Diante da necessidade de responder algumas indagações relacionadas a epidemiologia da Estomatite Vesicular, principalmente aquelas que dizem respeito a ocorrência de surtos em locais onde existem coleções d\'água, foi desenvolvido um modelo de transmissão do VSA utilizando a água como via de transmissão, a tilápia nilótica, inoculada intraperitonealmente, como fonte de infecção e o cobaio como hospedeiro susceptível. O objetivo da utilização deste modelo biológico de transmissão do Vírus da Estomatite Vesicular foi de avaliar o papel desempenhado pelos peixes no ciclo epidemiológico, propor um modelo de ciclo epidemiológico do VSA, destacando o papel da água como via de transmissão e padronizar uma técnica de RT-PCR para a detecção do VSA, em amostra de tecidos. Através do modelo desenvolvido, fica demonstrado que estes peixes eliminaram partículas virais na água, decorridos 13 dias pós-inoculação e que esta última se caracteriza como via de transmissão, possibilitando a infecção dos hospedeiros susceptíveis (cobaios) através de inoculações experimentais em coxim plantar. A tilápia nilótica pode ser considerada como uma fonte de infecção, por ser capaz de eliminar um agente infeccioso no meio ambiente e através de uma via de transmissão este agente alcançou o hospedeiro susceptível; os peixes podem ser inseridos no ciclo epidemiológico da Estomatite Vesicular como fonte de infecção, sendo capazes de eliminar na água partículas virais infectantes, destacando o papel da água como via de transmissão; fica padronizada uma técnica de RT-PCR dirigida ao gene codificador da proteína RNA-polimerase, útil para a detecção direta do Vírus da Estomatite Vesicular Alagoas e Indiana em amostras de tecidos. / A model of transmission of Vesicular Stomatitis was developed to Vesicular Stomatitis Alagoas (VSA) serotype employing water as a way of transmission, the Nile tilapia intraperitoneal inoculated as a source of infection and guinea pigs as susceptible hosts aiming to answer many questions concerning Vesicular Estomatitis epidemiology, as the risk of disease on farms with dose relationship with riverine areas and the role of fishes in the epidemiological cycle of the disease. Furthermore, a RT-PCR assay was developed to detect VSA in tissue samples. According to the experimental transmission, fishes eliminated virus into the water after 13 days pos-infection and a model to VSA epidemiological cycle is proposed in which water was characterized as a way of transmission, carrying the virus to the susceptible host through experimental inoculation and the Nile tilapia should be thought as a source of infection, once it was able to eliminate the infective agent into the environment. A useful tool to the diagnosis of both Indiana and Alagoas serotypes was developed.

Diante da necessidade de responder algumas indagações relacionadas a epidemiologia da Estomatite Vesicular, principalmente aquelas que dizem respeito a ocorrência de surtos em locais onde existem coleções d\'água, foi desenvolvido um modelo de transmissão do VSA utilizando a água como via de transmissão, a tilápia nilótica, inoculada intraperitonealmente, como fonte de infecção e o cobaio como hospedeiro susceptível. O objetivo da utilização deste modelo biológico de transmissão do Vírus da Estomatite Vesicular foi de avaliar o papel desempenhado pelos peixes no ciclo epidemiológico, propor um modelo de ciclo epidemiológico do VSA, destacando o papel da água como via de transmissão e padronizar uma técnica de RT-PCR para a detecção do VSA, em amostra de tecidos. Através do modelo desenvolvido, fica demonstrado que estes peixes eliminaram partículas virais na água, decorridos 13 dias pós-inoculação e que esta última se caracteriza como via de transmissão, possibilitando a infecção dos hospedeiros susceptíveis (cobaios) através de inoculações experimentais em coxim plantar. A tilápia nilótica pode ser considerada como uma fonte de infecção, por ser capaz de eliminar um agente infeccioso no meio ambiente e através de uma via de transmissão este agente alcançou o hospedeiro susceptível; os peixes podem ser inseridos no ciclo epidemiológico da Estomatite Vesicular como fonte de infecção, sendo capazes de eliminar na água partículas virais infectantes, destacando o papel da água como via de transmissão; fica padronizada uma técnica de RT-PCR dirigida ao gene codificador da proteína RNA-polimerase, útil para a detecção direta do Vírus da Estomatite Vesicular Alagoas e Indiana em amostras de tecidos. / A model of transmission of Vesicular Stomatitis was developed to Vesicular Stomatitis Alagoas (VSA) serotype employing water as a way of transmission, the Nile tilapia intraperitoneal inoculated as a source of infection and guinea pigs as susceptible hosts aiming to answer many questions concerning Vesicular Estomatitis epidemiology, as the risk of disease on farms with dose relationship with riverine areas and the role of fishes in the epidemiological cycle of the disease. Furthermore, a RT-PCR assay was developed to detect VSA in tissue samples. According to the experimental transmission, fishes eliminated virus into the water after 13 days pos-infection and a model to VSA epidemiological cycle is proposed in which water was characterized as a way of transmission, carrying the virus to the susceptible host through experimental inoculation and the Nile tilapia should be thought as a source of infection, once it was able to eliminate the infective agent into the environment. A useful tool to the diagnosis of both Indiana and Alagoas serotypes was developed.

