A avicultura brasileira é atualmente uma atividade de grande sucesso. A utilização de sistemas de planejamento associados a novas tecnologias, reflete-se no extraordinário crescimento da atividade. A produção brasileira de frango ultrapassou a marca anual de 10 milhões de toneladas, em 2007. O Brasil está entre os três maiores produtores de frango no ranking mundial, junto com Estados Unidos e China. Haja vista a importância que a avicultura representa para o país, pela geração de benefícios sociais e econômicos, o risco que a Doença de Newcastle (DNC) constitui para a avicultura brasileira é enorme. Um surto desta doença em um centro de produção avícola representaria um risco à economia e incidiria de forma negativa nos níveis de consumo de proteína de qualidade e economicamente acessível à população. A fim de estabelecermos um monitoramento do vírus da Doença de Newcastle (NDV) em aves selvagens, livres ou de cativeiro, e aves domésticas não vacinadas, residentes em regiões de elevada confluência migratória aviária no Brasil e em pingüins ao redor da Estação Antártica Comandante Ferraz (EACF), coletamos amostras de swabs orais e cloacais para a posterior análise por PCR em Tempo Real (qPCR), além de sangue para testes sorológicos, tendo como objetivos maiores, contribuir para o fortalecimento dos serviços de defesa sanitária animal, aumentar a capacidade de investigação, e finalmente, atualizar e harmonizar normas e procedimentos para a prevenção e controle da DNC, referenciando-se nas recomendações da Organização Mundial de Sanidade Animal (Office International des Epizooties - OIE). Das 1072 aves amostradas em diferentes regiões do Brasil, 8 (0,75%) apresentaram resultado positivo para o NDV por qPCR, sendo 5 (62,5% das positivas) delas provenientes da região Norte, 2 (25% das positivas) do Nordeste e 1 (12,5% das positivas) da região Sul do Brasil. Na Antártica, dos 100 pingüins estudados, 2 apresentaram resultado positivo para o NDV por qPCR e em cerca de 33,3% dos soros testados foi detectada a presença de anticorpo pelo teste de Inibição da Hemaglutinação (HI). Todas as amostras positivas foram re-analisadas por qPCR específico para cepas mesogênicas e/ou velogênicas, resultando negatividade, corroborando os dados que certificam o Brasil como sendo livre da Doença de Newcastle. / Brazilian chicken meat exports ended 2007 with shipments of 3,3 million tons, which represented a 21% increase in comparison with 2006. These results were the best in the history of the poultry sector in Brazil. The production reached 10.2 million tons, a result that kept the country as the worlds largest chicken meat exporter and the third largest producer, only behind USA and China. Thus the risk of an introduction of the Newcastle disease virus (NDV) into domestic poultry is enormous and will play severe consequences for the economy and poltry industries. In order to provide the surveillance of the NDV in wild, free or captive, and non vaccinated domestic birds from some regions of migratory birds confluence in Brazil and in penguins around the brasilian Antarctic Station Comandante Ferraz, we collected oral and cloacal swabs, for the viral detection by Real Time PCR (qPCR), and blood for the serological test (hemaglutination inhibition test HI). A total of 1072 birds were sampled in diferent regions of Brazil, where 8 (0.75%) shown positive results to NDV, in which 5 (62.5% of positive) were from North region, 2 (25% of positive) were from Northeast and 1 (12,5% of positive) was from South. In the Antarctic 100 penguins were studied, in which 2 were detected the NDV (2% of total). HI test showed that 33.3% of penguins were seropositives for NDV, indicating their previous contact with the pathogen. All the positive samples by qPCR were repeated with primers projected to detect only virulent strains and no sample was positive, indicating the absence of velogenic strains in Brazil. The epidemiological profile was richer with the isolament of positive samples in embrioned chiken eggs specific patogen free and latter nucleotidic genomic sequencing of isolate.

