Dissertations / Theses on the topic 'Alphavirus infections'

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 thérapeutiques<br>Ross 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

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.<br>National Institute for Health Research<br>Revisión por pares

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 humaines<br>Humans 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

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.

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 virus<br>The 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

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.<br>Thesis (PhD Doctorate)<br>Doctor of Philosophy (PhD)<br>Institute for Glycomics<br>Griffith Health<br>Full Text

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 monoterpènes<br>Essential 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

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.

La République du Congo (RC) ou nos travaux ont eu lieu, est un pays d’Afrique Centrale, il partage ses frontières avec la République démocratique du Congo, la République centrafricaine, le Gabon, le Cameroun, et l’Angola (Cabinda). Dans ces pays la circulation des arboviroses est documentée. En RC, il y avait peu ou pas de documentation sur les arboviroses avant nos travaux. Nous avons réalisé des études de séroprévalence des arboviroses de différentes familles chez des donneurs de sang Congolais. Nous avons aussi étudié l’épidémie de chikungunya ayant sévit en RC en 2011. Nos travaux ont permis de mettre en évidence des taux de séroprévalence élevés pour les pathogènes principaux incriminés: 47,2% pour Dengue, 27,8% pour Yellow fever, 24,4% pour West Nile, 38,8% pour Chikungunya et 7,9% pour Rift Valley fever. Ces taux de séroprévalence élevés prouvent la circulation de ces virus au Congo, bien qu’aucune épidémie n’ait été encore déclarée pour certains. Nous avons également isolé et caractérisé génétiquement une souche nommée "Brazza_MRS1", appartenant au lignage East Central and Southern African, issue de l’épidémie due au virus chikungunya de 2011. La RC a connu plusieurs épidémies dues au virus Ebola. Nous avons tenté de mieux caractériser la circulation des filovirus chez les donneurs de sang asymptomatiques par une étude de séroprévalence. Les taux de séroprévalence IgG anti Ebola virus, observés étaient de 2,5% en général (1,6% à Brazzaville, 4% à Pointe-Noire et 4% en milieux ruraux). Les facteurs de risques identifiés étaient l’exposition aux chauves-souris (p&lt;0.001) et aux oiseaux (p = 0.04). Le taux de séroprévalence IgG anti Marburg virus était de 0,5%<br>The Republic of Congo (RC) where our work took place is a Central African country, sharing borders with the Democratic Republic of Congo, Central African Republic, Gabon, Cameroon, and Angola (Cabinda). In these countries the circulation of arboviruses is documented. In RC, there was little or no documentation on arboviruses prior to our work. We conducted studies of arbovirus seroprevalence in Congolese blood donors for different virus families. We also studied the epidemic caused by the chikungunya virus that prevailed in RC in 2011.Our work have highlighted the high rate of seroprevalence for incriminated major pathogens: 47.2% for Dengue, 27.8% for Yellow Fever, 24.4% for West Nile, 38.8% for Chikungunya and 7.9% for Rift Valley fever. These high seroprevalence rates indicate that these viruses actively circulate in Congo, although no epidemic has yet been reported for some viruses. We have also isolated and genetically characterized a strain named "Brazza_MRS1", belonging to the East Central and Southern African lineage, after the chikungunya epidemic in 2011. The RC has experienced several outbreaks caused by the Ebola virus. We have performed a filovirus seroprevalence study to attempt to better characterize the circulation of filoviruses in asymptomatic Congolese blood donors. The observed rate of seroprevalence of anti Ebola IgG was 2.5% overall (1.6% in Brazzaville, 4% in Pointe-Noire and 4% in rural areas). Identified epidemiological risk factors were the exposure to bats (p &lt;0.001) and birds (p = 0.04). The seroprevalence rate of Marburg virus IgG was low (0.5%)

Arthritogenic alphaviruses such as Ross River virus (RRV) and chikungunya (CHIKV), Sindbis-like viruses (SINV), Barmah Forest virus (BFV), ) Mayaro virus (MAYV) and o’nyong-nyong virus (ONNV) are responsible for outbreaks of debilitating rheumatic joint disease during infection. CHIKV is typically notorious in central Africa, India, South-East Asia and Europe, while other alphaviruses such as RRV and BFV are endemic to Australia. In Australia, there are approximately 4000 cases of RRV reported annually, while the number of CHIKV cases continues to rise in epidemic regions. In 2014, CHIKV invaded several Caribbean islands in the Americas, with a current estimated 1.1 million autochthonous transmission cases as of 16th January 2015. During disease outbreak, more than 95% of patients experience intense persisting pain, generally in knees and the joints of extremeties. Despite the high occurrence rheumatic joint symptoms during alphavirus infection, there are limited studies on the skeletal pathologies due to the lack of routine clinical surveillance. Hence, very little is known about the impact of alphavirus infection on the bone remodelling process and warrants further investigation.<br>Thesis (PhD Doctorate)<br>Doctor of Philosophy (PhD)<br>Institute for Glycomics<br>Science, Environment, Engineering and Technology<br>Full Text