La SUMOylation est une modification post-traductionnelle qui gouverne divers processus cellulaires incluant immunité innée et défense antivirale. Des effecteurs de la synthèse d’IFN, de son signal de transduction ainsi que des facteurs de restriction sont modifiés par SUMO (Small Ubiquitin Modifier). Par ailleurs, certains virus exploitent la machinerie SUMO afin de contrecarrer les mécanismes de défense antivirale suggérant l’implication de SUMO dans l’interface virus et défense antivirale. A l’aide d’un modèle cellulaire exprimant les différents paralogues SUMO1, SUMO2 ou SUMO3 ou en diminuant l’expression de l’unique enzyme de conjugaison à SUMO, Ubc9, nous avons montré un effet différentiel de SUMO sur deux virus de la famille des Rhabdoviridae (virus de la stomatite vésiculaire (VSV) et le virus de la rage) et sur la réponse aux IFN alpha et IFN gamma. Le premier axe de recherche a permis de montrer que l’expression de SUMO inhibe la synthèse de l’IFN suite à l’infection par le VSV et le virus de la rage, rendant les cellules plus sensibles à l’infection par le virus de la rage. IRF3 est conjuguée à SUMO, ce qui corrèle avec l’inhibition de sa phosphorylation et l’inhibition de la synthèse d’IFN beta. En revanche, bien que la synthèse de l'IFN soit diminuée, l’expression de SUMO confère la résistance au VSV et inhibe sa transcription primaire. L’activité anti-VSV de SUMO est abolie par la déplétion de MxA. L’effet de SUMO est médié par sa capacité à augmenter l’oligomérisation et la stabilité de MxA. Par ailleurs, ce travail a permis d’identifier MxA comme nouvelle cible de la machinerie SUMO. MxA interagit avec SUMO de manière covalente sur la lysine K48 et de manière non covalente avec SUMO1. Le second axe de recherche a permis d’identifier SUMO comme un nouveau régulateur de la réponse aux IFN. La SUMOylation de STAT1 inhibe sa phosphorylation, régulant négativement le signal de transduction et par conséquent la transcription et la réponse biologique en réponse à l’IFN gamma. En revanche, l’expression de SUMO n’altère ni le signal de transduction ni la transcription en réponse à l’IFN alpha.Par ailleurs, dans les cellules exprimant SUMO3, l’IFN gamma et l’IFN alpha induisent la SUMOylation de PML par SUMO3 ce qui entraîne sa dégradation via le protéasome et inhibe les réponses biologiques médiées par PML. Ce travail a permis de montrer un rôle central de SUMO dans l’immunité intrinsèque et innée, médié par la SUMOylation de protéines cellulaires telles qu’IRF3, MxA, STAT1 ou PML. / SUMOylation modulates several cellular process including innate immunity and antiviral defense. Many key regulators involved in IFN induction, IFN signaling as well as various restriction factors are SUMOylated. Using cells stably expressing the different paralogs of SUMO; SUMO1, SUMO2 or SUMO3 and cells depleted of the only known SUMO conjugating enzyme, Ubc9, we show a differential effect on two viruses from Rhabdoviridae family (Vesicular Stomatitis Virus (VSV) and rabies virus) and on the response to IFN alpha and IFN gamma. First, we report that SUMO expression inhibits VSV- and rabies virus-induced IFN synthesis. Consequently, SUMO expression renders cells more sensitive to rabies virus infection. Overexpression of SUMO leads to IRF3 SUMOylation correlating rabies viral infection with both the inhibition of IRF3 activation and IFN beta synthesis. However, although SUMO inhibits VSV-induced IFN, SUMO confers resistance to VSV by inhibiting VSV primary transcription. Furthermore, the anti-VSV effect of SUMO is abolished in MxA depleted cells. Mechanistically, SUMO enhances MxA oligomerization resulting in the stabilization of the MxA protein. We also identified MxA as a new target of SUMO machinery. MxA interacts covalently with SUMO2/3 on lysine K48 and non-covalently with SUMO1. We then investigated the various roles of SUMO at different steps of the JAK/STAT pathway, including STAT activation, transcriptional response and IFN-induced biological effects, identifying SUMO as a new regulator of IFN response. The overexpression of SUMO leads to STAT1 SUMOylation and to a decrease in IFN-induced STAT1 phosphorylation resulting in an inhibition of IFN-gamma-induced transcription and biological responses. In contrast, SUMO expression does not alter IFN alpha signaling and transcriptional response. In addition, in SUMO3 expressing, IFN gamma;and IFN alpha induce SUMOylation of PML by SUMO3 inducing its degradation via the proteasome and inhibition of biological responses mediated by PML. Taken together our results show that SUMO plays a crucial role in innate and intrinsic immunity mediated by SUMOylated proteins such as IRF3, MxA, STAT1 or PML.

Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 130-160).

