinomys em áreas silvestres, as evidências encontradas no presente estudo sugerem a circulação de MAYV em área urbana de MT. Contudo, o ciclo de transmissão do vírus no estado não foi elucidado. A evidência de circulação do MAYV em indivíduos febris durante epidemia de dengue em área urbana deve ser motivo de atenção das autoridades locais de saúde pública para a eventual circulação silenciosa de outros arbovírus no estado.
Introduction: The Alphavirus genus, Togaviridae family, comprises arboviruses of medical importance reported in tropical areas worldwide. In the Americas, the most important alfaviruses are the equine encephalitis group and Mayaro virus (MAYV). In Brazil, MAYV has been reported in outbreaks of febrile illness mainly in the North region of the country. The aim of this study was to investigate the epidemiological situation of alfaviruses in febrile patients during a dengue outbreak in Mato Grosso (MT). Material and methods: Between 2011 and 2012 in MT, 604 serum samples collected from patients suspected of acute febrile illness were submitted to Duplex-RT-PCR followed by Multiplex-semi-nested-PCR for MAYV, Aura virus and East, West and Venezuelan equine encephalitis viruses. Positive samples were confirmed twice in independent tests and, PCR products were submitted to nucleotide sequencing. Positive samples were also submitted to Real time RT-PCR (RT-qPCR) and inoculation in cell culture. The samples were also investigated for flaviviruses in a parallel study. Amostras positivas foram submetidas a RT-PCR em tempo real (RT-qPCR) e isolamento viral em cultura de células. Todas as amostras foram também investigadas para flavivirus em um estudo paralelo. Results: 15/604 (2.5 %) patients from Cuiabá (9), Várzea Grande (3), Nossa Senhora do Livramento (1) and Sorriso (2) were positive for MAYV; 12 (80 %) are co-infected with DENV-4 and 3 (20 %) are single infections with MAYV. Co-infected patients presented a wider variety of clinical manifestations. Among 13 samples tested by RT-qPCR, 10 (76.9 %) presented viral load ranging from log 0,965-3,321 copies/μL. Discussion: Sporadic infections with MAYV were identified during a massive Dengue outbreak in MT in residents of urban areas without recent history of travel or visit to rural or sylvatic areas. The occurrence of MAYV infections in neighboring states, including cities affected by the Cuiabá-Santarém highway and seroprevalence in Xavante Indians from MT, corroborate the evidence of MAYV circulation in MT. Despite MAYV is transmitted mainly by Haemagogus janthinomys in sylvatic areas, the evidence found in this study suggests the circulation of MAYV in urban areas of MT. However, the transmission cycle of MAYV in MT remains to be determined. The evidence of MAYV circulation in febrile individuals during a dengue outbreak in urban areas should cause concerns in the local public health authorities about the eventual silent circulation of arboviruses in the state.
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Mai, Junbo. "Alphavirus nsP2 interacts with Host Pathways for Viral Minus-Strand Synthesis and Replication Complex Stability." University of Toledo Health Science Campus / OhioLINK, 2009. http://rave.ohiolink.edu/etdc/view?acc_num=mco1262819232.
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Boussier, Jeremy. "Chikungunya Virus Superinfection Exclusion and Defective Viral Genomes : Insights into Alphavirus Regulation of Genetic Diversity." Thesis, Sorbonne Paris Cité, 2018. http://www.theses.fr/2018USPCC181/document.
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Les arbovirus (dont le virus chikungunya, CHIKV) sont responsables de millions d'infections chaque année ; aucun vaccin n'est encore approuvé, et les traitements disponibles restent limités. De part leur circulation constante entre le moustique et l'humain, leur adaptation rapide à différents hôtes est un facteur clé pour leur pathogenèse. Le taux d'erreur particulièrement élevé de leur polymérase ARN permet une rapide diversification génique qui conduit à la génération d'un nuages de mutants, appelée quasi espèce. Les quasi espèces contiennent non seulement des génomes mutés, mais aussi des ARN recombinés à partir de deux génomes d'origine différente, ainsi que des génomes avec de grandes délétions, incapables de se répliquer sans l'aide d'un autre virion qui doit infection la même cellule, nommés génomes viraux défectifs (GVD). Une régulation étroite de la taille du nuage de mutants est clé pour une pathogenèse efficace : si trop petit, le potentiel adaptatif du virus sera impacté ; au contraire, une quasi-espèce trop grande peut mener à l'accumulation rapide de mutations délétères pour le virus. Alors que la régulation du paysage mutationnel est atteinte grâce à un taux d'erreur de la polymérase viral finement contrôlé, la recombinaison et la réplication des génomes défectifs sont influencés par le potentiel de co-infection des cellules cibles. Dans ce contexte, l'exclusion de la surinfection (ESI), un processus par lequel l'infection par un premier virus inhibe l'infection par un second virus, peut influer le dynamique de la quasi-espèce. Bien que décrite dans la plupart des familles virales, les mécanismes à l'origine de l'ESI restent mal caractérisés. Dans ce travail, je montre que CHIKV exclut une infection future par CHIKV, mais aussi par le virus Sindbis et le virus de la grippe A, mais non par le virus du Nil occidental. Je démontre que l'exclusion de CHIKV se situe au niveau de la pénétration du génome viral dans le cytoplasme, puis de sa réplication, mais n'influence ni l'attachement du virion ni la traduction de son génome. Je montre également que l'ESI est indépendant de l'action des interférons de type I, et qu'elle n'est médiée ni par la transcription cellulaire, ni par un facteur soluble. De plus, l'exclusion n'est pas due à une unique protéine virale, suggérant un potentiel rôle de la réponse cellulaire à l'infection.Déterminer l'influence des pressions immunologiques dans l'établissement de la quasi-espèce peut aider à une meilleure compréhension de l'interaction entre évolution virale et réponse immunitaire. Bien que la caractérisation non biaisée des mutations ponctuelles fût le fruit de nombreux travaux, les GVD restent peu caractérisées, en particulier chez les alphavirus. Dans la deuxième partie de ce travail, je développe des outils bio-informatiques pour isoler rapidement les GVD de données de séquençage à haut débit, et analyse les avantages et les inconvénients d'un ajout d'une étape de pré-amplification pour détecter et quantifier les GVD. À l'aide de ces outils, je fournis ensuite la première description complète des GVD produits par des passages séquentiels de CHIKV en culture cellulaire. En particulier, je montre que le type de GVD générés est très dépendants du type cellulaire, avec des motifs de séquences et des cadres de lecture ouverts différents lorsque les cellules hôtes sont des cellules de mammifère ou d'insecte. Ces résultats soulignent le role de l'environnement cellulaire dans le modelage de la quasi-espèce, et des GVD en particulier. Des travaux futurs aideront à dévoiler les mécanismes sous-jacents à cette interaction et pourraient permettre la conception de nouvelles stratégies thérapeutiques ciblant les dynamiques des quasi-espècesArboviruses such as chikungunya virus (CHIKV) are responsible for millions of yearly infections, with no approved vaccines and limited treatments. Because they circulate between mosquitoes and humans, their fast adaptation potential to different hosts is key to pathogenesis. To achieve genome diversification, they rely on the error-prone nature of their self-encoded RNA-dependent RNA polymerase, which quickly generates a cloud of mutants, termed quasispecies. Quasispecies contain not only mutated genomes, but also shuffled genomes of different parental origin (through a process known as recombination), as well as genomes with large deletions, unable to replicate without the co-infection with a full-length helper genome, and thus termed defective viral genomes (DVGs). A tight regulation of the mutant cloud size is key to pathogenesis: if too small, it will limit the adaptation potential of the virus, whilst too big a quasispecies may lead to the fast accumulation of deleterious mutations. While regulation of the mutational landscape is achieved through the finely tuned error rate of the viral polymerase, recombination and DVG replication are influenced by the co-infection potential of the target cells.In this context, superinfection exclusion (SIE), a process by which infection by a first virus prevents infection by a second, closely related virus, can regulate quasispecies dynamics. While described in most viral families, mechanisms underlying SIE remain poorly characterised. Here, I show that CHIKV infection excludes subsequent infection by CHIKV, Sindbis virus and influenza A virus, but not West Nile virus. I demonstrate that CHIKV exclusion occurs at two steps, impacting independently viral penetration and replication, but does not directly influence binding, nor viral protein translation. I further show that SIE is interferon independent, and does not rely on host cell transcription nor on soluble cellular factors. Moreover, exclusion is not mediated by the action of a single CHIKV protein, suggesting that a cellular response may be at play. Assessing how different immunological pressures can shape quasispecies landscape may prove useful to a more thorough understanding of the interplay between viral evolution and the immune response. Although the unbiased study of point mutations has received much attention, less is known about the characteristics of DVGs, especially in alphaviruses. In the second part of this work, I develop bioinformatics tools to quickly isolate DVGs from next-generation sequencing data, and assess the advantages and drawbacks of pre-amplification steps to detect and quantify DVGs. Using these tools, I provide the first unbiased description of the DVG landscape generated through serial passaging of CHIKV in cell culture. In particular, I show that the DVG landscape is highly dependent on the cell type, with sequence patterns and open reading frames differing between DVGs generated in mammalian and insect cells. These results highlight the role of the cellular environment in shaping quasispecies, and DVGs in particular. Future work will help uncover the mechanisms underlying this crosstalk and may prove useful for the design of treatments targeting quasispecies dynamics
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Aguilar-Luis, Miguel Angel, Valle-Mendoza Juana del, Isabel Sandoval, Wilmer Silva-Caso, Fernando Mazulis, Hugo Carrillo-Ng, Yordi Tarazona-Castro, et al. "A silent public health threat: emergence of Mayaro virus and co-infection with Dengue in Peru." BioMed Central Ltd, 2021. http://hdl.handle.net/10757/655809.
