As aves podem carregar um grande número de patógenos. As aves migratórias, por viajarem longas distâncias, são as principais responsáveis pela disseminação de agentes infecciosos. Entre os agentes, destacam-se os vírus, como por exemplo o retrovírus da Reticuloendoteliose aviária (REV), amplamente distribuído; e o vírus da febre do Oeste do Nilo (WNV), uma virose reemergente, com caráter zoonótico. Os principais sintomas da Reticuloendoteliose aviária incluem anemia, doença de Runting e síndrome não neoplásica aguda. Já o agente etiológico da Febre do Nilo Ocidental, é o Flavivirus West Nile (WNV).. As aves são seus hospedeiros definitivos e os humanos são hospedeiros acidentais, podendo manifestar quadro febril, e em menor porcentagem, meningite e encefalite. Mosquitos dos gêneros Culex e Aedes spp são os principais transmissores do vírus. Ao contrário do REV que não dispõe de evidências de sua circulação no Brasil, há evidências do WNV em aves e equinos e mais recentemente, em humanos. O objetivo desse trabalho foi investigar a presença do REV e do WNV em aves silvestres e de cativeiro da cidade de São Paulo e do Norte do estado do Pará. Sangue, soro e swab de cloaca foram coletados, totalizando mais de 1000 amostras. Através de técnicas moleculares foi possível detectar a presença do REV em 74 amostras (16%), todas do estado do Pará. O sequenciamento parcial dessas amostras e sua filogenia sugeriu que a migração de aves EUA-Brasil possa ter sido a rota utilizada. Através de ELISA anti-IgG de WNV, 4 amostras de São Paulo foram positivas. Apresentamos a primeira evidência do REV no país e sugerimos a presença do WNV no estado de São Paulo. / Birds can carry a large number of pathogens. The migratory birds are most responsible for the spread of infectious agents due to long distance travels. Among these pathogens, the most notable are viruses, such as the avian Reticuloendotheliosis retrovirus (REV), widely distributed; and the West Nile virus (WNV), a reemerging zoonotic disease. The main symptoms of avian reticuloendotheliosis include anemia, Runting\'s disease and acute nonneoplastic syndrome. The etiological agent of West Nile fever is Flavivirus West Nile (WNV). Birds are their definitive hosts and humans are accidental hosts, which generaly present febrile symptoms, but at less proportion,, meningitis and encephalitis. Mosquitoes of the genus Culex and Aedes spp are the main vectors of the virus. Differently from the REV that has no evidence of its circulation in Brazil, there is evidence of WNV in birds and horses and more recently in humans. The objective of this work was to investigate the presence of REV and WNV in wild birds and captive birds from the city of São Paulo and Northern from Pará State. Blood, serum and cloacal swab were collected, resulting in more than 1000 samples. Through molecular techniques it was possible to detect the presence of REV in 74 samples (16%), all from the State of Pará. The partial sequencing of these samples and their phylogeny suggested that the migration of US-Brazil may have been the route for the virus entry. Through anti-WNV IgG ELISA, 4 samples from São Paulo were positive. We present the first evidence of REV in the country and suggest the presence of WNV in the state of São Paulo.

Submitted by Tatiana Oliveira (tsilva@icict.fiocruz.br) on 2012-05-31T16:28:56Z No. of bitstreams: 1 ana_cecilia_r_cruz_ioc_bp_0001_2005.pdf: 4732352 bytes, checksum: ea13b3487709eb14b3bda0c2d649e513 (MD5) / Made available in DSpace on 2012-05-31T16:28:56Z (GMT). No. of bitstreams: 1 ana_cecilia_r_cruz_ioc_bp_0001_2005.pdf: 4732352 bytes, checksum: ea13b3487709eb14b3bda0c2d649e513 (MD5) Previous issue date: 2005 / Fundação Oswaldo Cruz.Instituto Oswaldo Cruz. Rio de janeiro, RJ, Brasil / A febre do dengue está amplamente distribuída por todas as áreas tropicais do mundo. Em certas áreas endêmicas, as formas graves da doença, febre hemorrágica do dengue (FHD) e síndrome de choque do dengue (SCD), ocorrem com freqüência. Outras manifestações clínicas, como, por exemplo, encefalopatias, encefalites, mielite transversa, também tem sido observadas. O nosso objetivo foi a caracterização genética e biológica de amostras de vírus VDEN2 e 3, com ênfase no caráter de neurovirulência do vírus VDEN2. As amostras de VDEN 2 e 3 utilizadas foram isoladas na Serviço de Arbovírus do Instituto Evandro Chagas a partir de soro de pacientes com quadro clínico de dengue (FD e FHD) e manifestações neurológicas. Os vírus padrão foram o VDEN2 Nova Guiné C e 44-2, VDEN3 H87. As 11 seqüências do vírus VDEN2 quando comparados entre si e com 16 outras seqüências do VDEN2 apresentaram percentual médio de identidade nucleotídica e aminoácido de 89,5 e 99,8% respectivamente. Os estudos filogenéticos realizados mostram os isolados brasileiros pertencem ao genótipo III de origem Asiática. As 10 seqüências do vírus VDEN3 quando comparados entre si e com 16 outras seqüências do VDEN3 apresentaram percentual médio identidade nucleotídica e aminoácido de 89,6 e 100% respectivamente.Os estudos filogenéticos realizados mostram que estão agrupados ao genótipo III também de origem Asiática. O virus VDEN2 