O Vírus Respiratório Sincicial Humano (hRSV) é o patógeno mais comumente associado à doença do trato respiratório inferior em lactentes e crianças. Altas taxas de admissão hospitalar, freqüência de casos e severidade da doenças foi demonstrado em crianças abaixo de dois anos de vida. A freqüência de casos positivos durante o ano de 2004 foi de 43% (188/435) das amostras coletadas no Hospital Universitário/USP na cidade de São Paulo e os isolados brasileiros do grupo A e B agruparam-se nos genótipos previamente caracterizados: GA2, GA5, SAB1, SAB4 e BA like, respectivamente. Palivizumab (PZ) é atualmente o único anticorpo monoclonal disponível para uso em humanos para infecções causadas pelo HRSV. Foi observado o surgimento de escapes mutantes ao PZ in vivo e in vitro, sendo que estas mutações no gene F determinam resistência ao palivizumab. Nós avaliamos através de seqüenciamento da região F1 a ocorrência de escapes mutantes em aspirados de nasofaringe. Realizamos RT-PCR para amplificação de fragmentos do gene F e as seqüências de nucleotídeos foram determinadas. As 30 seqüências analisadas não revelaram mutações ao PZ e através desses dados podemos aferir que o PZ usado profilaticamente em grupos específicos da população é eficaz. / The Respiratory Syncytial Virus (RSV) is the most common cause of lower respiratory tract disease in infants and young children. RSV has high rates of hospital admission and the frequency and severity of infections caused by RSV were assessed in children 2 years of age. In our study the frequency of RSV detection during 2004 was 43% (188/435) of the samples collected from Hospital University/USP in São Paulo city. Partial sequences of G protein gene of 45 isolates from group antigenic A and 8 isolates from antigenic group B were clustered into previously characterized genotypes: GA2,GA5,SAB1, SAB4 e BA like, respectively. Palivizumab (PZ) is the only monoclonal antibody currently available for uses in humans against RSV infectious disease. Here we evaluated the potential for PZ-resistant RSV mutants to arise in clinical samples. Samples from aspirates nasopharyngeal, reverse-PCR-amplified F gene fragments, and the nucleotide sequences were determined. In thirty sequences no revealed F gene mutations. This work shows that palivizumab prophylaxis is safe and efficacious.

Environ 40 pour cent du protéome humain est composé d'importantes régions dépliées. Ces protéines intrinsèquement désordonnées (PID) n'adoptent pas de structures secondaires et tertiaires stables mais échantillonnent un vaste paysage conformationnel. Malgré cela, elles sont aujourd'hui connues pour intervenir dans de nombreux processus biologiques ou pathologiques. À l'instar des eucaryotes, les virus -- surtout les virus à ARN -- ont eux aussi recours aux propriétés particulières des PID pour effectuer les interactions nécessaires à leur réplication. Les paramyxovirus, comme le virus de la rougeole, sont des virus à ARN simple brin de polarité négative et environ 10 pour cent de leur génome de 15 à 18 kilobases code pour des régions dépliées. Cette thèse détaille l'étude de deux protéines virales directement impliquées dans la réplication, la nucléoprotéine et la phosphoprotéine. Elles interagissent l'une avec l'autre et sont composées à la fois de régions dépliées et repliées. Des données à résolution atomique ont été obtenues en spectroscopie par Résonance Magnétique Nucléaire (RMN) en ce qui concerne les parties désordonnées, et en cristallographie pour ce qui est des parties repliées. Les résultats apportent un nouvel aperçu du rôle du désordre conformationnel dans la transcription et la réplication des paramyxovirus. / About 40 percent of the human proteome contains large disordered regions. These intrinsically disordered proteins (IDPs) do not adopt stable secondary and tertiary structures, but sample a large conformational space. In spite of that, they are now known to be involved in many physiological as well as pathological processes. Following the example of eukaryotes, viruses -- especially RNA viruses -- benefit from the particular features of IDPs in their replication machinery. Paramyxoviruses, that includes Measles virus, are single stranded, negative sense RNA viruses and about 10 percent of their 15 to 18 kilobase RNA genome is known to encode for disordered regions. This thesis focuses on the study of two different proteins of paramyxoviruses, namely the nucleoprotein and the phosphoprotein that are directly involved in the replication of the viral genome. They interact with each other and are composed of folded and disordered domains. Atomic resolution information is obtained about the structure and dynamics of these proteins using a combination of Nuclear Magnetic Resonance (NMR) spectroscopy measurements for the disordered parts and X-ray crystallography for the folded domains. The results provide novel insight into the role of conformational disorder in transcription and replication of paramyxoviruses.