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Objective: To describe frequency and clinical characteristics of MAYV infection in Piura, as well as the association of this pathogen with DENV. Results: A total of 86/496 (17.3%) cases of MAYV were detected, of which 54 were MAYV mono-infection and 32 were co-infection with DENV, accounting for 10.9% and 6.4%, respectively. When evaluating monoinfection by MAYV the main groups were 18–39 and 40–59 years old, with 25.9% and 20.4% respectively. Co-infections were more common in the age group 18–39 and those > 60 years old, with 34.4% and 21.9%, respectively. The most frequent clinical presentation were headaches (94.4%, 51/54) followed by arthralgias (77.8%, 42/54). During the 8-month study period the most cases were identified in the months of May (29.1%) and June (50.0%).National Research Foundation of Korea
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Wiley, Michael R. "New tools for the study of virus-vector interactions in mosquitoes." Diss., Virginia Tech, 2012. http://hdl.handle.net/10919/77343.
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Mosquito-borne diseases continue to be a burden to global health. The viruses that cause these diseases are maintained in nature through a biological transmission cycle involving susceptible vertebrate and mosquito hosts. While knowledge of the interactions occurring between mosquito-borne viruses and vertebrates is considerable, much less is known about the interactions of these viruses with their disease vectors. Studies with Drosophila melanogaster have been important in understanding how insects respond to viral infections. However, mosquitoes and the viruses they vector have co-evolved during a long period of time. Unfortunately, many of the genetic advantages of a fly model are not available when working with mosquitoes. Nevertheless, a sequenced genome, and molecular tools such as high-throughput sequencing and RNAi knockdown are helping to bridge these gaps. Here we describe several additional tools for the study of virus-vector interactions in the mosquito.Ph. D.
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Richartz, Rosaria Regina Tesoni de Barros. "Detecção de anticorpos inibidores de hemaglutinação para Alphavirus em soros de equinos do Estado do Parana." reponame:Repositório Institucional da UFPR, 2012. http://hdl.handle.net/1884/27683.
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Resumo: Foi realizado um inquérito sorológico, em 816 soros de eqüinos de diversas regiões do Estado do Paraná, em 1991, para detectar anticorpos inibidores de hemaglutinação para os vírus das encefalomielites eqüinas leste, oeste e venezuelana. Os resultados obtidos foram de uma prevalência de 30,4% para o vírus da encefalomielite eqüina oeste (WEE), 19,2% para o vírus da encefalomielite eqüina venezuelana (VEE) e 15,3% para o vírus da encefalomielite eqüina leste (EEE). Dos 816 soros analisados pelo teste de inibição da hemaglutinação (Hl), 300 reagiram positivamente, resultando em uma incidência de 36,76% de encefalomielite eqüina no Estado do Paraná. A região de Umuarama apresentou 19% do total de soros positivos, seguida das regiões de Curitiba (14%), Ponta Grossa (12%), Jacarezinho (9%), Irati (7%), Campo Mourão (7%), Guarapuava (7%), Ivaiporã (6%), Francisco Beltrão (4%), Maringá (4%), Paranavaí (4%), União da Vitória (3%), Toledo (3%), Cascavel (1%) e Pato Branco (0%).
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Jaffar-Bandjee, Marie-Christine. "Étude de la physiopathologie de l'infection Chikungunya en phase aiguë et chronique chez l'homme." Phd thesis, Université de la Réunion, 2010. http://tel.archives-ouvertes.fr/tel-00671277.
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Chikungunya est un alphavirus transmis par les moustiques (Aedes) et qui provoque de la fièvre, des éruptions cutanées, des myalgies et des arthralgies. La maladie (CHIKVD) est transitoire, mais des formes sévères menant à des arthrites chroniques incapacitantes ont été signalées. Nous avons dans un premier temps étudié prospectivement les paramètres cliniques et immunologiques associés à la maladie chez des patients hospitalisés et identifiés comme étant 'guéris' ou 'chronique' à M12 après l'infection. Dans la deuxième partie, nous avons observé in vitro les mécanismes et le rôle de l'apoptose dans le processus infectieux permettant au virus de persister dans les sanctuaires tissulaires. En phase aiguë, une forte réponse immune dominée par une activation des cellules NK/dendritique/cellules T, la production d'anticorps spécifiques et une faible production de cytokines Th1 > Th2 a été observée mais sans aucune différence significative entre les deux groupes. Cependant, la virémie initiale s'est révélée beaucoup plus élevée dans le groupe chronique est nous avons pu identifier du matériel viral dans les macrophages du tissu synovial d'un patient chronique post-CHIKVD (M18). Dans la deuxième partie de l'étude, nous avons constaté que CHIKV est capable d'induire l'apoptose par la voie intrinsèque et extrinsèque et également par un mécanisme 'bystander'. De plus, nous avons observé que le CHIKV présent dans des corps (blebs) apoptotiques était capable d'infecter les cellules voisines (Hela et macrophage MM6). Notre étude a permis de mettre en évidence pour la première fois que CHIKV contrôle et détourne à son profit les mécanismes de défense anti-infectieux.
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Carrera, J. P., Zulma M. Cucunuba, Karen Neira, Ben Lambert, Yaneth Pitti, Jesus Liscano, Jorge L. Garzon, et al. "Endemic and epidemic human alphavirus infections in eastern Panama: An analysis of population-based cross-sectional surveys." American Society of Tropical Medicine and Hygiene, 2020. http://hdl.handle.net/10757/655503.
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Madariaga virus (MADV) has recently been associated with severe human disease in Panama, where the closely related Venezuelan equine encephalitis virus (VEEV) also circulates. In June 2017, a fatal MADV infection was confirmed in a community of Darien Province. We conducted a cross-sectional outbreak investigation with human and mosquito collections in July 2017, where sera were tested for alphavirus antibodies and viral RNA. In addition, by applying a catalytic, force-of-infection (FOI) statistical model to two serosurveys from Darien Province in 2012 and 2017, we investigated whether endemic or epidemic alphavirus transmission occurred historically. In 2017, MADV and VEEV IgM seroprevalences were 1.6% and 4.4%, respectively; IgG antibody prevalences were MADV: 13.2%, VEEV: 16.8%, Una virus (UNAV): 16.0%, and Mayaro virus: 1.1%. Active viral circulation was not detected. Evidence of MADV and UNAV infection was found near households, raising questions about its vectors and enzootic transmission cycles. Insomnia was associated withMADVand VEEV infections, depression symptoms were associated with MADV, and dizziness with VEEV and UNAV. Force-of-infection analyses suggest endemic alphavirus transmission historically, with recent increased human exposure to MADV and VEEV in Aruza and Mercadeo, respectively. The lack of additional neurological cases suggests that severe MADV and VEEV infections occur only rarely. Our results indicate that over the past five decades, alphavirus infections have occurred at low levels in eastern Panama, but that MADV and VEEV infections have recently increased-potentially during the past decade. Endemic infections and outbreaks of MADV and VEEV appear to differ spatially in some locations of eastern Panama.National Institute for Health Research
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Grau, Nina. "Études structurales des conformations post-fusion des protéines d'enveloppe (E1) des alphavirus de la forêt de Semliki, du Chikungunya et de Sindbis : implications pour le mécanisme de fusion." Electronic Thesis or Diss., Sorbonne Paris Cité, 2017. http://www.theses.fr/2017USPCB141.