isolado a partir de caso mielite transversa (Bel61082) foi caracterizado pelo sequenciamento nucleotidico da região estrutural assim como em testes de neurovirulência em camundongos recém-nascidos. A susceptibilidade de culturas primárias de neurônios e astrócitos, células de glioma humano, também foi estudada para fins de caracterização do caráter neurovirulento do vírus.O estudo de neurovirulência em infecção de camundongo mostrou sinais aparentes de doença apenas após a inoculação da cepa NGC, mas a replicação do vírus Bel 61082 pode ser detectada por RT-PCR em suspensão de cérebro de camundongo. Obtivemos evidências experimentais de apoptose nas células neuronais infectadas com VDEN2 NGC e Bel61082. No sequenciamento nucleotídico, a principal alteração observada está localizada na proteína M 28, região esta conhecida como pró-apoptótica. / The dengue fever is widely distributed in all tropical areas of the world. In certain endemic areas, severe disease, dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS), occur frequently. Other clinical manifestations such as, for example, encephalopathies, encephalitis, transverse myelitis, has also been observed. Our objective was to characterize genetic and biological samples of viruses VDEN2 and 3, with emphasis on the neurovirulence of the virus VDEN2. The samples of DENV 2 and 3 used were isolated in the Service of the Instituto Evandro Chagas Arboviruses from the serum of patients with clinical dengue (DF and DHF) and neurological manifestations. Viruses were standard VDEN2 New Guinea C and 44-2, VDEN3 H87. The 11 sequences of the virus VDEN2 compared among themselves and with 16 other sequences showed VDEN2 average percentage of nucleotide and amino acid identity of 89.5 and 99.8% respectively. Phylogenetic studies performed show the Brazilian isolates belonged to genotype III of Asian origin. The 10 sequences of the virus VDEN3 compared among themselves and with 16 other sequences showed VDEN3 average percentage nucleotide and amino acid identity of 89.6 and 100% respectively.The phylogenetic studies carried out show that they are grouped to genotype III also of Asian origin. The virus isolated from VDEN2 case transverse myelitis (Bel61082) was characterized by nucleotide sequencing of the framework region as well as tests neurovirulence in mice newborn. The susceptibility of primary cultures of neurons and astrocytes, human glioma cells was also investigated for the characterization of the character neurovirulent vírus.O study of infection in mouse neurovirulence showed no apparent signs of disease after inoculation only NGC strain, but virus replication 61,082 Bel can be detected by RT-PCR on mouse brain suspension. We obtained experimental evidence of apoptosis in neuronal cells infected with NGC and VDEN2 Bel61082. In nucleotide sequencing, the main change observed is located at 28 M protein, an area known as pro-apoptotic.

Introdução: Arbovírus são vírus transmitidos por artrópodos, pertencendo, principalmente, aos gêneros Flavivirus (Flaviviridae), Alphavirus (Togaviridae) e Orthobunyavirus (Bunyavirus). Os Flavivirus, em sua maioria, são associados a zoonoses, causando doenças humanas febrís, febres hemorrágicas e encefalites. Inclusive, causam epidemias que são sério problema de saúde pública. Este estudo mostra uma pesquisa de Flavivirus em culicídeos, de diferentes regiões do país, utilizando uma técnica para identificação viral por RT-PCR com primers Flavivirusespecíficos e uma Multiplex-nested-PCR com primers espécie-específicos. Métodos: Culicídeos foram capturados, quantificados, identificados, agrupados em lotes por espécie e congelados. No laboratório, os animais foram macerados e tiveram o RNA extraído. Estes extratos foram submetidos a RT-PCR gênero-específica e à Multiplex-nested-PCR, para detecção e identificação dos vírus a nível de espécie. Resultado: De 3317 culicídios adultos e 571 larvas coletados em 4 diferentes regiões do Brasil, Sul, Sudeste, Norte e Nordeste, fez-se 246 lotes de mosquitos e desses foi possível obter amplicon sugestivo de Flavivirus em 16 (6,5%). Em 3 lotes contendo larvas de Aedes albopictus obteve-se amplicon sugestivo de vírus do dengue tipo 3. Também, em 13 lotes contendo Haemagogus leucocelaenus, Aedes aegypti e Aedes albopictus foi possível obter amplicons sugestivos de vírus do dengue tipos 1 e 2. Dos amplicons obtidos, 4 tiveram nucleotídios seqüenciados o que permitiu confirmar a presença dos vírus do dengue tipo 3 e Cacipacoré. Conclusão: O trabalho permitiu concluir que: a metodologia de RT-PCR para Flavivirus seguida de Multiplex-nested-PCR espécie-específica foi adequada para detecção e identificação destes vírus em culicídios; amplificaram-se genomas de Flavivirus em 6,5% dos lotes de culicídios estudados; vírus do dengue tipo 1 e tipo 2 foram encontrados infectando Aedes aegypti de Santos em 1999, Manaus em 2005-2006 e Foz do Iguaçu; vírus do dengue tipos 2 e 3 foram encontrados em Aedes albopictus de Santos em 1999 e Manaus em 2005-2006, sugerindo que este mosquito participe na transmissão de dengue; vírus do dengue tipo 3 foi encontrado em larvas de Aedes albopictus mostrando transmissão