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 virus<br>Alphaviruses 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

Depuis 2007, le Gabon est régulièrement confronté à des infections par les virus Chikungunya (CHIKV) et Dengue (DENV). Au total, près de 4300 prélèvements provenant de patients se présentant avec un syndrome fébrile algique, en phase clinique aiguë, ont pu être collectés et analysés pour la période de 2007 à 2010. En effet, deux importantes épidémies concomitantes de CHIKV et de DENV ont sévi au Gabon (i.e. Libreville en 2007 et Franceville en 2010). Entre ces deux flambées épidémiques, de nombreux cas sporadiques d'infection à CHIKV ou à DENV ont continué à être enregistrés à travers le pays. Des cas de co-infection à CHIKV/DENV ont également été diagnostiqués lors des deux flambées épidémiques. Ces deux arbovirus se sont ainsi propagés en quelques années dans un mouvement de nord-ouest à sud-est à travers le pays. L’étude plus avancée des cas de co-infection à CHIKV/DENV a pu démontrer que ce phénomène pouvait survenir soit de manière simultanée soit séquentielle au cours du repas sanguin d'Aedes albopictus, principal vecteur du CHIKV et du DENV au Gabon.(…)En conclusion, ce travail de thèse décrit précisément la survenue brutale d’épidémies imputables à plusieurs arbovirus circulant simultanément au Gabon et responsables de nombreux cas cliniques se présentant sous la forme d'un syndrome fébrile algique. La co-circulation de ces virus suggère l’apparition d’une dynamique de type épidémique/endémique et implique un problème de santé publique latent dans cette région d’Afrique, voire dans l’ensemble de la sous-région d’Afrique Centrale<br>Following to the first simultaneous Chikungunya (CHIKV) and Dengue (DENV) viruses outbreak in 2007, an active surveillance of febrile syndromes was set up in Gabon, a central African country. During a three-year period, we observed a rapid spread of CHIKV and DENV in a southward movement from north-west to south-east of the country. Indeed, CHIKV and DENV have disseminated within a non-immune population, widely favored by the extraordinary capacity of Aedes albopictus vector to colonize diverse environments and to replace local mosquito’s species. In 2010, a second outbreak occurred in Gabon with further CHIKV/DENV co-infections in both human and mosquito. This is the first documented evidence of co-infection in a wild-caught Aedes albopictus. Additionally, an underlying Zika (ZIKV) virus epidemic transmission by the same invasive vector was retrospectively recorded during the outbreak in 2007. These data reveal an unusual ZIKV natural life cycle, occurring in an urban environment and potentially representing a new arboviral emerging threat from Aedes albopictus.(…)In conclusion, these data highlighted the recent introduction and rapid dissemination of CHIKV and DENV in Gabon. The Aedes albopictus vector has shown its extraordinary capacity to sustain epidemic transmissions, leading to arboviral co-circulations (i.e. CHIKV, DENV-2, DENV-1, DENV-3, ZIKV) and notably to some CHIKV/DENV-2 co-infection cases. This multiple arboviral circulation suggests an epidemic/endemic dynamic in Gabon, involving a latent public health problem in this region of Africa

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.

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.

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.

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%).<br>National Research Foundation of Korea<br>Revisión por pares

The current situation of global warming has a serious impact on the control of arthropod-borne infectious diseases. Climate change has led to an increase in conducive breeding habitats for mosquitoes. This change in climate has contributed to the widespread distribution of mosquito-transmitted arthritogenic alphaviruses such as Ross River virus (RRV) and chikungunya virus (CHIKV). RRV is currently endemic to Australia and Papua New Guinea, while CHIKV causes global outbreaks. The recent CHIKV outbreak in the Americas has taken the world by surprise and has affected more than 1 million individuals as of early 2015. To date, there are no specific therapeutics strategies available for the treatment of alphaviral diseases, largely due to the ill-defined innate immune responses elicited by these viruses. This thesis describes new insights into the alphavirus-elicited immune response at the cellular and molecular levels using in vitro, in vivo and ex vivo experimental approaches. In these studies, we identified the differential role of macrophages in modulating the RRV disease progression through an atypical IL-10-dependent M1/M2 polarisation of inflammatory monocytes. RRV-induced differentiation of M1 macrophages triggered the pathogenic inflammatory processes giving rise to the onset of disease, while M2 macrophages were shown to play a protective role.<br>Thesis (PhD Doctorate)<br>Doctor of Philosophy (PhD)<br>Institute for Glycomics<br>Science, Environment, Engineering and Technology<br>Full Text