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Contexte : Le virus du Chikungunya (CHIKV), de la Forêt de Semliki (SFV) et de Sindbis (SINV) appartiennent au genre Alphavirus et sont transmis par des moustiques. CHIKV a provoqué récemment des épidémies planétaires causant de graves maladies. Les alphavirus sont enveloppés d'une membrane lipidique et infectent les cellules par voie d'endocytose ; le milieu acide des endosomes induit la fusion de l'enveloppe virale et de la membrane endosomique, fusion catalysée par la protéine d'enveloppe E1. Celle-ci appartient à la classe II des protéines de fusion, et est repliée en trois domaines (I, II et III) organisés en longueur avec I au centre et II et III de chaque côté, le domaine III se prolongeant par une tige ("stem") suivi par le segment trans-membranaire (TM) ancré dans la membrane virale. Le long domaine II expose à son extrémité une "boucle de fusion" hydrophobe, qui s'insère dans la membrane cible avant un changement de conformation de E1. E1 s'organise alors en trimères sur la membrane virale, ses trois sous-unités se pliant ensuite au milieu pour amener les domaines III sur le coté de chaque trimère, ainsi que le "stem" et le TM de façon à juxtaposer cette région avec le peptide de fusion. Ceci force les membranes virale et cellulaire l'une contre l'autre, provoquant leur fusion. Méthodes: Nous avons produit les ectodomaines recombinants de E1 (I, II, III et "stem") de ces trois alphavirus avec des mutations dans la boucle de fusion de façon à les rendre solubles sous la forme trimérique, post-fusion. Nous les avons cristallisé et déterminé leurs structures par cristallographie aux rayons X. Résultats: Notre travail montre que la conformation des protéines de fusion de E1 de ces trois alphavirus est clairement distincte, malgré la forte conservation de séquence parmi le genre viral. Ces différentes conformations ne pouvaient pas être prédites par modélisation basée sur leur homologie. Les structures montrent qu'elles gardent un « core » trimérique commun formé par le domaine I, la base du domaine II et le domaine III. Cependant le sous-domaine distal du domaine II, avec la boucle de fusion, adopte des conformations différentes (fermée, intermédiaire et ouverte) en fonction des virus, et interagit avec le "stem" qui connecte les segments TM. Conclusion: Nos résultats montrent l'importance de conduire des études structurales comparatives sur des virus similaires, et permettent d'expliquer des résultats d'inhibition de la fusion membrane non-concordants entre ces virus. Ils permettent ainsi de jeter une nouvelle lumière sur le mécanisme de fusion des alphavirusBackground: Chikungunya virus (CHIKV), Semliki Forest virus (SFV) and Sindbis virus (SINV) are mosquito-borne alphaviruses. CHIKV has caused important outbreaks of severe disease during the last decade. Alphavirus are enveloped viruses that enter cells via low-pH triggered fusion in the endocytic pathway. The envelope protein E1 is a class II fusion protein and is responsible for driving membrane fusion for entry. The E1 ectodomain is folded into three structured domains (I, II and III), arranged as a rod with domain I at the center and domains II and III at either side. Domain II is elongated and exposes a hydrophobic "fusion loop" at its distal end. The fusion loop inserts into the target membrane, and a subsequent conformational change brings the fusion loop into proximity with the viral-membrane-anchored segment of the protein, via a relocation of the C-terminal domain III and the downstream "stem" segment, which connects to the viral trans-membrane anchor at the C-terminal end. This results in a "hairpin" conformation of the protein that draws the two membranes together for fusion. Methods : We made the recombinant E1 ectodomain using a mutant of the fusion loop, to allow for solubility, and determined the crystal structures under acidic conditions, in their trimeric hairpin conformation. Results : Our work shows that the post-fusion trimers of E1 from the three alphaviruses studied adopt a strikingly different conformation in spite of their high sequence similarity, a change that could not have been predicted by homology modeling from the available structure of E1 from Semliki Forest virus truncated of part of the stem. They show that while the internal trimer core, composed of domain I and the domain II base, is maintained in the same conformation, and domain III flips to the sides of the trimer to form the hairpin in the same way, the downstream "stem" region and the domain II "tip" pack in a different way, resulting in three alternative forms: closed, intermediate, and open, depending on the virus. Conclusion : E1 is a very conserved protein in alphaviruses, and the available structure was believed to be a model for all alphaviruses. Our results highlight the importance of carrying out comparative structural studies, and help explain contradictory results in the use of domain III and the stem region for inhibition of the fusion step with different alphaviruses. These results will have an impact to better understand the conformational change and the role of the "stem" region for membrane fusion, and allow the development of fusion inhibitors in the future
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Rulli, Nestor Ezequiel, and na. "Ross River Virus Infection: Disease Mechanisms and Potential Treatment." University of Canberra. School of Health Sciences, 2007. http://erl.canberra.edu.au./public/adt-AUC20080227.091948.
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Ross River virus (RRV) is a mosquito-borne alphavirus and the aetiological agent of
epidemic polyarthritis (EPA). Arthropod borne-Alphaviruses that are related to RRV,
such as Chikungunya virus, Sindbis virus and Barmah Forest virus, are usually
associated with epidemics of infectious arthritides in different parts of the world. In
humans, RRV-induced disease symptoms include fever, rash, myalgia and pain and
stiffness of the joints. Muscle and joint pain are the most debilitating symptoms in RRV
patients and the best treatment available is non-steroidal anti-inflammatory drugs
(NSAID). Previous studies in mice have demonstrated that RRV infection results in
inflammation of skeletal muscle and joints and that macrophages play a primary role in
disease.
The present study was carried out to further elucidate the underlying mechanisms
mediating RRV-induced muscle and joint pathology. Previous studies have reported
that encephalitic alphaviruses trigger apoptosis of brain cells in mice and that blocking
apoptosis reduces mortality rates. In the present study, the ability of RRV to induce
muscle apoptosis was investigated in vitro, using a murine myoblast cell line (C1C12),
and in vivo, using a mouse model of RRV disease. RRV-infected C1C12 myofibres
displayed an array of morphological and biochemical makers of apoptosis. Apoptosis
was also observed in the skeletal muscle of RRV-infected C57BL/6J mice. Blocking
apoptosis by general caspase inhibition resulted in milder disease symptoms, reduced
myofibre damage and decreased inflammation of muscle and joint tissues. The total
number of cell infiltrates as well as the number of macrophages infiltrating muscle was
significantly reduced by the treatment with a caspase inhibitor.
The effects of RRV infection on the skeletal system were also investigated. Primary
human osteoblast cells were infected with RRV and monitored for viral-induced
cytopathic effect. Osteoblasts supported rapid virus growth and, by 48 hours after
infection, succumbed to viral-induced necrosis. In addition, histological examination of
bone tissue from RRV-infected C57BL/6J mice showed clear evidence of bone
resorption. Tibias from infected mice showed an increased number of activated
osteoclasts, a reduction in bone density and thinning of cortical bone.
The expression of host factors involved in inflammatory responses and bone
remodelling was studied in RRV-infected myofibres and osteoblast cell cultures and in
the muscle and joint tissues from infected mice. RRV-infected muscle cells and tissue
showed elevated mRNA levels for the chemokines CCL-2, CCL3, CCL5 and CXCL1,
all of which are known to mediate the migration of monocytic cells. With the exception
of CXCL1, these chemokines were also found to be up-regulated in RRV-infected
osteoblast cultures and in joint tissues from infected mice. Muscle and joint tissue from
infected mice also showed elevated mRNA levels for type I and type II interferons,
TNF- and NOS2. In addition, joint tissues from infected animals contained high levels
of IL-6 and IL-1, two cytokines known to mediate bone remodelling.
Finally, the therapeutic potential of the drug bindarit was investigated using the mouse
model of RRV disease. Bindarit is a known inhibitor of CCL-2 and TNF- and has
been found to prevent protein denaturation. Treatment with bindarit resulted in mice
developing milder disease symptoms, reduced muscle damage and decreased
inflammation of muscle and joint tissues. In particular, bindarit significantly reduced
macrophage infiltration into skeletal muscle tissue.