vertical do vírus; vírus do dengue tipo 1 foi encontrado infectando Haemagogus sp. sugerindo existência de ciclo silvático deste vírus; Aedes aegypti do Amazonas estavam infectados com o vírus Cacipacoré. / Introduction: Arbovirus are rodent-borne viruses mostly from Flavivirus (Flaviviridae), Alphavirus (Togaviridae) e Orthobunyavirus (Bunyavirus) genus. Flavivirus, are commonly zoonotic and can cause febrile illness, haemorrhagic fever and encephalitis. Flavivirus outbreaks occur in Brazil and are a major public health problem. We show here a research looking for Flavivirus infections in Culicidae by a RT-PCR using Flavivirus-especific primers and a Multiplex-nested-PCR using specie-specific primers for virus identification. Methods: Culicidae were captured, quantified, identified, pooled based on the specie and frozzen. In the laboratory, the animals were crushed and had the RNA extracted. These extracts were tested by a Flavivirus genus-specific RT-PCR followed by a specie-specific Multiplex-nested-PCR. Results: From 3317 captured adult Culicidae and 571 collected larvae in 4 different regions of Brazil, 246 pools were obtained and from these, Flavivirus indicative amplicons were obtained in 16 (6.5%). Amplicons of dengue type 3 were obtained from 3 pools of Aedes albopictus larvae. It was also possible to obtain indicative amplicons of dengue types 1 and 2 in 13 pools of Haemagogus leucocelaenus, Aedes aegypti and Aedes albopictus. Besides, 4 amplicons had the nucleotides sequenced, confirming the mosquito infection by dengue type 3 and Cacipacoré viruses. Conclusion: The technique combining a Flavivirus genus-specific RT-PCR followed by a specie-specific Multiplex-nested-PCR was suitable for detection of these viruses in the mosquitoes; Flavivirus infecting Culicidae were detected in 6.5% of the analyzed mosquito pools; dengue virus type 1 and type 2 were found infecting Aedes aegypti from Santos (1999), Manaus (2005-2006) and Foz do Iguaçu cities; dengue type 2 virus was found in Aedes albopictus from Santos city (1999) and Manaus city (2005-2006), suggesting that this mosquito could be participating on dengue transmition; dengue type 3 virus was found in Aedes albopictus larvae showing the vertical transmission of this virus; dengue type 1 virus was found infecting Haemagogus sp. what suggests on the existence of a sylvatic maintenance cycle of this virus; Aedes aegypti from Amazonas state were found infeted by Cacipacoré virus.

INTRODUÇÃO: Em grandes cidades, como São Paulo, a pequena porcentagem de matas existentes é representada pelos parques municipais. Estas áreas, além de representarem ambientes de lazer para as pessoas, albergam uma enorme diversidade biológica, desde mosquitos até aves e mamíferos. A interação destas espécies favorece a circulação de Flavivirus, causadores de importantes doenças humanas, que têm nas aves um importante reservatório. Atribui-se às aves migratórias o papel de carreadoras dos vírus, já que as mesmas percorrem longas distâncias para completar seu ciclo biológico. A chegada de aves do Hemisfério Norte ocorre em alguns Parques, o que favoreceria a dispersão de alguns vírus, como o Vírus do Nilo Ocidental, já que nestas áreas estão presentes mosquitos potencialmente vetores. OBJETIVOS: identificar infecção por Flavivírus nas aves dos parques municipais de São Paulo. MÉTODOS: De Março de 2012 a Janeiro de 2013, foram coletadas amostras de swab de cloaca, orofaringe e sangue de aves capturadas em redes de neblina de duas áreas do município de São Paulo: Parque Anhanguera e Fazenda Castanheiras/APA Bororé Colônia. As aves foram anilhadas e liberadas após a coleta do material. Foi realizada técnica de RT-PCR em tempo real, utilizando iniciadores genéricos que amplificam fragmento do gene NS5 de Flavivirus. Amostras positivas foram encaminhadas para sequenciamento. RESULTADOS: Foi capturado um total de 231 aves, em sua maioria da Ordem Passeriformes. De um total de 463 amostras, nenhuma amostra apresentou presença de RNA viral. DISCUSSÃO: Em se tratando de alguns Flavivírus, os passeriformes são considerados os reservatórios mais competentes no ciclo de transmissão, pois atingem altos níveis de viremia. Cabe ressaltar que algumas espécies desta ordem, de ocorrência na cidade de São Paulo, já foram identificadas como portadoras dos vírus Rocio e Ilhéus. A ausência de positividade é esperada, pois embora altamente sensível, a técnica de PCR depende do estado de viremia das aves, que é curta. CONCLUSÃO: Os parques municipais são áreas que aproximam aves, mosquitos e humanos, pelo papel ambiental e de lazer que os mesmos representam. Este fato classifica estas áreas como locais com potencial de transmissão de Flavivirus, o que torna importante a continuação este estudo, aumentando as áreas de abrangência, para conhecer os Flavivírus circulantes e realizar vigilância para vírus que podem ocasionar problemas de Saúde Pública / INTRODUCTION: In big cities, as São Paulo, the little percentage of existing forests is represented by the municipal parks. These áreas, besides acting as entertainment environments to the users, promote a huge biodiversity, including