O Vírus Respiratório Sincicial Humano (hRSV) é o patógeno mais comumente associado à doença do trato respiratório inferior em lactentes e crianças. Altas taxas de admissão hospitalar, freqüência de casos e severidade da doenças foi demonstrado em crianças abaixo de dois anos de vida. A freqüência de casos positivos durante o ano de 2004 foi de 43% (188/435) das amostras coletadas no Hospital Universitário/USP na cidade de São Paulo e os isolados brasileiros do grupo A e B agruparam-se nos genótipos previamente caracterizados: GA2, GA5, SAB1, SAB4 e BA like, respectivamente. Palivizumab (PZ) é atualmente o único anticorpo monoclonal disponível para uso em humanos para infecções causadas pelo HRSV. Foi observado o surgimento de escapes mutantes ao PZ in vivo e in vitro, sendo que estas mutações no gene F determinam resistência ao palivizumab. Nós avaliamos através de seqüenciamento da região F1 a ocorrência de escapes mutantes em aspirados de nasofaringe. Realizamos RT-PCR para amplificação de fragmentos do gene F e as seqüências de nucleotídeos foram determinadas. As 30 seqüências analisadas não revelaram mutações ao PZ e através desses dados podemos aferir que o PZ usado profilaticamente em grupos específicos da população é eficaz. / The Respiratory Syncytial Virus (RSV) is the most common cause of lower respiratory tract disease in infants and young children. RSV has high rates of hospital admission and the frequency and severity of infections caused by RSV were assessed in children 2 years of age. In our study the frequency of RSV detection during 2004 was 43% (188/435) of the samples collected from Hospital University/USP in São Paulo city. Partial sequences of G protein gene of 45 isolates from group antigenic A and 8 isolates from antigenic group B were clustered into previously characterized genotypes: GA2,GA5,SAB1, SAB4 e BA like, respectively. Palivizumab (PZ) is the only monoclonal antibody currently available for uses in humans against RSV infectious disease. Here we evaluated the potential for PZ-resistant RSV mutants to arise in clinical samples. Samples from aspirates nasopharyngeal, reverse-PCR-amplified F gene fragments, and the nucleotide sequences were determined. In thirty sequences no revealed F gene mutations. This work shows that palivizumab prophylaxis is safe and efficacious.

A avicultura brasileira é atualmente uma atividade de grande sucesso. A utilização de sistemas de planejamento associados a novas tecnologias, reflete-se no extraordinário crescimento da atividade. A produção brasileira de frango ultrapassou a marca anual de 10 milhões de toneladas, em 2007. O Brasil está entre os três maiores produtores de frango no ranking mundial, junto com Estados Unidos e China. Haja vista a importância que a avicultura representa para o país, pela geração de benefícios sociais e econômicos, o risco que a Doença de Newcastle (DNC) constitui para a avicultura brasileira é enorme. Um surto desta doença em um centro de produção avícola representaria um risco à economia e incidiria de forma negativa nos níveis de consumo de proteína de qualidade e economicamente acessível à população. A fim de estabelecermos um monitoramento do vírus da Doença de Newcastle (NDV) em aves selvagens, livres ou de cativeiro, e aves domésticas não vacinadas, residentes em regiões de elevada confluência migratória aviária no Brasil e em pingüins ao redor da Estação Antártica Comandante Ferraz (EACF), coletamos amostras de swabs orais e cloacais para a posterior análise por PCR em Tempo Real (qPCR), além de sangue para testes sorológicos, tendo como objetivos maiores, contribuir para o fortalecimento dos serviços de defesa sanitária animal, aumentar a capacidade de investigação, e finalmente, atualizar e harmonizar normas e procedimentos para a prevenção e controle da DNC, referenciando-se nas recomendações da Organização Mundial de Sanidade Animal (Office International des Epizooties - OIE). Das 1072 aves amostradas em diferentes regiões do Brasil, 8 (0,75%) apresentaram resultado positivo para o NDV por qPCR, sendo 5 (62,5% das positivas) delas provenientes da região Norte, 2 (25% das positivas) do Nordeste e 1 (12,5% das positivas) da região Sul do Brasil. Na Antártica, dos 100 pingüins estudados, 2 apresentaram resultado positivo para o NDV por qPCR e em cerca de 33,3% dos soros testados foi detectada a presença de anticorpo pelo teste de Inibição da Hemaglutinação (HI). Todas as amostras positivas foram re-analisadas por qPCR específico para cepas mesogênicas e/ou velogênicas, resultando negatividade, corroborando os dados que certificam o Brasil como sendo livre da Doença de Newcastle. / Brazilian chicken meat exports ended 2007 with shipments of 3,3 million tons, which represented a 21% increase in comparison with 2006. These results were the best in the history of the poultry sector in Brazil. The production reached 10.2 million tons, a result that kept the country as the worlds largest chicken meat exporter and the third largest producer, only behind USA and China. Thus the risk of an introduction of the Newcastle disease virus (NDV) into domestic poultry is enormous and will play severe consequences for the economy and poltry industries. In order to provide the surveillance of the NDV in wild, free or captive, and non vaccinated domestic birds from some regions of migratory birds confluence in Brazil and in penguins around the brasilian Antarctic Station Comandante Ferraz, we collected oral and cloacal swabs, for the viral detection by Real Time PCR (qPCR), and blood for the serological test (hemaglutination inhibition test HI). A total of 1072 birds were sampled in diferent regions of Brazil, where 8 (0.75%) shown positive results to NDV, in which 5 (62.5% of positive) were from North region, 2 (25% of positive) were from Northeast and 1 (12,5% of positive) was from South. In the Antarctic 100 penguins were studied, in which 2 were detected the NDV (2% of total). HI test showed that 33.3% of penguins were seropositives for NDV, indicating their previous contact with the pathogen. All the positive samples by qPCR were repeated with primers projected to detect only virulent strains and no sample was positive, indicating the absence of velogenic strains in Brazil. The epidemiological profile was richer with the isolament of positive samples in embrioned chiken eggs specific patogen free and latter nucleotidic genomic sequencing of isolate.

