Les arbovirus sont un groupe de virus transmis par des vecteurs arthropodes contaminant chaque année plusieurs centaines de millions de personnes à travers le monde. De nombreux facteurs environnementaux, comme les xénobiotiques peuvent influencer la propagation des infections virales, la susceptibilité de l'hôte et la sévérité de l'infection. Parmi eux, le cadmium, métal toxique est connu pour moduler la résistance de l'hôte lors des infections par les arbovirus. Ces travaux de thèse se sont portés sur l'étude de l'alphavirus Sindbis et du flavivirus Zika infectant des cellules épithéliales humaines. Les résultats de cette thèse ont permis de démontrer que les cellules A549 étaient permissives à la souche épidémique de 2013 du virus Zika. Le virus se réplique efficacement et induit une apoptose de type mitochondriale dans les cellules A549. La réponse immunitaire innée des cellules a été caractérisée et le rôle des interférons de type I a été mis en avant. Ces résultats peuvent contribuer à une meilleure compréhension des mécanismes de pathogénicité de ce virus chez l'homme. Par la suite, les effets d'une exposition au cadmium lors des infections par les virus Sindbis et Zika ont été évalués. L'infection par le virus Zika n'a pas été modulée par l'exposition au cadmium in vitro. Mais de façon surprenante dans le cas de l'infection des cellules HEK 293, le cadmium a protégé les cellules des effets cytopathiques induits par le virus Sindbis. En présence de cadmium, l'apoptose induite par le virus a été inhibée et la réplication du virus Sindbis a été réduite. / Arboviruses are a group of viruses transmitted by arthropod vectors infecting hundreds of millions of people each year worldwide. Many environmental factors including xenobiotics can influence the spread, the susceptibility and the outcome of viral infections. Among them, cadmium, a toxic metal is known to affect the host resistance against arboviral infection. In this thesis, our work has been focused on studying the infection capability of the Sindbis alphavirus and the Zika flavivirus in human epithelial cells. The results demonstrated that A549 cells was permissive to the 2013 epidemic strain of Zika virus. The virus replicates efficiently leading to mitochondrial apoptosis. The innate immune response was characterized and the crucial role of type I interferon was highlighted. These results may contribute to a better understanding of Zika virus pathogenesis in humans. Thereafter, the effects of cadmium exposure on Sindbis virus and Zika virus infection was evaluated. Zika virus infection was not modulated by cadmium in vitro. Interestingly, cadmium protected the HEK 293 cells from the cytopathic effect induced by Sindbis virus. Cadmium exposure inhibited the apoptosis and reduced the viral replication.

Depuis la fin du 19ème siècle, les épidémies dues aux arbovirus constituent un problème de santé publique majeur en Nouvelle-Calédonie et dans la région Pacifique. Ces cinq dernières années, la fréquence des épidémies de dengue a augmenté et l’introduction des virus du chikungunya et du Zika (ZIKAV) a démontré que cette région du monde pouvait être un hot spot d’émergence des arboviroses. Ces trois virus sont transmis par des moustiques du genre Aedes dont le principal vecteur de la région Pacifique est Aedes aegypti. Mis à part des données sur l’épidémiologie de la dengue dans la région, peu d’informations étaient alors disponibles sur les vecteurs ou sur leurs capacités à transmettre les arbovirus. Or ces données sont cruciales pour mieux comprendre l’incidence particulière de ces épidémies dans la région.Dans ce travail de thèse, nous avons tout d’abord caractérisé génétiquement le vecteur Ae. aegypti de la région Pacifique et montré une différence génétique modérée de ce vecteur à l’échelle régionale. Nous avons ensuite, pour la première fois, évalué la compétence vectorielle d’Ae. aegypti pour les virus de la dengue 1 (DENV-1) et du ZIKAV. Les résultats obtenus montrent une faible transmission du DENV-1 et des taux de transmission hétérogènes du ZIKAV en fonction des différentes lignées virales.Les interactions entre le génotype du vecteur et le génotype viral semblent donc revêtir une importance particulière dans notre contexte. Les résultats obtenus au cours de ce travail doctoral devraient permettre une meilleure évaluation du risque de survenue d’une épidémie d’arbovirose en Nouvelle-Calédonie. / Since the end of the 19th century, epidemics due to arboviruses have been a major public health problem in New Caledonia and the Pacific region. Over the past five years, the incidence of dengue epidemics