mosquitoes, birds and mammals. These species interaction promotes Flavivirus circulation, viruses responsible for important human diseases. The avian species are important reservoirs for these viruses, specially the migrating birds that can fly for long distances, carrying these viruses to several areas. In some parks, the arrival of migrating birds from the North Hemisphere is documented, fact that can support some viruses dispersion, for example, the West Nile Vírus, considering that in these areas potencial vector for this virus can be found. OBJECTIVES: detect Flavivirus infection in birds captured in municipal parks of São Paulo city. METHODS: from March, 2012 to January, 2013, oropharyngeal and cloacal swabs, and blood samples were collected from birds captured in mist-net webs located in two municipal areas: Anhanguera Park and Castanheiras Farm/APA Bororé Colônia. The birds were ringed and released after samples collection. The real time RT-PCR was performed, using generic primers which amplify NS5 gene fragment. Positive samples were forwarded to sequence analysis. RESULTS: a total of 231 birds were capture, in which the majority belongs to Passeriforms order. Of 463 samples collected, all samples were negative for the viral RNA presence. DISCUSSION: when talking about Flaviviruses, the passeriforms are considered the most competent reservoirs in the transmission cycle, because they can achieve great viremia levels. Some passeriforms species, endemic in São Paulo city, were identified as Rocio and Ilhéus viruses carriers in previous studies. The negativity is expected, once the Real Time RT-PCR, although highly sensitive, depends on the viremia duration, which is short in avian species. CONCLUSION: the public parks are areas which encloses birds, mosquitoes and the human being, for the environmental and entertainment role they play. This fact classifies these parks as areas with great potencial of Flavivirus transmission, ressalting the great importance to continue this study, increasing the number of areas, to detect the circulating Flavivirus and to perform surveillance for viruses which can cause public health problems

Introdução: Dos arbovírus transmitidos no Brasil, os Flavivirus apresentam destaque por causarem o maior número de infecções e doenças no homem e pela gravidade das doenças que provocam. A cidade de São Paulo possui áreas verdes, representadas por parques municipais em que existem lagos, aves e mamíferos. Esses locais são frequentados pela população e podem servir de refúgio para mosquitos que infestam a área urbana. Alguns trabalhos revelam a ocorrência de espécies de mosquitos conhecidas como vetoras de Flavivirus em tal cidade, o que pode favorecer o aparecimento de casos e transmissão de doenças causadas por este tipo de vírus. No entanto, a presença de Flavivirus infectando tais artrópodes não é conhecida. Objetivo: Identificar presença de Flavivirus em mosquitos (Diptera: Culicidae) provenientes de áreas verdes da cidade de São Paulo. Método: Sete parques municipais localizados em diferentes regiões da cidade foram selecionados para o estudo. Foram realizadas coletas de mosquitos mensais em cada parque no período de março de 2011 e fevereiro de 2012. As armadilhas utilizadas foram: aspirador, Shannon e CDC-CO2. Os mosquitos foram transportados para o laboratório em gelo seco, identificados em mesa fria e agrupados em \"pools\" de até 10 fêmeas não ingurgitadas, segundo espécie, data e local de coleta. Fêmeas ingurgitadas foram armazenadas individualmente. As amostras foram submetidas à extração de ácidos nucleicos, seguida de RT-PCR em tempo real para amplificação de fragmento de aproximadamente 200pb do gene NS5 de Flavivirus. Amostras positivas foram encaminhadas para sequenciamento e análises filogenéticas. Resultados: Dentre as 5.213 fêmeas não ingurgitadas (818 pools) e as 778 fêmeas ingurgitadas coletadas, ocorreu presença de onze gêneros de Culicídeos, sendo mais frequentes Culex e Aedes. Dezenove pools de fêmeas não ingurgitadas obtiveram resultado positivo para Flavivirus. Análises filogenéticas mostraram presença de RNA relacionado à Aedes flavivirus (AEFV) em dois pools de Aedes e à Culex flavivirus (CxFV) nos demais pools, todos de Culex. Conclusões: CxFV e AEFV estão presentes em mosquitos dos gêneros Culex e Aedes, respectivamente, coletados em parques municipais da cidade de São Paulo. Embora flavivírus patogênicos ao homem não tenham sido encontrados no presente trabalho, recomenda-se vigilância de flavivírus nas áreas estudas, visto que elas possuem presença de mosquitos vetores e hospedeiros vertebrados, ou seja, elementos necessários para a transmissão destes vírus. / Introduction: Among the arboviruses transmitted in Brazil, Flavivirus are noteworthy once they cause the largest number of infections and diseases in humans and also because they can cause serious illnesses. In the city of São Paulo there are green spaces, represented by city parks in which lakes, birds and mammals are found. The population visits those places, which can also be a refuge for mosquitoes that infect the urban area. Some studies reveal the occurrence of mosquitoes species