Le virus chikungunya est un arbovirus, transmis par les moustiques du genre Aedes, qui provoque des arthralgies invalidantes et parfois des rhumatismes chroniques. Dans une première partie nous avons décrit les aspects ambulatoires cliniques, biologiques et virologiques du chikungunya (CHIK) de la phase aiguë jusqu'au 300ème jour lors de l’épidémie de la Réunion en 2006. Des scores d’aide au diagnostic ont été élaboré et une étude de la diversité virale intra-hôte a été réalisée. Pour compléter nos premiers résultats nous avons étudié une épidémie survenue en République du Congo en 2011. La description clinique était similaire à celle identifiée lors de l’épidémie de la Réunion. L’évaluation du score clinique ne permettait pas de le proposer comme outil diagnostique à l’échelle individuelle mais apparaissait comme un bon marqueur pour le suivi de la courbe épidémique. Une étude de séroprévalence et une analyse phylogénétique complètent ce travail. Le dernier travail porte sur l’utilisation de la chloroquine à la phase aiguë du CHIK lors d’une prise prophylactique chez le singe et lors d’un essai clinique chez l’homme. Le principal effet de ce type de traitement semble lié son action immuno-modulatrice ; en prise préventive il provoque une exacerbation de la symptomatologie aiguë tandis qu’en prise à la phase précoce de la maladie il augmente le risque d’évolution vers des arthralgies chroniques. En conclusion nous avons réalisé une description des formes ambulatoires du CHIK, identifié des facteurs de risques de formes chroniques, proposé des scores d’aide au diagnostic et argumenté la contre-indication de l’utilisation de la chloroquine à la phase aiguë du CHIK<br>Chikungunya virus (CHIKV) is an arthropod-borne virus transmitted by Aedes mosquitoes that cause debilitating arthralgia and possible chronic rheumatism. In the first part we describe the clinical, biological and virological presentation of outpatients with chikungunya disease (CHIK) from the acute stage to the chronic stage at day 300, during the outbreak in the Reunion Island in 2006. We elaborated scores for CHIK diagnosis and we also analysed the intra-host genetic diversity.To complete our first results, we investigated a CHIKV outbreak, which occurred in the Republic of Congo in 2011. The clinical presentation was similar to the first description of the Reunion island outbreak. We assessed the clinical score which appeared to be unusable at the individual level but was still relevant to follow the epidemic curve. This work was completed by seroprevalence and phylogenetic analyses.The last study presented in this thesis focused on the use of chloroquine during the acute stage of CHIK in a non-human primate (NHP) model (prophylactic use) and during a clinical trial (therapeutic use). The main effect of chloroquine treatment at the acute stage of CHIK appeared to be related to its immuno-modulatory action; in prophylactic taking, chloroquine exacerbated acute symptoms while treatment during the early stages of the disease increased the risk of acquiring chronic arthralgia.In conclusion, we provide a detailed description of CHIK outpatients and identify risk factors for the chronic stage of the disease. We propose tentative diagnostic scores and we firmly establish that the use of chloroquine at the acute phase of CHIK is contraindicated

Arthropod-borne-viruses (arboviruses) pose a global threat due to their ability to be transmitted by hematophagous insects to vertebrate hosts resulting in a range of serious infectious diseases. Sindbis virus (SINV) is the prototype arbovirus of the genus Alphavirus in the family Togaviridae. The purpose of this study was to investigate the use of a fluorescent tagged reporter virus in both in vitro and in vivo environments. The fluorescent protein GFP was inserted between the Capsid and PE2 in the genome of TR339; SINV TaV-GFP (Wm. Klimstra Lab). This virus construct should have the same infectivity and virulence as wild type TR339, leaving a fluorescent ‘path’ in infected cells that may reveal virus transit. Virus stocks were grown in BHK-21 vertebrate cells and C7-10 mosquito cells. Two Aedes albopictus mosquito cell lines, C7-10 and C6/36, were then challenged with vertebrate and mosquito grown reporter virus. Evidence of GFP were seen as early as 6 hours post infection (p.i.) in all samples. Infected C7-10 cells with the vertebrate grown reporter virus were fixed for 1 hour in chilled 4% buffered paraformaldehyde; GFP was shown to be resilient to both fixation and light quenching. Ultimately, Ae. aegypti mosquitoes were challenged with a viremic bloodmeal at a titer of 107 PFU/ml and midguts were dissected over several days. The presence of GFP was observed in midgut columnar epithelial cells as early as day 3 p.i. and remained localized even at day 30 p.i. This is in agreement with published work on the interaction of TR339 in Ae. aegypti gut, signaling this viral construct as a means to visualize wild-type infection.