has increased and the introduction of the chikungunya and Zika viruses (ZIKAV) has demonstrated that this region of the world is to be considered as a hot spot for the emergence of arboviruses. These three viruses are transmitted by mosquitoes of the genus Aedes, whose main vector in the Pacific region is Aedes aegypti. With the exception of some data concerning the dengue virus epidemiology in the region, little was known about vectors or on their ability to transmit arboviruses. These data are crucial to increase our knowledge about the specific appearance of these epidemics in the region.During my PhD, I first genetically characterized the vector Ae. aegypti from the Pacific region and demonstrated a moderate but significant genetic differentiation. We were also the first to evaluate Ae. aegypti vector competence for dengue virus 1 (DENV-1) and for different ZIKAV strains. The results have shown a low transmission efficiency for DENV-1 and heterogeneous results for the ZIKAV transmission, these differences were linked to the ZIKAV genetic lineage.The interactions between the vector genotype and the viral genotype seem to be particularly important in our context. The results obtained during this PhD work should allow a better assessment of the risk of an arbovirus outbreak in New Caledonia as well as the development of prevention strategies.

Parmi les virus émergents transmis par des moustiques (arbovirus), le genre flavivirus est fortement représenté avec les virus Dengue, Zika, et le virus West Nile (WNV). Le WNV est responsable de nombreux cas de maladies neuroinvasives sévères, parfois mortelles, chez l'humain et les chevaux. Ce virus représente donc un problème de santé publique humaine et animale. Il n'existe pour le moment aucun vaccin humain ni aucun traitement spécifique anti-WNV.Parmi les déterminants viraux essentiels à l'infection par les flavivirus, la glycoprotéine non-structurale NS1 possède des propriétés multifonctionnelles. La forme sNS1, sécrétée dans le milieu extracellulaire, est fortement impliquée dans la dérégulation du système immunitaire de l'hôte. Ces mécanismes participent à l'évasion du virus à la réponse antivirale et, paradoxalement, à la pathogenèse observée dans les formes sévères de la maladie. L'essentiel de ces données concernant le virus de la Dengue, nous souhaitions étudier les propriétés fonctionnelles, in vitro, de la protéine sNS1WNV au cours de l'infection de cellules épithéliales, gliales et neuronales de mammifères. En effet, la structure des protéines sNS1 de flavivirus étant très similaire, notre hypothèse suppose un rôle de sNS1WNV dans les infections neuroinvasives.Si la protéine sNS1WNV ne semble pas moduler les étapes de l'infection virale, elle est cependant à l'origine d'un remodelage du cytosquelette d'actine dans les cellules épithéliales. Elle est aussi impliquée dans l'activation de voies antivirales chez les cellules neuronales non infectées. D'autre part, en ciblant sNS1 et la protéine d'enveloppe E du WNV, nous avons pu isoler, par criblage de molécules aRep (protéines artificielles à motifs répétés), des ligands de haute affinité pour ces déterminants viraux. Ces nouvelles molécules, capables de se lier spécifiquement aux protéines sNS1 et E, ont le potentiel pour servir de base au développement de nouveaux outils de diagnostics et d'agents thérapeutiques antiviraux / Among emerging mosquito-borne viruses (arboviruses), flaviviruses like Dengue, Zika and West Nile virus (WNV) are very often involved in outbreaks. WNV causes several neuroinvasive diseases, which can be lethal, in humans and horses each year. This virus is a threat for both, human and animal public health. Furthermore, there is no human vaccine currently or any specific antiviral treatments against WNV.Among viral factors which are essential for flavivirus infection, the nonstructural glycoprotein NS1 is a multifunctional protein. The secreted form sNS1, is released in the extracellular medium from infected cells and is strongly involved in immune system dysregulation. The functions of sNS1 play roles in immune escape and, paradoxically, in pathogenesis which is observed in severe forms of the disease. Because most of this data are about Dengue Virus, we would like to study, in vitro, functional properties of the sNS1WNV during infection of epithelial, glial and neuronal mammalian cells. Based on the high sNS1 protein structure similarities among flaviviruses, our hypothesis suggests a role of sNS1WNV in neuroinvasive infections.The sNS1WNV protein doesn’t seem to modulate viral infection steps. However, it is involved in actin cytoskeleton remodeling in epithelial cells. sNS1WNV is also involved in the activation of antiviral response pathways in non-infected neuronal cells. On the other hand, by targeting sNS1 and envelope protein E of WNV, we performed a screening of aRep molecules (artificial proteins with alphahelicoïdal repeats) and isolated ligands with high affinity for these viral factors. Because this new type of molecules is able to specifically bind to sNS1 and E, they have potential to be used for the development of new diagnostic tools and antiviral therapeutic agents