known to be vectors of Flavivirus in this city, what can contribute to the emergence of cases and transmission of diseases caused by this kind of virus. However, the presence of Flavivirus infecting those arthropods is not known. Objective: To Identify infection by Flavivirus in mosquitoes (Diptera: Culicidae) collected in São Paulo\'s city parks. Method: Seven city parks located in different places of the city were selected for the study. Monthly mosquito collections were carried out in each park from March 2011 to February 2012. The traps employed were aspirator, Shannon and CDC-CO2. The mosquitoes were transported to the laboratory on dry ice. Identification was performed in a chill table. Up to 10 non-engorged females were pooled according to specie, date and place of collection. Engorged females were stored individually. Nucleic acids were extracted and submitted to real time RT-PCR that amplifies a 200pb fragment of NS5 gene of Flavivirus. Positive samples were sequenced and phylogenetic analyses were performed. Results: Eleven genus of Culicidae were found among the 5,213 non engorged females (818 pools) and the 778 engorged females. Culex and Aedes were the most frequent collected genus. Nineteen non engorged female pools had positive results for the presence of Flavivirus RNA. Phylogenetic analyses showed the RNA found was related to Aedes flavivirus (AEFV) in two pools of Aedes and to Culex flavivirus (CxFV) in the other pools, all of them consisting of Culex. Conclusion: CxFV and AEFV are present in mosquitoes Culex and Aedes, respectively, collected in São Paulo\'s city parks. Flavivirus of medical importance were not detected. Although, once there are species of mosquitoes that can act as vectors of pathogenic Flavivirus and vertebrate hosts, which are required for the transmission cycle of those virus, surveillance is recommended in the studied areas.

As aves podem carregar um grande número de patógenos. As aves migratórias, por viajarem longas distâncias, são as principais responsáveis pela disseminação de agentes infecciosos. Entre os agentes, destacam-se os vírus, como por exemplo o retrovírus da Reticuloendoteliose aviária (REV), amplamente distribuído; e o vírus da febre do Oeste do Nilo (WNV), uma virose reemergente, com caráter zoonótico. Os principais sintomas da Reticuloendoteliose aviária incluem anemia, doença de Runting e síndrome não neoplásica aguda. Já o agente etiológico da Febre do Nilo Ocidental, é o Flavivirus West Nile (WNV).. As aves são seus hospedeiros definitivos e os humanos são hospedeiros acidentais, podendo manifestar quadro febril, e em menor porcentagem, meningite e encefalite. Mosquitos dos gêneros Culex e Aedes spp são os principais transmissores do vírus. Ao contrário do REV que não dispõe de evidências de sua circulação no Brasil, há evidências do WNV em aves e equinos e mais recentemente, em humanos. O objetivo desse trabalho foi investigar a presença do REV e do WNV em aves silvestres e de cativeiro da cidade de São Paulo e do Norte do estado do Pará. Sangue, soro e swab de cloaca foram coletados, totalizando mais de 1000 amostras. Através de técnicas moleculares foi possível detectar a presença do REV em 74 amostras (16%), todas do estado do Pará. O sequenciamento parcial dessas amostras e sua filogenia sugeriu que a migração de aves EUA-Brasil possa ter sido a rota utilizada. Através de ELISA anti-IgG de WNV, 4 amostras de São Paulo foram positivas. Apresentamos a primeira evidência do REV no país e sugerimos a presença do WNV no estado de São Paulo. / Birds can carry a large number of pathogens. The migratory birds are most responsible for the spread of infectious agents due to long distance travels. Among these pathogens, the most notable are viruses, such as the avian Reticuloendotheliosis retrovirus (REV), widely distributed; and the West Nile virus (WNV), a reemerging zoonotic disease. The main symptoms of avian reticuloendotheliosis include anemia, Runting\'s disease and acute nonneoplastic syndrome. The etiological agent of West Nile fever is Flavivirus West Nile (WNV). Birds are their definitive hosts and humans are accidental hosts, which generaly present febrile symptoms, but at less proportion,, meningitis and encephalitis. Mosquitoes of the genus Culex and Aedes spp are the main vectors of the virus. Differently from the REV that has no evidence of its circulation in Brazil, there is evidence of WNV in birds and horses and more recently in humans. The objective of this work was to investigate the presence of REV and WNV in wild birds and captive birds from the city of São Paulo and Northern from Pará State. Blood, serum and cloacal swab were collected, resulting in more than 1000 samples. Through molecular techniques it was possible to detect the presence of REV in 74 samples (16%), all from the State of Pará. The partial sequencing of these samples and their phylogeny suggested that the migration of US-Brazil may have been the route for the virus entry. Through anti-WNV IgG ELISA, 4 samples from São Paulo were positive. We present the first evidence of REV in the country and suggest the presence of WNV in the state of São Paulo.