Human metapneumovirus (HMPV) is a respiratory pathogen in the Paramyxoviridae family that infects nearly 100% of the world population. This enveloped RNA virus causes severe viral respiratory disease in infants, the elderly, and immunocompromised patients worldwide. Despite its prevalence and importance to human health, no therapies are available against this pathogen. Entry of paramyxoviruses into host cells generally requires the coordinated activity of the attachment glycoprotein, G, which interacts with a cell receptor, and the fusion glycoprotein, F, which promotes subsequent fusion of viral and cellular membranes. However, HMPV F is the primary viral protein mediating both binding and fusion for HMPV. Previous work that showed HMPV F mediates attachment to heparan sulfate proteoglycans (HSPGs), and some HMPV F fusion activity can be promoted by acidic pH. The work presented here provides significant advances in our understanding of the fusion and binding events during HMPV infection. We demonstrated that low pH promotes fusion in HMPV F proteins from diverse clades, challenging previously reported requirements and identifying a critical residue that enhances low pH promoted fusion. These results support our hypothesis that electrostatic interactions play a key role in HMPV F triggering and further elucidate the complexity of viral fusion proteins. Additionally, we characterized the key features of the binding interaction between HMPV and HSPGs using heparan sulfate mimetics, identifying an important sulfate modification, and demonstrated that these interactions occur at the apical surface of polarized airways tissues. We identified differences in particle binding related to the presence or absence of the HMPV G and SH glycoproteins. Lastly, we characterized paramyxovirus infection in cystic fibrosis bronchial epithelial cells, identifying a potential specific susceptibility to HMPV infection in these individuals. The work presented here contributes to our understanding of HMPV infection, from mechanisms of early events of entry to clinical scenarios.

RNA:protein interactions are central in many cellular processes, including activation of innate immune responses against microbial infection. Their study is essential to better understand the diverse biological events that occur within cells. However, isolation of RNA:protein complexes is often laborious and requires specialized techniques. This thesis is concerned with attempts to develop an improved purification protocol to isolate specific RNA:protein complexes. Taking advantage of the specific interaction of the Pseudomonas aeruginosa PP7 protein with its cognate RNA binding site, termed the PP7 recognition sequence (PRS), the aim was to identify cellular proteins involved in activating cell-signalling pathways, including the interferon-induction cascade, following viral infection with stocks of parainfluenza virus 5 (PIV5) rich in copyback defective interfering (DI) particles. Copyback DI genomes are powerful inducers of IFN and, here, I show they also activate the induction of IL-6, IL-8 and TNFα; cytokines that also have antiviral properties. Following the successful cloning of the PRS into a copyback DI genome, we investigated conditions for optimal in vitro capture of DI-PRS:protein complexes by PP7 on Dynabeads. When tested, the protocol led to the successful capture of ILF3 and PKR, two dsRNA binding proteins induced by IFN. We further developed a tap-tagging system to minimize the presence of non-specifically bound proteins to Dynabeads that may interfere with future mass spectrometry analysis. To isolate DI-PRS RNA:protein complexes from infected cells, attempts were made to rescue replicating DI-PRS genomes in the context of wild type PIV5. Similarly, efforts were made to isolate influenza A virus RNPs that contained the PRS in the neuraminidase (NA) gene from infected cells using the PP7-based protocol developed. However, for reasons discussed, unfortunately RNA:proteins complexes were not successfully purified from infected cells in either case.