This thesis has contributed to the understanding of RRV pathogenesis. It has identified
novel mechanisms of RRV-induced muscle and bone pathology and provided further
evidence that associate pro-inflammatory host factors to RRV disease. This work has
also demonstrated that bindarit should be considered as a candidate for treating RRV
disease in humans.
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Chanel, Vos Chantal. "Etude du rôle de l' acide aminé histidine 230 dans la boucle ij de la protéine E1 du virus de la Forêt de Semliki dans la fusion membranaire virale." Paris 7, 2005. http://www.theses.fr/2005PA077060.
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Benmaamar, Rim. "Development and optimization of alphaviral systems for membrane protein functional expression." Strasbourg, 2009. http://www.theses.fr/2009STRA6037.
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Morin, Benjamin. "Etude structurale et fonctionnelle de protéines de virus à ARN impliquées dans la réplication virale et la réponse cellulaire à l'infection." Aix-Marseille 2, 2009. http://theses.univ-amu.fr.lama.univ-amu.fr/2009AIX22102.pdf.
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A l’heure actuelle, les études structurales et fonctionnelles sur les virus émergents se limitent à quelques virus dont l’impact sociétal est établi et prévisible. Pourtant, le monde des virus à ARN est très vaste, et présente des potentialités d’émergences en pleine augmentation. Au cours de mon doctorat, j’ai étudié 2 types de protéines impliquées dans la réplication de tels pathogènes viraux. La protéine L des virus à ARN négatif (ARN(‐)) est la polymérase indispensable à la transcription et à la réplication de leur génome. Du fait de la difficulté de production de ces protéines sous forme entière, très peu de données structurales et fonctionnelles sont disponibles. J’ai alors recherché des domaines solubles de ces protéines et résolu la première structure cristallographique d’un domaine d’une protéine L. J’ai ensuite démontré que celui‐ci est un domaine de type endonucléase présent dans l’ensemble des virus à ARN(‐) segmenté, et probablement impliqué dans la stabilisation des ARN messagers viraux. Ces résultats font alors de ce domaine une cible majeure pour la recherche d’antiviraux dirigée spécifiquement contre ces virus. Lors d’une infection virale, la cellule est capable de mettre en place des réponses antivirales, dont la voie de réponse à l’interféron (IFN) via l’oligoadénylate synthétase (OAS)/Ribonucléase L(RNase L). Cependant, les virus ont des moyens de se protéger contre la réponse cellulaire à l’infection. Les connaissances actuelles sur les domaines macro viraux suggèrent qu’ils pourraient interagir avec des 2’‐5’ oligoadénylates (2‐5As), effecteurs de cette voie de réponse à l’IFN. J’ai alors mis en place une méthode de production à grande échelle des 2‐5As, puis cristallisé le domaine macro nsp3 du virus Chikungunya en complexe avec un trimère de 2‐5A. Ces travaux ouvrent une voie de recherche sur les relations entre les virus et un des mécanismes de défense contre l’infection virale, la voie OAS/RNase LThe structural and functional studies of emergent viruses are restricted to few viruses whose overall impact is already established and predictable. Though, the RNA virus world is incredibly wide and increasingly presents new possibilities of emerging pathogens. During my PhD I studied 2 types of proteins involved in replication of these interesting viral pathogens. The L protein of negative strand RNA viruses ((‐)RNA) is the essential RNA polymerase for transcription and replication of the viral genome. Because of the difficulty of producing crystals of the entire protein, very few structural and functional data are available. I generated and studied soluble domains of these proteins and solved the first crystallographic structure of a L protein. I found that it harbors an endonuclease fold conserved in all segmented (‐)RNA viruses, with a putative role in stabilization of viral mRNAs. These results make this domain a suitable target for antiviral research specifically directed against these viruses. During a viral infection the cell is able of generate an antiviral response, the Interferon (IFN) response pathway, which occurs via oligoadenylate synthetase (OAS) /Ribonuclease L (RNase L) involvement. However, viruses use different ways to protect themselves from this cellular response. The actual knowledge on the conserved viral macro domains suggests that they could interact with 2’‐5‘ oligoadenylates (2‐5As), which are signalling molecules in the induction of the IFN response pathway. I developed a method to produce 2‐5As on a large scale. Then I crystallized the macro nsp3 domain of Chikunguya virus in complex with a 2‐5A trimer. These studies open perspectives of research about the relation between viruses and one of the defense mechanism against viral infection, that involving OAS and RNase L
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Fitzpatrick, Kelly Ann. "Luminex microsphere immunoassay offers an improved method in testing for antibodies to Eastern Equine Encephalitis virus in sentinel chicken sera." [Tampa, Fla] : University of South Florida, 2008. http://purl.fcla.edu/usf/dc/et/SFE0002480.
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Liu, Xiang. "Ross River Virus Interaction with the Type I IFN Pathways." Thesis, Griffith University, 2016. http://hdl.handle.net/10072/367506.
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Ross River virus (RRV) belongs to the genus Alphavirus and is a medically important arbovirus that causes musculoskeletal disease in humans with symptoms such as arthralgia, arthritis and myalgia. Disease symptoms consistent with RRV infection were first recorded in 1928 in Australia. Currently, with approximately 5,000 cases of RRV infection reported each year in Australia, RRV is the most widely spread arbovirus throughout the South Pacific region. At present there are no specific therapeutics or vaccines available. RRV disease is treated with analgesics and non-steroidal anti-inflammatory drugs to provide symptomatic relief. Therefore, it is important to investigate RRV disease mechanisms so as to better understand disease pathogenesis, which could lead to identifying potential targets for therapeutic intervention. The host Type I interferon (IFN) system is the primary innate antiviral defence mechanism. The antiviral effects of type I IFN act to both suppress viral replication and modulate innate and adaptive immune responses during viral infection. However, the interplay between the host type I IFN responses and alphavirus infection is currently poorly understood. This thesis focuses on the role of type I IFN system in RRV infection and disease pathogenesis.Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
Institute for Glycomics
Science, Environment, Engineering and Technology
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Ralambondrainy, Miora. "Caractérisation chimique et biologique de trois huiles essentielles répulsives issues de la biodiversité régionale contre l'alphavirus du Ross River." Thesis, La Réunion, 2017. http://www.theses.fr/2017LARE0018/document.
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Les huiles essentielles de citronnelle (Cymbopogon citratus), de géranium (Pelargonium graveolens) et de vétiver (Vetiveria zizanioides) sont utilisées partout dans le monde pour leur activité répulsive contre les principaux vecteurs (moustiques, tiques) de maladies infectieuses chez l'Homme (paludisme, chikungunya, …). L'application cutanée de ces produits naturels pour éviter le contact avec un vecteur n'avait pas été encore envisagée comme moyen de limiter les premiers stades de l’infection par l'agent pathogène transmis par le vecteur. Pour vérifier cette hypothèse, les travaux ont été consacrés à la mise en place d'un cadre structuré pour la réévaluation chimique et biologique des trois huiles essentielles sur le modèle du virus du Ross River (alphavirus) de la même famille que le virus du Chikungunya. La caractérisation chimique des huiles essentielles avec une technique de haute résolution (GC×GC/TOF-MS) a permis d'établir leur profil chémotypique précis. L'utilisation de marqueurs spécifiques (clones moléculaires du virus) a permis d'établir l'inhibition de la réplication virale en fonction des conditions d'application des huiles essentielles de géranium et citronnelle. Ces résultats suggèrent l'intérêt d’une huile essentielle répulsive dans les premiers stades d'une infection par un vecteur. À ce titre, l'étude comparative établit la haute valeur ajoutée de l'huile essentielle de géranium et oriente la recherche de nouveaux anti-infectieux naturels vers des complexes riches en monoterpènesEssential oils of citronella (Cymbopogon citratus), geranium (Pelargonium graveolens) and vetiver (Vetiveria zizanioides) are used worldwide as topical repellent against the main vectors (mosquitoes, ticks) of human infectious diseases (Malaria, chikungunya, …). Skin treatment with these natural products, initially to avoid contact with the vector had not yet been considered as a way to disrupt the early stages of infection when the repelling action fails. To check this hypothesis, a structured framework has been performed for the chemical and biological re-evaluation of the three essential oils. The latter was tested against Ross River virus (alphavirus) that belongs to the same family of Chikungunya virus. Analysis of essential oils using a high-resolution technique (GC × GC / TOF-MS) resulted in a more accurate chemotypical profile of the local production. The use of specific markers (molecular clones of the virus, Saclay CEA) allowed to establish the inhibition of viral replication depending of the conditions of geranium and citronella essential oils application. These results suggest the great interest of an essential oil topical repellent in the early stages of a vector infection. The comparative study established the high value of geranium essential oil and gave future direction to the discovery of new anti-infectious solutions from monoterpenes-rich natural complexes
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Brehin, Anne-Claire. "La protéine OAS3 de la voie interferon inhibe la réplication du virus chikungunya dans les cellules humaines." Paris 7, 2008. http://www.theses.fr/2008PA077082.