Les flavivirus ayant un impact en médecine vétérinaire sont largement distribués dans le monde (à l’exemple de la fièvre West Nile (WNF) présente sur les cinq continents ou de l’Encéphalite japonaise (EJ) en Asie du Sud-Est) et sont responsables de maladies à dominante neurologique chez l’homme et/ou le cheval.La virémie étant généralement brève lors de ces infections virales, les méthodes de diagnostic utilisées sont essentiellement sérologiques. Or le chevauchement fréquent des aires de répartition des flavivirus complique le diagnostic sérologique. En effet, des réactions sérologiques croisées entre flavivirus sont observées lors de l’utilisation de méthodes de diagnostic usuelles telles que l’ELISA et l’immunofluorescence (IF). Les résultats sérologiques doivent donc être confirmés par la méthode fastidieuse de séroneutralisation (SNT) virale avec les différents flavivirus existants dans la région. De plus, le risque d’émergence sur un territoire donné de nouveaux flavivirus comme le virus Zika au Brésil ou en Amérique du Nord ne peut être exclu.Nous avons donc développé dans la première partie de ce travail une nouvelle stratégie de diagnostic sérologique multiplexe à l’aide de la méthode d’immuno-essai (MIA) sur billes. Sachant que la glycoprotéine d’enveloppe soluble (sE) des flavivirus est divisée en 3 domaines (D) structuraux, DI, DII et DIII et que les épitopes du DIII sont spécifiques de chaque flavivirus, des protéines EDIII recombinantes de différents flavivirus d’intérêt ont été synthétisées en système d’expression Drosophila S2 et leur antigénicité a été testée sur sérums équins et ovins. Nous avons obtenu des résultats très encourageants en démontrant que l’utilisation des EDIII associée avec la capacité de multiplexage de la méthode MIA apparaît comme une réponse adaptée au défi du diagnostic sérologique des infections à flavivirus.Nous avons en outre utilisé la même méthode de multiplexage mais avec des antigènes NS1 du WNV pour mettre en place un test DIVA (Differentiating Infected from Vaccinated Animals) permettant de différencier les chevaux infectés par le WNV des chevaux vaccinés avec un vaccin recombinant WNV.Un autre écueil en médecine vétérinaire est le traitement des affections à flavivirus. En effet l’arsenal thérapeutique est limité et le traitement est avant tout symptomatique. Nous avons dans la seconde partie de ce travail testé in vitro une molécule antivirale à large spectre, le sr1057 sur nos virus d’intérêt (WNV et JEV). Cette molécule qui provient d’une stratégie de criblage développée par l’Institut Pasteur a probablement pour cible la cellule de l’hôte car elle est capable d’inhiber des virus très différents, à génome à ARN de polarité positive ou négative et ADN.Les résultats que nous avons obtenus avec ce composé sont en demi-teinte pour les flavivirus testés avec une efficacité démontrée pour le JEV mais plus modeste pour le WNV. Ils n’excluent cependant pas une possible utilisation de cet antiviral en médecine vétérinaire équine car une activité inhibitrice in vitro sur les virus de l’Herpes équin de type I et de l’artérite virale a été confirmée par d’autres collaborateurs. / Flaviviruses with a major impact in veterinary medicine are widely distributed (e.g. West Nile fever (WNF) has spread across the five continents and Japanese Encephalitis (JE) is reported in South-East Asia) and are mainly responsible for neurological diseases in humans and/or horses.After flavivirus infection, viremia in mammal hosts is generally short and consequently indirect methods are mostly used to diagnose flavivirus infections. However, frequent spatial overlapping in their circulation areas renders the interpretation of serological assays difficult. Indeed, cross-reactions between flaviviruses are observed in rapid serological tests such as in ELISA and immunofluorescence assays (IFA). Serological assay results should thus be confirmed by the tedious comparative virus neutralization test (VNT) using a panel of viruses known to circulate in the area. Moreover, the risk of emergence of new flaviviruses such as reported recently with the Zika virus in Brazil or in North America should be considered when studying flavivirus epidemiology.In the first section, a new strategy aiming at improving the serological diagnosis of flavivirus infections was developed using the multiplexing capacity of microsphere immunoassays (MIA). The flavivirus soluble envelope (sE) glycoprotein ectodomain is composed of three domains (D), e.g. DI, DII and DIII, with EDIII containing virus-specific epitopes. Recombinant EDIIIs of different flaviviruses were synthesized in the Drosophila S2 expression system. The microspheres coupled with rEDIIIs were assayed with equine and ovine sera from natural and experimental flavivirus infections or non-immune samples. Very encouraging results have been obtained and this innovative multiplex immunoassay based on flavivirus rEDIIIs appears to be a powerful alternative to ELISAs and VNTs for veterinary diagnosis of flavivirus-related diseases.MIA with WNV nonstructural 1 protein were also implemented to differentiate Infected from Vaccinated Animals (DIVA). Such a DIVA approach was only successful when horses had been immunized with a recombinant canarypox vaccine, while horses receiving inactivated WNV vaccine developed immune responses close to the ones induced after natural infection.Another pitfall in veterinary medicine is the lack of therapeutics for viral diseases and specifically for flaviviruses. The therapeutic arsenal is indeed rather limited and animals are generally administered supportive treatments only. In the second part, the results of the in vitro testing of a broad spectrum antiviral named sr1057 on WNV and JEV replication are presented. This chemical, identified from a unique screening strategy developed by Pasteur Institute, is probably targeting the host cell and was found to inhibit the replication of varied RNA and DNA viruses belonging to different virus families. The sr1057 compound was not as efficient at inhibiting the replication of flaviviruses as for other RNA+ viruses, with a modest antiviral effect demonstrated for WNV and a higher efficacy on JEV. This antiviral presents however potentials for applications in equine veterinary medicine because it efficiently inhibited equine herpes virus-1 and equine arteritis virus in vitro, as clearly shown by other collaborators.