As an outcome of very active arbovirus monitoring programs that began in Australia in the 1950s, some of the most diverse and unusual rhabdoviruses in the world have been isolated from this continent. These novel rhabdoviruses represent an important and valuable pool of highly diverse viruses; however, most of them have remained poorly characterised. In light of the significant disease potential of numerous rhabdoviruses, the characterisation of novel rhabdoviruses is indispensable for threat assessment to livestock, wildlife and humans and preparedness for outbreaks. The genetic characterisation of novel viruses is also an essential step for the development of molecular detection assays for improved monitoring and investigations into unidentified disease cases. In this study, the complete genomes of four novel rhabdoviruses have been sequenced and a fifth is close to completion. The substantial new data generated has significantly extended the understanding of the biology and evolution of the Rhabdoviridae. Wongabel virus (WONV), isolated from the biting midge Culicoides austropalpalis, was found to contain a unique genome structure encoding ten genes, including five novel genes (Chapter 2). Analysis by western blotting suggested that four out of the five novel genes were expressed in infected cell cultures. Ngaingan virus (NGAV), isolated from Culicoides brevitarsis, was found to have the largest genome of any rhabdovirus sequenced to date, and with thirteen genes has the largest number of genes of any (-) ssRNA virus sequenced to date (Chapter 3). Seven of the thirteen genes are novel. Similar to viruses in the genus Ephemerovirus (bovine ephemeral fever virus and Adelaide River virus), NGAV contains a second glycoprotein with an unknown function. Phylogenetic analysis places this virus alongside WONV and the north-American bird and mosquito-associated Flanders virus within the Hart Park group that remains to be classified by the ICTV. Screening of various wildlife and livestock sera collected in northern Australia indicated a strong association of NGAV with macropods. Tibrogargan virus (TIBV) and Coastal Plains virus (CPV) were isolated from cattle and Culicoides brevitarsis (TIBV). Past serological surveys reported both viruses to be highly prevalent in cattle in northern Australia and demonstrated that the two viruses share a relatively close relationship at the antigenic level. The genomic analyses revealed that these two viruses have a unique genome organization, with three additional genes (Chapter 4). These additional genes are highly diverged at the sequence level but the encoded putative proteins share a significant conservation of secondary structure elements. The sequencing of these two related viruses has provided a unique opportunity to gain insights into the characteristics and evolution of novel proteins in two different rhabdoviruses. Phylogenetic analyses showed that TIBV and CPV form an independent cluster which does not appear to belong to any of the current genera, but which is most closely related to the genus Ephemerovirus based on N protein analysis. Although neither virus has been associated with disease, a serological survey of various animal sera collected in northern Australia showed that these viruses are currently highly prevalent in sentinel cattle and buffalo. Oak Vale virus (OVRV) was isolated from mosquitoes, Culex edwardsi and Ochlerotatus vigilax, from two geographically diverse regions of Australia located approximately 3000 km apart. The genome of OVRV was found to contain only one novel gene (Chapter 5). Comparatively, the genome of this virus is much less complex than the others in this study, but this virus displays considerable divergence from all other rhabdoviruses. A high seroprevalence for this virus was found in the feral pig population in northern Australia. The data generated from this study represents a considerable increase in the quantity of genetic data available for this viral family, and has revealed the existence of a large number of previously unidentified genes, highlighting that that the potential for complexity within the prototype genomic model of a rhabdovirus is much greater than previously thought. The novel nature of the additional genes provides grounds for further research into rhabdovirus evolution. Analysis of this new data suggests that these viruses cannot be classified into existing genera under the current criteria and it is clear that the taxonomy of the Rhabdoviridae requires revision. The observation that these viruses are currently circulating in livestock and wildlife in northern Australia accentuates the need for closer monitoring of animals and the need for further study of this diverse and fascinating group of viruses.

Le virus de la rougeole est un virus à ARN négatif, membre de la famille des Paramyxoviridae. Le complexe de réplication viral comprend trois protéines principales : la polymérase à ARN ARN-dépendante (L), la nucléoprotéine (N) et la phosphoprotéine (P). Cette thèse présente une étude structurale, par des méthodes biochimiques et biophysiques, de la phosphoprotéine et de la nucléoprotéine produites dans la bactérie Escherichia coli. J'ai étudié les déterminants de la polymérisation de N et identifié un variant intéressant pour l'étude de N par cristallographie aux rayons X. J'ai aussi mis au point la purification d'un complexe de N et P, prélude à son étude structurale. Par ailleurs, j'ai montré que la partie N-terminale de P est intrinsèquement désordonnée. J'ai pu étendre ce résultat aux protéines P de nombreux virus apparentés par une étude bio-informatique. J'ai ensuite démontré l'importance du désordre structural au sein du complexe réplicatif des Paramyxoviridae #à la fois par son abondance et d'un point de vue fonctionnel#. Au-delà de leur intérêt pratique immédiat, ces résultats indiquent que la machinerie réplicative de ces virus pourrait constituer un système modèle pour l'étude du désordre structural dans les protéines. De plus, ils soulèvent de nombreuses questions et sont donc un prélude à une refonte de notre vision du complexe réplicatif de ces virus dont l'étude revêt une importance majeure en santé humaine.