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Chez l'Homme, l'infection par le virus chikungunya (CHIK), un membre du genre Alphavirus de la famille des Togaviridae, se manifeste classiquement par des arthralgies aiguës. La flambée inattendue de fièvre chikungunya dans les îles de l'Océan Indien en 2006 mis en exergue la nécessité de comprendre la pathogénie de cette maladie peu étudiée. La caractérisation moléculaire de plusieurs isolats cliniques collectés sur l'Ile de La Réunion mis en évidence l'émergence du variant viral E1-226V associé à l'adaptation au vecteur Ae. Albopictus. Pour poursuivre cette caractérisation, nous avons produit des outils spécifiques pour la détection du virus CHIK comme une forme soluble de la glycoprotéine d'enveloppe E2 (gp-E2) ainsi que des anticorps monoclonaux murins spécifiques de la gp-E2 du virus. Chez les individus infectés par les alphavirus dont le virus CHIK, l'infection virale est contrôlée par l'IFN-α/β. Qui stimule la production d'un ensemble de molécules antivirales. Nos travaux suggèrent qu'une famille de ces gènes, les 2',5'-Oligoadenylate Synthétases (OAS), joue un rôle primordial dans l'immunité innée anti-arbovirale. Nous avons étudié si l'isoforme OAS3 humaine joue un rôle contre l'infection par le virus CHIK. Les cellules épithéliales humaines surexprimant l'OASS inhibent efficacement la croissance du virus CHIK aussi bien que d'autres alphavirus comme les virus Sindbis et Semliki Forest. Cette activité anti-alphavirale empêche l'accumulation des ARN viraux et des protéines virales. En conclusion, l'activité de la protéine OAS3 représente une importante voie anti-alphavirale par laquelle l'IFN-α/β contrôle l'infection du virus CHIK dans les cellules humainesHumans infected with chikungunya virus (CHIKV), a member of the Alphavirus genus of the Togaviridae family, typically experience acute illness with incapaciting polyarthralgia. The unexpected outbreak of chikungunya fever in the Indian Ocean islands in 2006 highlights the need to understand this disease pathogenesis not well studied. Several clinical isolates collected in La Reunion Island were characterized at the molecular level. Our study emphasized the emergence of the viral variant E1-226V associated with adaptation to the vector Ae. Albopictus. The production of specific tools for the CHIK virus detection was necessary to pursue this characterization. We produced a soluble form of the envelope E2 glycoprotein (gp-E2) in Drosophila S2 cells, as well as mouse monoclonal antibodies specific of the virus gp-E2. In people infected by alphavirus such as CHIK virus, the viral infection is controlled by IFN-α/βwhich stimulates the production of a set of antiviral molecules. Our laboratory had shown that the 2', 5'-Oligoadenylate Synthetases (OAS) genes, inducible by IFN-α, play a critical role in antiviral immunity against arboviruses. Whether the OAS3 human form may play a role in the protective innate immunity to CHIKV was investigated. Human epithelial cells respond to ectopic OAS3 protein expression by inhibiting CHIKV growth as efficiently as that of other alphaviruses such as Sindbis and Semliki Forest viruses. The OAS-mediated inhibition of CHIKV growth was attributable to a dramatic reduction in viral RNA and protein levels. In conclusion, OAS3 activity represents an important antialphaviral pathway by which IFN-α/β controls CHIKV infection in human cells
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Human, Stacey. "Characterization of zoonotic flavi- and alphaviruses in sentinel animals in South Africa." Diss., University of Pretoria, 2011. http://hdl.handle.net/2263/30726.
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In South Africa (SA), the arboviruses West Nile virus (WNV), Wesselsbron virus (WSLV), Sindbis virus (SINV) and Middelburg virus (MIDV) are considered the most important flavi- and alphaviruses. Clinical presentation and importance of these viruses as animal pathogens in SA remains ambiguous. Although widely endemic in SA, lineage 2 (L2) WNV has rarely been associated with cases of neurological disease and was therefore assumed to be non-pathogenic. However, fatal encephalitis in a foal was diagnosed as L2 WNV in SA, 1996, leading to the thought that L2 cases were possibly being missed. As the above-mentioned arboviruses have the same transmission vectors, Culex mosquitoes for WNV and SINV and Aedes mosquitoes for WSLV and MIDV, co-screening for these viruses is important. We hypothesise that horses could be used as sentinels for virus activity in SA and cases of unexplained neurological disease or fever in animals overlooked, rather than being non-existent. To this end, the study aimed to screen horses displaying unexplained neurological disease or fever with Flavivirus family-specific RT-PCR. Additionally, samples were screened with an Alphavirus family-specific RT-PCR to determine whether co-circulating viruses could be responsible for neurological symptoms in horses. The results would aid in establishing the molecular epidemiology and disease description of each virus, virus distribution and disease seasonality in SA. In total 261 clinical specimens were collected from horses displaying these symptoms (2008 - 2010). Samples were screened with Flavi- and Alphavirus differential diagnostic RT-PCR and acute serum was screened for WNV-IgM and neutralizing antibodies. Serological screening (WNV haemagglutination inhibition, WNV IgG and/or WNV neutralization) identified 62 suspected WNV cases while 34 cases could be confirmed by RT-PCR (16/34), WNV IgM and neutralization assays (18/34) and virus isolation. Neurological disease made up 91% (31/34) of the cases, mortality was calculated at 44% (15/34). Phylogenetically 12/16 RT-PCR positives grouped with L2 SA strains. The first detection of L1 WNV and horse-associated abortion in SA was reported when a pregnant mare aborted her foetus in Ceres, Western Cape. The first cases of WSLV-associated disease in horses were identified by sequencing Flavivirus RT-PCR positive products from 2 horses displaying severe neurological disease; one being fatal. This suggests missed cases in the past. To elucidate virulence factors of WSLV, a human encephalitic strain AV259, was subjected to Roche FLX454 full-genome sequencing and compared to a previously sequenced febrile strain (H177). Several structural amino acid changes occurred in proteins NS2A, NS4B and NS5 of AV259; necessary for Flavivirus replication. Phylogenetically AV259, clinical horse strains and WSLV strains previously isolated from animals, humans and arthropods were similar. Additionally and in concurrence with other studies, WSLV clusters with Sepik virus (SEPV) within the YFV group of the Flaviviridae family. Alphavirus screening identified 17 cases; 6/17 SINV and 11/17 MIDV. SINV-WNV co-infections resulted in fatal neurological disease; remaining SINV cases recovered after displaying fever and/or mild neurological disease. MIDV symptoms varied from “three-day-stiffness” to severe neurological symptoms, with 2 fatalities. Co-infections with equine encephalosis and Shuni virus were identified. MIDV strains identified in this study were phylogenetically distinct from older strains. Results highlight the use of horses as sentinels for virus activity and suggest that these arboviruses may have been previously missed as horse pathogens in Africa. These viruses should be considered as the aetiological agents in animals displaying unexplained neurological or hepatic disease, fevers or abortions. Awareness of flavi- and alphaviruses and the disease manifestation they may have in horses was illustrated. These findings suggest that a WNV vaccine may be beneficial for horses in SA.Dissertation (MSc)--University of Pretoria, 2011.
Medical Virology
Unrestricted
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Lee, Wai Suet. "Discovery of Novel Markers of Virus Transmission by Mosquitoes." Thesis, Griffith University, 2019. http://hdl.handle.net/10072/390018.