Le virus Chikungunya (CHIKV), arbovirus en pleine ré-émergence, a envahi rapidement de nombreuses zones géographiques du monde. La propagation mondiale de ce virus constitue une menace pour la santé humaine car il n'y a pas de vaccin ou d'agents antiviraux appropriés pour contrôler l'infection virale. La transmission du virus s’effectue lors de la piqure d’un moustique infecté du genre Aedes, qui injecte sa salive contenant le virus dans la peau de l’hôte humain. Afin de contrôler la dissémination du virus, il est primordial de développer des recherches sur l’identification de molécules antivirales et de comprendre les mécanismes moléculaires impliqués dans les interactions hôte-virus et/ou vecteur-virus-hôte. En utilisant différentes stratégies moléculaires et cellulaires, nous avons étudié le potentiel antiviral de l'Imipramine, une molécule déjà commercialisée et qui a la capacité de perturber le transport du cholestérol intracellulaire. Nous avons démontré que cette molécule est capable d'inhiber la réplication du CHIKV dans les fibroblastes cutanés humains. Nous avons mis en évidence que l'Imipramine affectait à la fois les étapes de fusion et de réplication pendant le cycle de réplication du virus. En outre, la molécule a également fortement inhibé la réplication de plusieurs Flavivirus comme le virus Zika (ZIKV), le virus du Nil occidental et le virus de la Dengue. Nous avons également déterminé le profil protéomique global des fibroblastes humains infectés par le CHIKV ou le ZIKV. Cela nous a permis de mettre en évidence les modulations significatives de plusieurs protéines stimulées par l'interféron et de protéines impliquées dans à la défense anti-virale dans les cellules infectées. Plus important encore, nos résultats montrent pour la première fois le rôle de la protéine SAMHD1 dans l'infection des fibroblastes cutanés par les arbovirus. Enfin, compte tenu des fortes interactions entre l’hôte, le vecteur et le CHIKV, l'effet de la salive du moustique Ae. Aegypti sur l'infection virale a été étudié. À notre connaissance, cette étude est la première à montrer l’importance de la salive d’Ae. aegypti sur la facilitation de l’infection du CHIKV, dans des fibroblastes cutanés, à travers la régulation des gènes impliqués dans la réponse interféron de type I. / Chikungunya virus (CHIKV) is a re-emerging mosquito-borne alphavirus that has been spread worldwide. The dissemination of this virus is a threat to human health since there is no approved vaccine or appropriate antiviral agents to control viral infection. The global expansion of the virus is preceded by biting of infected Aedes mosquitos, which injects saliva containing the virus into the skin of the human host. Searching for effective antiviral compounds and understanding of the molecular mechanisms involved in host-virus or vector-virus-host interactions are crucial for controlling viral spread.Using different molecular and cellular strategies, we demonstrated that the FDA approved drug, imipramine, which has the capability to disturb intracellular cholesterol transport inhibits CHIKV replication in human skin fibroblasts. Imipramine was found to affect both the fusion and replication steps of the viral life cycle. Moreover, it also strongly inhibited the replication of several Flaviviridae family members, including Zika, West Nile and Dengue virus. We have also determined the global proteomic profile of Chikungunya and Zika virus infected human skin fibroblasts, and found that several interferon-stimulated proteins and antiviral response proteins are significantly up-regulated in the infected cells. More importantly, our results also provided for the first time a role of SAMHD1 in arbovirus infection of human skin fibroblasts. Finally, we demonstrated that Aedes aegypti saliva enhances CHIKV replication in human skin fibroblasts. To our knowledge, this is the first report showing the importance of Aedes aegypti saliva on promoting CHIKV infection via down regulation of the genes involving type I IFN secretion in the infected human cutaneous cells.