Emerging arthropod-borne viruses, such as alphaviruses and bunyaviruses, represent a serious threat to human and animal health worldwide, and for most of them, vaccines and specific treatments are unavailable. Viral host cell entry can be divided into several entry checkpoints, and the most important checkpoints for low pH-dependent enveloped viruses, such as bunyaviruses and alphaviruses, include receptor binding at the cell surface and, followed by endocytosis, low pH dependent membrane fusion from within intracellular compartments. A more thorough understanding of the detailed mechanisms allowing the viruses to pass these checkpoints is a pre-requisite for the design of viral entry inhibitors. This thesis reports the in vitro analysis of native alphavirus-receptor interactions, with the help of electron cryo-microscopy and icosahedral reconstruction of virus-recaptor complexes, using the prototypic alphavirus Semliki Forest virus (SFV) and the C-type lectin DC-SIGN. Together with results from collaborative work on SFV glycosylation, this study provides progress in defining the binding sites of DC-SIGN at the surface of SFV. Second, an in vitro system for phlebovirus fusion was developed using standard fluorometry, and has been characterized with the help of electron cryo-microscopy. It was discovered that negatively charged phospholipids with a conical shape, including the late endosomal phospholipid BMP, allow efficient phlebovirus fusion in vitro, thereby providing a possible rationale for phlebovirus fusion in late endosomes. Furthermore, electron cryo-microscopy of phlebovirus-liposome complexes allowed the capture of early stage fusion intermediates and laid the basis for possible future higher resolution studies of these fusion intermediates.

Afin de modéliser l'évolution de l’infection par le chikungunya (CHIK), son impact, et les facteurs pronostiques de chronicité, nous avons travaillé en trois parties. L'impact à long terme de l’épidémie de CHIK en 2005-2006 à la Réunion a été estimé en calculant la proportion de patients en phase chronique au cours du temps et la charge globale de morbidité du CHIK par la méthode des années de vie ajustées sur l'invalidité (méthode DALY de l’OMS, qui prend en compte les années de vie perdues en raison de la mortalité prématurée et des années de vie vécues avec une incapacité). Ainsi entre 51,2 et 65,3% des patients étaient estimés symptomatiques après 1 an et 0% à15,2% après 5 ans. Le total d’années de vie en bonne santé perdues à la Réunion a été estimé à 65-73/1000 personnes, 55,5% des pertes concernant la population active (les 20 à 60 ans), et 86% étant dues à la persistance de rhumatismes post-CHIK (phase chronique). Les facteurs pronostiques de la persistance de rhumatismes et de l’altération de la qualité de vie (QdV) à long terme (30 mois) ont été étudiés dans une cohorte des gendarmes dont 25% étaient infectés (CHIK+). Etre CHIK+, avoir des comorbidités et un moral déprimé pendant la phase aiguë étaient prédictifs de la persistance d’arthrite comme d’arthralgies. De plus, la présence d’arthralgies ou arthrite à six mois était très prédictive de la persistance des mêmes rhumatismes à 30 mois<br>To model the evolution of chikungunya virus (CHIK) infection, its impact and the prognostic factors of post-CHIK rheumatism and quality of life, we worked in three parts. The long-term impact of the 2005-2006 CHIK outbreaks in Reunion Island was estimated by calculating the proportion of chronic patients over time and the global burden of CHIK using the Disability Adjusted Life Years (DALY) method. This method sums the years of life lost due to premature mortality and the years lived with disability. Between 51.2 and 65.3% of patients were estimated chronic after 1 year and 0%-15.2% after 5 years. The global disease burden of CHIK was estimated 65-73 DALYs/1000 persons, 55.5% concerning the active population (20-60 years old), and 86% due to persistence of post-CHIK rheumatisms. Prognostic factors of the long-term (30 months) rheumatisms and impaired quality of life (QoL) were studied in a cohort of French army policemen (25% CHIK infected: CHIK+). Being CHIK+, suffering of comorbidity and having depressed mood during the acute stage were predictive for both persistent arthritis and arthralgias at 30 months. In addition, suffering of either arthralgias or arthritis at six months was predictive of the same symptoms at 30 months. Determinants of impaired QoL were CHIK infection and comorbidity, in addition to older age, work-stoppage during the acute infection and arthritis at 6 months for the QoL physical component, and depressed mood at 6 months for the mental health component.Association between the severity of initial CHIK-stages and recovery were studied using multiple correspondence analysis (MCA)