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Mosquito-borne diseases are responsible for significant human morbidity and mortality throughout the world. Current vector control strategies have been impeded by mosquitoes acquiring resistance to insecticide. Therefore, development of new vector control strategies is urgently needed to complement current strategies. In this thesis, efforts have focused on characterizing the glycan-lectin interactions of Ross River virus (RRV; Togaviridae: Alphavirus) with their mosquito vectors. RRV is the most common arbovirus in Australia that causes clinical manifestations including arthralgia and myalgia. Many studies have shown the importance of viral surface glycans in mediating viral entry into host cells. Moreover, the viral surface glycans varies depending on the cells that they replicate in and this variation can affect the infectivity of virus. However, gaps remain in the role of viral glycans in virus host cell recognition. In Chapter 2, the surface glycans of RRV derived from two different cell lines, C6/36 (Ae. albopictus) and Vero (African green monkey kidney) were characterized using lectin array. Lectin array data revealed that RRV derived from two different cell lines exhibited similar glycan profiles. The glycan structures present on the surface of RRV are mannose, galactose, N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc). To investigate the importance of these viral surface glycans in mediating viral entry into host cells, six lectins targeting these glycan structures were assessed for their ability to bind and block RRV entry into host cells. Of these lectins, two mannose-binding lectins, GNA and ConA, showed inhibition of RRV entry into C6/36 and Vero cells. These results suggest the potential use of mannose-binding lectins to block RRV transmission by mosquitoes. It is well known that cell surface glycans or lectins play important role in viral entry. Therefore, comprehensive surface glycan profiles and carbohydrate-binding characteristics of lectins from five mosquito cell lines were established in Chapter 3. Using lectin and glycan arrays, our results showed differences between the glycan structures and carbohydrate-binding characteristics of mosquito cell lines. In particular, complex-type glycans were detected on the cell surface of Ae. albopictus and An. gambiae. The presence of complex-type glycans as authentic constituents of insect glycans is still controversial and this is an important finding as complex-type glycans play diverse roles in regulation of biological functions.
Zika virus (ZIKV) is primarily transmitted by the Aedes (Ae) mosquito, with Ae. aegypti and Ae. albopictus being the primary vectors. However, controversial findings on the potential of other mosquito species belonging to the genera of Anopheles (An) and Culex (Cx) have been reported. To date, the rationale underlying the specificity of ZIKV in infecting Aedes mosquitoes remain to be an unaddressed issue. In Chapter 4, we seek to characterize the susceptibility of seven cell lines derived from Ae, An and Cx mosquitoes towards ZIKV infection. Indeed, Ae cell lines were permissive to ZIKV infection and supported viral replication up to seven days post infection, while cells lines from An and Cx mosquitoes were unable to support replication. To specifically address if non-susceptible cell lines were due to the incompetence of ZIKV in establishing viral entry, a pseudoZIKV replicon system was utilized. Interestingly, while all Ae cell lines were highly susceptible to pseudoZIKV infection, the non-susceptible An. gambiae cell line (4a-3B) was also highly permissive to pseudoZIKV entry, in contrast to other An and Cx cell lines tested. Therefore, to identify the host factors involved in ZIKV replication in mosquito cells, RNA sequencing (RNAseq) analysis was performed on ZIKV-susceptible Ae and non-susceptible An cells after infection. Through comparative transcriptomics approach, we observed a differential regulation of attacin, an antimicrobial peptide (AMP) that may potentially play a critical role in modulating ZIKV replication in mosquito cells. Further investigations on the expression profile of different classes of AMPs including attacin, cecropin, defensin, diptericin and gambicin in Aedes; and attacin, cecropin, defensin and gambicin in Anopheles cells by qRT-PCR demonstrated that these AMPs were differentially regulated in ZIKV-susceptible and -resistant mosquito cells. These results suggest that the innate immunity may have a role to influence mosquito vector competence.
Species specificity often relies on a specific interaction between a virus ligand and its host cell receptor. Therefore, expression levels of the receptor largely determine the tropisms of viruses. In Chapter 5, we constructed a representative cDNA library from the ZIKV-susceptible Ae. aegypti Aag-2 cell line using the SMART (Switching Mechanism At 5’ end of RNA Template) cDNA synthesis technology. This library will be a useful tool to provide genetic resources for many applications using the proposed strategy including identification of the cellular surface receptors for ZIKV viral entry and host factors required for sustained ZIKV replication in Aedes cells.
Overall, this thesis provides in depth investigations into the glycan-lectin interactions between RRV and their mosquito vectors that may affect vector competence. Our study also identified the potential antiviral lectins that can block virus transmission. Finally, our study shed insights into the host factors involved in modulating ZIKV replication, providing a molecular platform for the future development of effective vector control and to evaluate the risk of emergence of a new vector for mosquito-transmitted viruses.Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
Institute for Glycomics
Griffith Health
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Pinto, Tatiana Priscilla Coelho. "Expressão e purificação da proteína E3 do vírus chikungunya (CHIKV)." Master's thesis, Expressão e purificação da proteína E3 do vírus chikungunya (CHIKV), 2013. http://hdl.handle.net/10362/10528.
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RESUMO: O vírus chikungunya (CHIKV) é um vírus de RNA, com invólucro, da família Togaviridae, transmitido por mosquitos Aedes spp. Distribuído por largas regiões de África e Ásia, causa grandes epidemias de artrite grave. A semelhança de sintomas com outras doenças como a dengue e a malária e a persistência de IgM específicas, dificultam o diagnóstico da infeção por CHIKV. A deteção no sangue de E3, uma glicoproteína viral secretada, a incluir num ensaio imunoenzimático poderá melhorar o diagnóstico nos países onde as técnicas de biologia molecular são de difícil acesso. Para testar a utilidade de E3 num ensaio de diagnóstico, esta deverá ser expressa em quantidade, purificada e usada para produção de anticorpos específicos.
Para expressar E3 numa forma solúvel, suscetível de ser purificada num único passo cromatográfico sem proteases, recorreu-se à estratégia da fusão com o domínio de ligação à quitina (CBD)-inteína (IMPACT™ System, NEB). A sequência codificadora de E3 foi amplificada a partir de RNA viral, clonada em pTYB21 e expressa em E. coli como uma proteína de fusão insolúvel de 64 kDa. A expressão a 12ºC induzida por IPTG 0,1 mM aumentou a solubilidade de CBD-inteína-E3. A aplicação de lisados celulares em colunas de quitina originou a retenção de CBD-inteína-E3 na matriz. Porém, a autoclivagem da inteína na coluna, induzida com reagentes tiol, foi pouco eficiente e mesmo a proteína E3 separada não eluiu da coluna. E3 foi ainda expressa em E. coli com uma cauda de seis histidinas (E3[His]6) por clonagem no vetor pET28b(+). Lisados celulares aplicados em colunas de níquel permitiram a eluição de uma proteína de 9 kDa, compatível com a massa molecular estimada para E3[His]6, ainda que com outros contaminantes proteicos. A identidade da proteína de 9 kDa será confirmada pela indução de anticorpos com esta preparação e reatividade daqueles com células infetadas com CHIKV.----------------ABSTRACT: Chikungunya virus (CHIKV) is an enveloped, positive strand RNA virus belonging to the family Togaviridae. Transmitted by Aedes spp mosquitoes, CHIKV causes large epidemics of severe arthritogenic disease in Africa and Asia and represents a serious threat in countries where vectors are present. Symptoms similarity with other diseases, e.g. dengue and malaria, along with CHIKV IgM persistence turns accurate CHIKV diagnosis a difficult task in low-income countries. Detection of E3, a small secreted viral glycoprotein, to be included in an immunoenzymatic test was envisaged as a possible improvement in CHIKV diagnosis. To test the diagnostic value of E3, recombinant E3 should be expressed and purified to generate antibodies.
In order to express CHIKV E3 in a soluble form amenable to purification by a single step affinity chromatography, the chitin binding domain (CBD)-intein fusion strategy without proteases (IMPACT™ System, NEB) was employed. The E3 coding sequence was amplified from viral RNA, cloned in pTYB21 and expressed in E. coli ER2566 as an insoluble 64 kDa CBD-intein-E3 fusion protein. Solubility was partially achieved by lowering the expression temperature to 12ºC and the inducer (IPTG) concentration to 0.1 mM. Clarified cell lysate loaded onto a chitin column allowed ligation of the fusion protein but the intein-mediated cleavage efficiency was low and E3 failed to elute from the column as demonstrated by SDS-PAGE. E3 was further expressed with a six histidine tag, E3[His]6, employing the pET System (Novagen). E3[His]6 was expressed in E. coli Rosetta (30ºC, 0.4 mM IPTG) as a 9 kDa protein. Soluble cell extracts in 20-40 mM imidazole, applied onto a nickel column and eluted with 500 mM imidazole yielded a protein preparation enriched in the 9kDa protein. The 9 kDa will be used as antigen to generate antibodies that upon reaction with CHIKV infected cells will confirm its identity.