Introducción: La fiebre de Oropouche es una enfermedad infecciosa causada por el virus Oropouche (OROV). El diagnóstico y predicción del cuadro clínico continúa siendo un gran desafío para los médicos quienes manejan a los pacientes con síndrome febril agudo. Se han asociado diversos síntomas con la infección por virus OROV en pacientes con síndrome febril; sin embargo, a la fecha no existe una regla de predicción clínica. Objetivo: Evaluar el rendimiento de un modelo de predicción basado únicamente en signos y síntomas para diagnosticar la infección por el virus de Oropouche en pacientes con síndrome febril agudo. Materiales y Métodos: Estudio de validación, que incluyó a 923 pacientes con síndrome febril agudo registrados en la base de datos de la Vigilancia Epidemiológica de tres zonas del Perú durante los años 2015-2016. Resultados: Un total de 97 (19.0%) pacientes fueron positivos para infección por Oropouche en el grupo de desarrollo y 54 (23.6%) para el grupo de validación. El área bajo la curva fue de 0.65 y la sensibilidad, especificidad, VPP, VPN, LR+ y LR- fueron de 78.2%, 35.1%, 27.6%, 83.6%, 1.20 y 0.62 respectivamente. Conclusión: El desarrollo de un modelo de predicción clínica para el diagnóstico de OROV basado únicamente en signos y síntomas no funcionó bien debido a que la clínica es inespecífica y se relaciona con otras infecciones arbovirales, lo cual dificulta la predicción del diagnóstico, especialmente en áreas endémicas de coinfección de estas enfermedades. Se recomienda la vigilancia epidemiológica de OROV utilizando pruebas como PCR molecular. / Background. Oropouche fever is an infectious disease caused by the Oropouche virus (OROV). The diagnosis and prediction of the clinical picture continue to be a great challenge for clinicians who manage patients with acute febrile syndrome. Several symptoms have been associated with OROV virus infection in patients with febrile syndrome; however, to date, there is no clinical prediction rule. Objective. To assess the performance of a prediction model based solely on signs and symptoms to diagnose Oropouche virus infection in patients with acute febrile syndrome. Materials and Methods. Validation study, which included 923 patients with acute febrile syndrome registered in the Epidemiological Surveillance database of three areas of Peru during the years 2015-2016. Results. A total of 97 patients (19%) were positive for OROV infection in the development group and 23.6% in the validation group. The area under the curve was 0.65 and the sensitivity, specificity, PPV, NPV, LR + and LR- were 78.2%, 35.1%, 27.6%, 83.6%, 1.20 and 0.62, respectively. Conclusions. The development of a clinical prediction model for the diagnosis of Oropouche based solely on signs and symptoms does not work well because the clinic is nonspecific and is related to other arbovirus infections, which makes it difficult to predict the diagnosis, especially in areas co-infection endemics of these diseases. Epidemiological surveillance of OROV using laboratory tests such as molecular PCR is recommended. / Tesis

Identifica Culicoides spp. como vectores del virus de Lengua Azul (VLA) en granjas de ovinos de la ciudad de Pucallpa, provincia Coronel Portillo, Ucayali. Las granjas se caracterizaron por la presencia de arbustos, árboles, humedales y charcos; así como por la crianza semiextensiva de ovinos de pelo. El estudio fue realizado en tres etapas: la primera consistió en el análisis serológico de 46 muestras de suero de ovinos procedente de tres granjas mediante la prueba de inmunodifusión en gel agar (IDGA) y ELISA de competición (ELISAc) a fin de seleccionar granjas seropositivas a VLA. En la segunda se capturó 7930 Culicoides spp. durante tres días consecutivos con cinco trampas tipo CDC de luz ultravioleta instaladas a 50m aprox de los rebaños desde las 18:00 horas hasta las 06:00 horas del día siguiente; con el fin de determinar la población de especies presentes en las granjas seleccionadas. Por último, se realizó un periodo de captura adicional, obteniendose 11 pools de 100 