Les hélicases sont des enzymes extrêmement répandues qui sont essentielles pour mener à bien l'ensemble des processus cellulaires impliquant des acides nucléiques. Ces protéines parviennent à annihiler les structures secondaires et tertiaires qui peuvent se former dans l'ADN ou l'ARN grâce à l'énergie puisée par l'hydrolyse de l'ATP. Les hélicases ne sont toutefois pas l'exclusivité des organismes eucaryotes et procaryotes, mais bon nombre de virus codent aussi pour des enzymes possédant cette fonction. La famille des Flaviviridae ne fait pas exception à ce groupe puisque l'ensemble des virus de cette famille code pour une hélicase nommée NS3. La famille des Flaviviridae est une famille virale d'une importance monumentale étant donné la présence de nombreux pathogènes humains parmi ses rangs. On y dénote le virus de l'hépatite C, le virus de la dengue et le virus du Nil occidental pour ne nommer que ceux-là. Ces virus ont aussi un autre point en commun, le manque criant d'une thérapie efficace pour traiter les patients atteints. La protéine NS3 et plus particulièrement son activité hélicase est une cible potentielle envisageable pour le traitement de ces virus étant donné l'aspect essentiel de cette activité dans le cycle de réplication de ceux-ci. L'étude présentée dans ce mémoire a pour dessein d'étudier le site d'hydrolyse de l'ATP de la protéine NS3 du virus du Nil occidental. Le but final est de découvrir les déterminants moléculaires qui mènent à la sélection du substrat à hydrolyser, tant au niveau du substrat qu'au niveau de la protéine. Du côté du substrat, une panoplie d'analogues de purines fut testée pour déterminer la capacité de ces molécules à compétitionner pour le site actif de la protéine ainsi que pour la faculté de la protéine à hydrolyser ces substrats. Du côté de la protéine, une gamme de mutants de NS3 fut exprimée et l'habileté de ces mutants à discriminer entre les différents substrats qui lui étaient présentés a été mesurée. Les acides aminés qui ont été sélectionnés pour être mutés l'ont été soit pour leur ressemblance à un motif de reconnaissance de l'ATP chez d'autres hélicases, soit pour leur proximité du substrat, déterminée grâce à un modèle de la protéine bâti sur la structure cristallisée de la protéine NS3 du virus de la dengue. L'ensemble des résultats obtenus a permis de dresser le portrait des atomes de l'ATP qui sont liés par la protéine en plus de mettre à jour plusieurs acides aminés qui sont impliqués directement ou indirectement dans la sélection du substrat par NS3. Il semble en effet que la présence d'un groupement donneur de liaison hydrogène soit nécessaire à la position six d'une purine pour permettre un bon niveau d'hydrolyse de la molécule, permettant ainsi à la protéine de faire la distinction entre l'ATP et le GTP. Aussi, l'apport insoupçonné dans le processus d'hydrolyse d'acides aminés tels que Arg202, Ans417 ou encore Arg185 a été démontré. Finalement, différentes expériences permettant d'élargir les connaissances sur la protéine NS3 ainsi que sur les hélicases en général sont proposées. Des recherches visant à déterminer les ressemblances et les différences au niveau de la discrimination entre sources d'énergie à utiliser entre les diverses protéines NS3 des Flaviviridae vont permettre d'amener à un autre niveau la compréhension du mécanisme d'hydrolyse de cette protéine. Des études s'appliquant à modifier différents acides aminés afin de façonner la protéine NS3 pour qu'elle hydrolyse des analogues particuliers ou qu'elle lie des acides nucléiques précis pourraient aussi permettre de mieux comprendre les bases de la reconnaissance des substrats de NS3 en plus de fournir éventuellement des outils moléculaires voués à des fonctions multiples.