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Ferreira, Ramos Ana Sofia. "Inhibitors of the mRNA capping machinery and structural studies on macro domains from alphaviruses." Thesis, Aix-Marseille, 2019. http://theses.univ-amu.fr.lama.univ-amu.fr/190708_FERREIRARAMOS_112plefdq222vlt303lhj860uuajmi_TH.pdf.
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Les alphavirus comme le virus Chikungunya et le virus de l'encéphalite équine vénézuélienne sont des arbovirus (ré)-émergents. Ils possèdent un mécanisme de coiffe de l’ARNm non conventionnel catalysé par nsP1 et nsP2 pour former une structure cap-0 (m7GpppN-) qui est cruciale pour la réplication. Le coiffage constitue une cible antivirale attractive. NsP1 catalyse trois activités: méthyltransférase, guanylylation de nsP1 (GT), et transfert sur l’ARNm. Nous avons développé un test pour cribler la chimiothèque de composés de Prestwick Chemical® contre l’activité GT de nsP1. 18 composés sont ressortis de ce crible et trois séries de composés ont été sélectionnées pour une caractérisation plus poussée. Ces composés inhibent peu une MTase cellulaire suggérant leur spécificité vis-à-vis de nsP1. Des analyses de relations structure/activité (SAR) ont également été initiées pour identifier les pharmacophores actifs. Ce travail montre que notre test permet de sélectionner des composés spécifiques ciblant le coiffage de l’ARNm des alphavirus. NsP3 consiste en un Macro domaine, un domaine de liaison au zinc et une région hypervariable. Le Macro domaine est essentiel à la réplication virale en fixant l’ADP-ribose (ADPr) et en dé-ribosylatant des protéines cellulaires. Nous avons effectué une étude structurale et fonctionnelle du Macro domaine du virus Getah (GETV) dont la séquence de la boucle catalytique présente des particularités. Cette étude a permis de caractériser plusieurs poses adoptées par l’ADPr dans le site actif. Ces poses peuvent représenter plusieurs instantanés du mécanisme de l’ADP-ribosylhydrolase, et mettent en lumière de nouveaux résidus à caractériserAlphaviruses such as Chikungunya virus and Venezuelan equine encephalitis virus (VEEV) are (re-)emerging arboviruses. They own an unconventional mRNA capping catalysed by nsP1 and nsP2 leading to the formation of a cap-0 structure (m7GpppN-), which is crucial for virus replication and constitutes an attractive antiviral target. NsP1 catalyses three activities: methyltransferase (MTase), guanylylation (GT) and guanylyltransferase (GTase). A high throughput ELISA was developed to monitor the GT reaction and screen the Prestwick Chemical library®. The IC50 was determined for 18 selected hit compounds. Three series of compounds were selected for further characterization. These compounds poorly inhibit a cellular MTase suggesting their specificity against nsP1. Analogue search and structural activity relationships (SAR) were also initiated to identify the active pharmacophore features. The results show that our strategy is a convenient way to select specific hit compounds targeting the mRNA capping of alphaviruses. NsP3 consists in a Macro domain at the N-terminal, a zinc binding domain and a C-terminal hypervariable region. The Macro domain is essential for the replication through ADP-ribose (ADPr) binding and de-ribosylation of cellular proteins. In order to better understand this mechanism, we initiated a structure-based study of Getah virus (GETV) Macro domain, which contains a peculiar substitution in the catalytic loop. By crystallographic studies we characterized several poses adopted by ADPr in the binding site. Together, these poses may represent several snapshots of the ADP-ribosylhydrolase mechanism, highlighting new residues to be further characterised
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Bouraï, Mehdi. "Caractérisation d'un interactome virus-hôte : l'exemple du virus du Chikungunya." Paris 7, 2011. http://www.theses.fr/2011PA077183.
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Récemment, la levée de nombreux verrous technologiques a permis le développement de la « génomique fonctionnelle », désignant l'approche systémique appliquée à la biologie moléculaire et cellulaire. Les virus, parasites intracellulaires obligatoires, interagissent avec de nombreux composants de la cellule afin de se répliquer. Mieux définir les interactions entre les protéines virales et cellulaires permet de mieux comprendre le cycle de réplication et la pathogenèse virale, et ouvre la voie à de nouvelles approches thérapeutiques innovantes. Au cours de mon travail de thèse, j'ai cherché à définir la carte d'interaction, ou interactome, du virus du chikungunya (CHIKV), virus dont les interactions avec la cellule à l'échelle moléculaire restent encore mal connues. J'ai pour cela utilisé des approches par double-hybride à haut débit en levure (HT-Y2H) et de validations en cellules de mammifère (notamment la technique de protein complémentation assay ou PCA). Nous avons criblé toutes les protéines matures du CHIKV contre 3 banques différentes d'ADNc humains, et une banque normalisée de 12. 000 ORFs (open reading frame) humaines entières. Nous avons ainsi mis en évidence 22 interactions dont la plupart impliquent la protéine non structurale 2 (nsP2) du CHIKV. Parmi les interacteurs cellulaires mis en évidence, nous avons pu montrer le rôle important joué par hnRNP-K (heterogeneous nuclear ribonucleoprotein K) et l'ubiquiline 4 dans la réplication du virus in vitro. Par ailleurs, nous avons démontré l'implication de la protéine TTC7B (tétratricopeptide 7B) dans l'activité d'inhibition transcriptionnelle induite par la protéine nsP2 du CHIKV. Les techniques utilisées au laboratoire m'ont également permis de participer à la caractérisation de trois interactions virus-hôte identifiées par un autre étudiant en thèse du laboratoire et jouant un rôle dans la réplication du virus de la rougeole (VR) et du virus parainfluenza humain (hPIV3). J'ai notamment pu cartographier avec précision des domaines peptidiques impliqués dans ces interactions, grâce à une technique adaptée du Y2H. Ce travail m'a donc permis d'appréhender les techniques actuelles permettant de définir les interactomes virus-hôte, et de proposer ainsi une carte d'interactions virus-hôte pour le CHIKV, mais aussi d'apporter des éléments de réponses quant aux mécanismes viraux impliquées dans le cycle réplicatif et la pathogenèse de ce virusThe lifting of many technological barriers in recent years has allowed the development of « functional genomics », an innovative systemic approach to molecular and cell biology. Viruses, being intracellular parasites, interact with several components of the cell to replicate. Thus, defining and improving our knowledge of the interactions between viral and cellular proteins ensures a better understanding of the viral replication cycle and pathogenesis and opens the pathway to new therapeutic approaches. In my thesis, I defined the interaction map, or interactome, of the chikungunya virus (CHIKV), a virus whose interactions with the cell at the molecular level have been poorly understood. For this, I performed high throughput two-hybrid approaches in yeast (HT-Y2H) and validations in mammalian cells (including protein complementation assay technique or PCA). We screened all the CHIKV mature proteins across three different human cDNA libraries and a normalized 12,000 human full-length open reading frames (ORF) library. We identified 22 interactions, the majority of which involve non-structural protein 2 (nsP2) of CHIKV. Among the identified cellular interactors, we showed the important role of hnRNP-K (heterogeneous nuclear ribonucleoprotein K) and ubiquilin 4 in virus replication in vitro. Furthermore, we demonstrated the involvement of the TTC7B protein (tétratricopeptide 7B) in the transcriptional inhibition activity induced by the nsP2 protein of CHIKV. Such techniques conducted in the laboratory also allowed me to participate in thé charaterization of three virus-host interactions identified by a fellow PhD student and contribute to researching the replication of measles virus (MV) and type 3 human parainfluenza virus (hPIV3). In particular, I was able to accurately map the peptide domains involved in these interactions, using a technique adapted from Y2H. This work has allowed me to not only understand the current techniques for defining virus-host interactomes and consequently produce a map of virus-host interactions for CHIKV, but also to shed some light on the viral mechanisms involved in the replication cycle and the pathogenesis of this virus
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Wolf, Stefan. "Novel Approaches in the Treatment of Virus- Induced Inflammatory Disease." Thesis, Griffith University, 2016. http://hdl.handle.net/10072/366853.