hembras cada uno (Culicoides insignis : n = 1000 y Culicoides spp.: n = 100) y se obtuvo 15 muestras de sangre de ovino para el análisis molecular por PCR anidado utilizando dos pares de cebadores específicos que amplifican el segmento 7 que codifica la VP7 de VLA. Las tres granjas fueron seropositivas a Orbivirus con frecuencias de 46.7, 81.3, 20%, de las cuales el 96% fueron positivas a VLA. Se identificaron 7930 ejemplares de Culicoides spp., de los cuales 7839 (98.9%) fueron hembras y 91 (1.1%) machos. La principal especie fue Culicoides insignis (94.8%) seguido por C. foxi (3.2%) y C. ocumarensis (1.3%); otras especies como C. pseudodiabolicus, C. hylas y C. leopoldoi fueron capturados en densidades menores de 0.5%. También se registró a Culicoides lutzi aún no reportado en el Perú. De los 11 pool de Culicoides spp. (C. insignis = 10, Culicoides spp.=1), solo en un pool de C. insignis se observó banda a nivel de 1070pb, asimismo una muestra de ovino fue positivo. Se concluye que Culicoides insignis fue la principal especie capturada, y se ha demostrado la presencia del ARN del VLA en Culicoides insignis y en ovinos seropositivos de las granjas en estudio. / Tesis

A febre Zika é uma enfermidade que afeta pessoas de países tropicais e subtropicais. O agente etiológico da doença é o vírus Zika (ZIKV), um flavivirus de genoma RNA de fita simples, transmitido principalmente por mosquitos do gênero Aedes. Devido a associação do ZIKV à microcefalia e síndrome de Guillain-Barré é necessário desenvolver estratégias para a prevenção da doença. Entre eles, a melhor medida profilática é a vacinação. A proteína NS3 tem sido relatada como um potencial ativador da imunidade celular contra o ZIKV e outros flavivírus, cuja atuação tem sido utilizada como um alvo contra infecções virais. Neste contexto, o objetivo deste projeto foi testar a eficiência da NS3 como ativadora da resposta imunológica e, em combinação com o vírus inativado, produzir uma vacina capaz de desenvolver proteção total contra a infecção viral. Uma sequência da NS3 foi clonada no vetor pET-26b, expresso em E. coli Rosetta e purificado por cromatografia de afinidade em coluna de níquel. A completa inativação do ZIKV foi realizada por meio de adição de formaldeído 0,05% e incubação por 3 dias. Combinações vacinais de NS3 e vírus inativado foram inoculados em vários grupos de camundongos 129 Sv/Ev e A129 nos dias 0 e 14. No dia 21, os camundongos A129 vacinados foram desafiados com o ZIKV selvagem e analisados segundo a perda de peso e sobrevivência por 21 dias. Os camundongos vacinados com NS3 e vírus inativado, na mesma formulação vacinal, tiveram 100% de proteção contra o ZIKV infeccioso. O sangue total e o baço foram coletados dos camundongos 129 Sv/Ev e A129 após 21 dias de uma nova imunização. Foi avaliado a produção de anticorpos específicos e neutralizantes contra ZIKV por ELISA e PRNT, mostrando que animais imunizados com a formulação vacinal \"vírus inativado com NS3\" produzem mais anticorpos contra o vírus, porém não foi detectado neutralização do vírus em nenhum grupo imunizado. O perfil de citocinas expressas por linfócitos do baço, analisado por FACS, sugere que a NS3 participa na modulação para uma resposta Th1. Esta abordagem possibilitou a avaliação de uma combinação vacinal que se mostrou capaz de prevenir a infecção com o ZIKV em camundongos susceptíveis / Zika Fever is a disease that affects many people in tropical and subtropical countries. The etiologic agent is Zika Virus (ZIKV), a single-stranded RNA flavivirus, transmitted mostly by Aedes mosquitoes, but sexual, congenital and blood transfusion transmission has also been reported. Due to the possible association of ZIKV to microcephaly and Guillain-Barré Syndrome, it is necessary to develop strategies for disease prevention. Among them, the best prophylactic measure is vaccination. The non-structural protein NS3 has been shown to stimulate cellular immunity response against others flaviviruses, and such activity has been used as a target against other flaviviruses infection. In this context, the