La faune sauvage a été depuis longtemps incriminée dans la survenue de zoonoses et joue le rôle de réservoir d'agents pathogènes (virus Nipah, Hendra, Ebola, Hantaan etc.) pour l'homme. Les îles tropicales et subtropicales du Sud-Ouest de l'Océan Indien (SOOI) constituent l'une des 34 régions reconnues comme « hotspot » de biodiversité au niveau mondial. Elles sont caractérisées par un très fort endémisme de la faune sauvage surtout sur l'Ile de Madagascar. Le caractère multi-insulaire de la région du SOOI, la diversité de ses biotopes et ses disparités biogéographiques et humaines offrent un champ d'investigation unique pour explorer « in natura » la dynamique évolutive des agents infectieux et les relations hôtes-virus. Nos travaux de recherche ont porté sur deux modèles de virus à ARN de polarité négative, les paramyxovirus et les lyssavirus. Le premier modèle viral nous a permis d'aborder les questions relatives à la dynamique de transmission virale au sein de communauté d'hôtes, plus particulièrement, les chauves-souris et les petits mammifères terrestres de Madagascar et d'identifier les facteurs agissant sur cette dynamique de transmission et de diversification virale, en particulier les facteurs bio-écologiques associés à leurs hôtes. Le second modèle viral, les lyssavirus, nous a permis de décrire sur l'ensemble des îles du SOOI échantillonés, la circulation virale dans ce système multi-insulaire diversifié, au sein des chauves-souris dont la plupart des espèces sont endémiques à cette région. Dans l'ensemble, nos investigations ont permis de mettre en évidence des échanges viraux (« host-switch ») importants entre chauves-souris, petits mammifères terrestres endémiques de Madagascar et les rongeurs introduits, le rôle de ces mammifères en tant que réservoir viral majeur et souligner le rôle disséminateur de Rattus rattus. Par ailleurs, nous avons pu identifier ce phénomène de « host-switch » comme étant le mécanisme macro-évolutif prépondérant et l'importance des facteurs biotiques et abiotiques à l'origine de la dynamique de transmission et de la diversification virale observée chez les paramyxovirus de chauves-souris de Madagascar. / Since many decades, the wild fauna has been incriminated as an important reservoir of many zoonotic pathogens (Nipah, Hendra, Ebola, Hantaan viruses etc.) at risk for humans. Tropical and subtropical islands of the South West Indian Ocean (SWIO) are part of the 34 areas of the world recognized as "hotspot" of biodiversity. They are characterized by a strong wildlife endemism especially on Madagascar. The multi-island structure of the SWIO region, the diversity of its biotopes and its biogeographical human disparities, offer a unique opportunity to investigate "in natura" the evolutionary dynamics of infectious agents and the host-virus relationships. Our research has focused on two models of negative RNA viruses, paramyxoviruses and lyssaviruses. The first virus model allowed us to address issues related to the dynamics of viral transmission within a host community, in particular, bats and small terrestrial mammals of Madagascar and to identify the driving factors, especially bio-ecological factors associated with their hosts, affecting the dynamic of transmission and of viral diversification. The second model allowed us to describe on the islands of the SWIO, the intense circulation of bats lyssaviruses in this multi-island system which bats are endemic to this region. Overall, our investigations highlighted (i) intense viral exchanges ("host-switch") between bats, endemic terrestrial small mammals and introduced rodents from Madagascar, (ii) the role of these mammals as major viral reservoir and (iii) the key role played by Rattus rattus as viral spreader. Furthermore, we identified both the phenomenon of "host-switch" as the major macro-evolutionary mechanism among bat paramyxoviruses from Madagascar and the importance of biotic and abiotic factors in shaping the transmission dynamics and viral diversification.

The family of paramyxoviruses has received growing attention as several new species have been identified recently, notably two different clusters in domestic cats, designated as feline morbillivirus (FeMV) and feline paramyxovirus (FPaV). Their phylogenetic origin and whether wild felids also harbor these viruses are currently unknown. Kidney samples from 35 guignas (Leopardus guigna), a wild felid from Chile, were investigated for paramyxoviruses using consensus-RT-PCR. In addition, thirteen serum samples of guignas were screened for the presence of FeMV-specific antibodies by an immunofluorescence assay (IFA). Viral RNA was detected in 31% of the kidney samples. Phylogenetic analyses revealed two well-supported clusters, related to isolates from domestic cats, rodents and bats. No significant histopathology changes were recorded in infected guignas. Serology identified two samples which were positive for FeMV-specific antibodies. Our study highlights the diversity of paramyxovirus infections in felids with special emphasis on guignas from Chile.

No description available.