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This PhD thesis combines four chapters on different fields of basic research and sets the focus on two circulating viruses of global concern, the orthomyxovirus influenza A virus (IAV) and the alphavirus Ross River virus (RRV). The first three chapters include swine influenza A virus (sIAV) surveillance for the detection and characterisation of IAV subtypes, an in vitro high throughput screening (HTS) on host micro RNAs (miRNAs) for the discovery of novel anti-IAV (H7N9) targets and their underlying mechanisms, and an approach to reduce disease pathogenesis in mice infected with H7N9 by targeting the pro-inflammatory factor CCL2. In a fourth chapter, drug repurposing with the interleukin-1 (IL-1) inhibitor anakinra was investigated to treat RRV-induced bone loss in mice. By combining these four chapters, a broad range of drug discovery is covered in this PhD thesis; Surveillance, HTS target discovery and the application of drug repurposing in animal models of viral diseases.Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
Institute for Glycomics
Science, Environment, Engineering and Technology
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Davis, Gustavo Henrique Nolasco Grimmer. "Estudos epidemiológicos sobre arbovírus em populações rurais e urbanas do estado do Amazonas." Universidade Federal do Amazonas, 2009. http://tede.ufam.edu.br/handle/tede/3430.
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Previous issue date: 2009-03-06FAPEAM - Fundação de Amparo à Pesquisa do Estado do Amazonas
Arbovirosis are currently recognized by the World Health Organization as a global problem. Most arbovirosis are summarized in diseases with acute and nonspecific symptoms such as fever, headache and muscle pain. Although self limited, these symptoms create relevant social and economic impacts. In Brazil, the arbovirosis caused by viruses belonging to the genus Alphavirus, Flavivirus and Orthobunyavirus and are the main cause of outbreaks or epidemics. This study aimed to study the circulation of arbovirus mayaro, venezuelan equine encephalitis virus, yellow fever virus, Saint Louis encephalitis virus and oropouche virus in a rural and an urban area of the State of Amazonas, Brazil. Therefore, the serology for detection of immunoglobulin G was used to assess the prevalence of antibodies against these viruses in 335 residents of a rural community in the state of Amazonas and PCR was used to assess the incidence of these viruses in 250 samples collected in urban area of Manaus. The results for serology suggest the movement of mayaro virus in the rural community. The seroprevalence detected in the samples was 41.5%. There was no significant relationship to risk for mayaro infection between genders (p value = 0.7760) or between age groups (p value = 0.9422). The positive serology detected among 39 children younger than 10 years indicates a recent infection. The factors of protection against mayaro infection detected were the use of mosquito net (p value = 0.0119) and the presence of animals in surrounding (p value = 0.0407). The risk factors identified for mayaro infection were the location of residence in towns near the forest (p value <0.0001) and presence of toilet in or near the home (p value = 0.0415). The serological results suggest that infection with mayaro occurred less than 10 years in the vicinity of residences analyzed. Molecular analysis of the samples collected in the urban area of Manaus not detected genomic fragments of arboviruses. Factors such as low viremia at the time of blood collection and storage of serum samples may have contributed to the non-detection of genomic fragments. However, the protocol for the detection of genomic fragments of arboviruses based on the PCR technique is already used in research centers and surveillance of Fundação de Medicina Tropical do Amazonas FMTAM and Instituto Leônidas e Maria Deane ILMD/FIOCRUZ.
As arboviroses são atualmente reconhecidas pela Organização Mundial da Saúde como um problema global. A maioria das arboviroses resume-se em afecções com sintomatologias agudas e inespecíficas, como febre, dores de cabeça e dores musculares. Embora sejam auto limitados, tais sintomas geram impactos sociais e econômicos relevantes. No Brasil, as arboviroses provocadas pelos por vírus pertencentes aos gêneros Alphavirus, Orthobunyavirus e Flavivirus são as principais causadoras de surtos ou epidemias. O presente estudo teve como objetivo estudar a circulação dos arbovírus mayaro, vírus da encefalite eqüina venezuelana, vírus da febre amarela, vírus da encefalite de Saint Louis e vírus oropouche em uma área rural e uma área urbana do Estado do Amazonas, Brasil. Assim sendo, a sorologia para detecção de imunoglobulinas G foi utilizada para avaliar a prevalência de anticorpos contra tais vírus em 335 moradores de uma comunidade rural do Estado do Amazonas e a PCR foi utilizada para avaliar a incidência de tais vírus em 250 amostras coletadas na área urbana de Manaus. Os resultados encontrados para a sorologia sugerem a circulação do vírus mayaro na comunidade rural. A soroprevalência detectada nas amostras foi de 41,5%. Não houve relação estatisticamente significativa de risco para a infecção por mayaro entre os gêneros (p valor=0,7760) ou entre as faixas etárias (p valor=0,9422). A sorologia positiva detectada entre 39 crianças menores de 10 anos indica uma infecção recente. Os fatores de proteção contra a infecção por mayaro detectados foram o uso de mosquiteiro (p valor=0,0119) e a presença de animais no peridomicílio (p valor=0,0407). Os fatores de risco detectados para a infecção por mayaro foram a localização do domicílio em vilas próximas à floresta (p valor<0,0001) e a presença de toalete dentro ou próximo ao domicílio (p valor=0,0415). Os resultados sorológicos sugerem que a infecção por mayaro ocorreu há menos de 10 anos nas proximidades das residências analisadas. A análise molecular das amostras coletadas na zona urbana de Manaus não detectou fragmentos genômicos de arbovírus. Fatores como baixa viremia no momento da coleta de sangue e estocagem das amostras de soro podem ter contribuído para a não detecção dos fragmentos genômicos. Contudo, o protocolo de detecção de fragmentos genômicos de arbovírus baseado na técnica de PCR está em uso nos centros de pesquisa e vigilância epidemiológica da Fundação de Medicina Tropical do Amazonas FMTAM e do Instituto Leônidas e Maria Deane ILMD/FIOCRUZ.
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Stretton, Clare. "Alphaviruses and host cell interactions." Thesis, University of Liverpool, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.441742.
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Morazzani, Elaine M. "Modulation of Alphaviruses by Small RNAs." Diss., Virginia Tech, 2011. http://hdl.handle.net/10919/39328.
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Mosquito-borne diseases remain a significant burden on global public health. Maintenance of mosquito-borne viruses in nature requires a biological transmission cycle that involves alternating virus replication in a susceptible vertebrate and mosquito host. Although infection of the vertebrate host is acute and often associated with disease, continual transmission of these viruses in nature depends on the establishment of a persistent, nonpathogenic infection in the mosquito vector. It is well known that invertebrates rely on small RNA pathways as an adaptive antiviral defense. The canonical antiviral response in these organisms involves dicer enzymes that cleave viral double-stranded RNA replicative intermediates (RIs) into small interfering RNAs (siRNAs; ~21-24 nucleotides). One strand of the siRNA duplex guides the targeting and destruction of complementary viral RNAs when loaded and retained in a multi-protein complex called the RNA-induced silencing complex. Here, we show that mosquito vectors mount a redundant double defense against virus infection mediated by two different small RNA pathways. Specifically, we demonstrate that in addition to a canonical antiviral response mediated by siRNAs, virus infection of the mosquito soma also triggers an antiviral immune pathway directed by ping-pong-dependent PIWI-interacting RNAs (piRNAs; ~24-30 nucleotides). The complexity of mosquito antiviral immunity has important implications for understanding how viruses both induce and modulate RNA-silencing responses in mosquito vectors.
In mammals, viral RIs induce a range of relatively nonspecific antiviral responses. However, it remains unclear if viral RIs also trigger RNA silencing in mammals. Mosquito-borne viruses represent an ideal model for addressing this question as their transmission cycles involve alternating replication in mammalian and invertebrate hosts. Although we report identifying a subset of virus-derived small RNAs that appear to be products of RNA silencing in two mammalian cell lines infected with the mosquito-borne chikungunya virus (CHIKV), our studies suggest these small RNAs have little biological relevance in combating virus infections. Thus, while the accumulation of virus-derived siRNAs is essential to the survival of mosquitoes infected with CHIKV, they appear to have little functional significance in mammalian antiviral immunity.Ph. D.