The RNA cap structure is a fundamental feature of most known eukaryotic mRNAs and some viral RNAs, It is important for the stability, transport and translation of mRNAs. It is co-transcriptionally synthesized via the action of 3 consecutive enzymatic reactions: (1) A RNA triphosphatase which cleaves off the 5' terminal phosphate of nascent RNAs; (2) A RNA guanylyltransferase which transfers a GMP moiety onto the acceptor RNA; (3) A RNA (guanine-N7) methyltransferase which methylates the cap guanine at the N7 position. Through the end of the 1990's until now, the crystal structures of several capping enzymes have been solved. However, these structures, although very insightful in themselves, failed to provide any instructive information on several key issues regarding enzyme-substrate interactions. For instance, one of the first breakthrough crystallographic studies in RNA capping chemistry led to the elucidation of the yeast RNA triphosphatase structure (the Cet1 protein). However, in the crystal structure, the Cet1 protein was bound to a sulphate molecule, which was hypothesised to be mimicking the product of the RNA triphosphatase reaction- a phosphate molecule.The inability to capture the RNA triphosphatase in complex with its ligands is probably on account of the inherent thermodynamic instability of this protein when bound to RNA or a nucleotide. A structural definition of the active site of the yeast RNA triphosphatase in complex with an appropriate substrate is still lacking. In addition, the elucidation of the structure of the RNA guanylyltransferase of the Paramecium bursaria chlorella virus -1 (PBCV-1) in several different conformations has been a landmark study which greatly contributed towards the understanding of the catalytic pathway of this model enzyme. On the other hand, despite the presence of the natural substrate-GTP, within the active site of the enzyme, the rationale behind the GTP specificity of RNA guanylyltransferase remains poorly understood. Moreover, a molecular mechanism for the RNA guanylyltransferase reaction is still missing. Finally, the importance of the RNA cap for the process of eukaryotic translation is undisputable. However, the relationship between the RNA cap and translation has been mostly studied indirectly through proteins which bind to the cap structure. Most studies pertaining directly to the impact of the binding of the RNA cap structure have been restricted to investigating the inhibitory potential of various cap analogues on the translation process. Studies on the effects of modified RNA caps at the 5' ends of RNAs have only started in the last few years, and more importantly, the necessity of the N7-methyl group on RNA cap analogues had not been addressed. This thesis therefore aims to provide a structural insight into the structural dynamics of enzyme-ligand(s) interactions of the model S. cerevisiae's RNA triphosphatase and the PBCV-1 RNA guanylyltransferase. In addition, we show that purine analogues can be a useful tool for the study of several cellular processes, such as RNA translation. In the process we have uncovered a novel class of RNA capping enzyme in the flavivirus genus of the Flaviviridae family of RNA viruses, thus providing a more succinct insight into the flaviviral replication complex.

Le genre Flavivirus regroupe plus de 70 espèces dont plusieurs sont des pathogènes humains de première importance responsables dans les formes les plus graves de manifestations hémorragiques ou d’encéphalites parfois mortelles. L’absence de traitements antiviraux spécifiques et l’augmentation croissante des flaviviroses, surtout dans les régions tropicales, justifient un effort de recherche et développement pour lutter contre ces maladies.Ce travail a abordé deux aspects de l’infection à flavivirus : un aspect épidémiologique et un aspect plus fondamental sur l’immunité innée et les contremesures flavivirales. L’étude épidémiologique a été menée en collaboration avec le CENETROP (Centro national de enfermedades tropicales) de Bolivie grâce à la contribution de l’IRD. De par l’analyse des différentes souches circulantes dans ce pays, elle a permis une meilleure compréhension de l’épidémiologie de la dengue et de la fièvre jaune et nous a fait prendre conscience de la variabilité génétique de ces virus. Devant le peu de données répertoriées en Bolivie, nos travaux serviront de référence pour comprendre les épidémies futures, peut-être améliorer les techniques de diagnostic et permettre le développement de stratégies de prévention adaptées et l’amélioration des politiques de lutte contre la fièvre jaune en Amérique du Sud. La cohabitation entre le virus et l’hôte immunocompétent est le résultat d’un équilibre subtil entre le taux de réplication virale et la clairance du système immunitaire pour garantir la survie des deux espèces. Chacun a évolué en développant des mécanismes de défenses contre l’autre. Notre second travail visait à analyser l’influence de la protéine flavivirale non structurale NS1 sur la réponse interféron de l’hôte. L’identification de stratégies virales d’évasion face à l’immunité de l’hôte et l’analyse de leurs fonctions dans l’infection virale permettrait de mieux comprendre le système immunitaire ainsi que l’interaction virus–hôte. Ceci aiderait au développement de nouvelles stratégies antivirales afin de traiter les pathologies associées à ces arbovirus. / The Flavivirus genus consists of sevevral human pathogens responsible for hemorragic syndrome or encephalitis. The absence of specific antiviral treatment and an increase in Flavivirus incidence has led to a greater research effort in fighting these diseases. The study takes an epidemiological and a fundamental approach in its analysis of the innate immune response to flavivirus infection as well as flaviviral adaptation to evade this response. The analysis of circulating strains in Bolivia has led to a better understanding of dengue and yellow fever and also an awareness of their genetic variability. Given the limited information available in Bolivia, our studies could be used as a reference to understand future epidemics, improve diagnostic methods and allow the development of prevention strategies to fight against yellow fever in south Africa. The relationship between virus and host results from a subtle balance between viral replication and immunity clearance allowing the survival of both species. Each one as developed defence mechanisms against the other. We also examined the role of the non structural protein NS1 in the interferon respons to Flaviviral infection. Knowledge on viral escape strategies from host immunity could help to develop antiviral treatment for these arbovirus diseases