aim of this project is to test the efficiency of NS3, in combination with the inactivated virus, to produce a vaccine capable of developing full protection against viral infection. The NS3 sequence was cloned into the pET-26b vector, expressed in E. coli Rosetta and purified by nickel column affinity chromatography. The complete inactivation of ZIKV was performed by addition of 0.05% formaldehyde and incubation for 3 days. Vaccine combinations of NS3 and inactivated virus were inoculated into 129 Sv/Ev and A129 mice on days 0 and 14. On day 21, vaccinated A129 mice were challenged with wild ZIKV and analyzed for weight loss and survival by 21 days. Mice vaccinated with NS3 and inactivated virus, in the same vaccine formulation, had 100% protection against infectious ZIKV. Whole blood and spleen cells were collected from 129 Sv/Ev and A129 mice after 21 days of new immunization. The production of specific and neutralizing antibodies against ZIKV was evaluated by Imunnofluorescence, ELISA and PRNT, showing that animals immunized with the vaccine formulation \"virus inactivated with NS3\" increased the antibodies\' level against the virus, but no virus neutralization was detected in any immunized group. The cytokine profile expressed by spleen lymphocytes, analyzed by FACS, suggests that NS3 participates in modulation for a Th1 response. This approach gave us the opportunity to evaluate this vaccinal combination that was shown to be able to prevent infection with ZIKV in susceptible mice

Realizou-se estudo analitico transversal relacionando caracteristicas individuais e familiares de 182 moradores pertencentes a 58 famílias de área de reserva ecológica à presença de infecções por arbovirus e hantavirus de interesse sanitário local. Pesquisou-se anticorpos para os antigenos dos virus Rocio, Ilhéus, da encefalite de st. Louis, das encefalites equinas do leste, oeste e venezuelana e Hantaan. Foram utilizados os testes de inibição de hemaglutinação, neutralização com redução de placas, imunofluorescência indireta e ensaio imunoenzimático com captura de anticorpos IgM (MAC-ELISA).· A associação estatistica foi pesquisada utilizando-se o teste de qui-quadrado e o grau de associação foi obtido calculando-se o odds ratio. Também foram pesquisadas a sensibilidade, a especificidade e os valores preditivos positivo e negativo das caracteristicas investigadas para avaliar seu poder de discriminar infectados de não infectados e de predizer infecções por arbovirus e hantavirus. A prevalência observada de anticorpos para arbovirus nos individuos foi de 26,9% e nas familias foi de 62,1%. Observou-se uma prevalência de 1,6% de anticorpos para hantavirus nos individuos e 5,2% nas familias. Não foram encontrados anticorpos para o virus da encefalite equina do oeste e nem anticorpos da classe IgM para os antigenos testados. Entre as caracteristicas estudadas, a idade, a ocupação, a naturalidade e o hábito de entrar na mata mostraram-se fatores de risco para infecções por arbovirus. Foram considerados fatores preditivos de infecção por arbovirus a presença de galinheiro anexo à casa, o hábito de criar galinhas e a presença de ratos no domicilio. Não foram observados fatores de risco ou de predição para infecção por hantavirus entre as caracteristicas estudadas. Os resultados obtidos sugerem que as caracteristicas, que mostraram ser fator de risco ou predição, poderiam ser usadas no diagnostico presuntivo preliminar de infecções por arbovirus e hantavirus, permitindo priorizar medidas de intervenção. O uso de um questionário ofereceria aos serviços de saúde ferramenta de simples aplicação e baixo custo, especialmente em condições de campo em áreas que, como a estudada, dispõe de escassos recursos humanos e laboratoriais. / In order to identify risk factors and predictive factors to arbovirus and hantavirus infections among individual and family characteristics, a cross-sectional study was carried out among 182 persons belonging to 58 families living at ecological reserve. The characteristics were associated with the presence of antibodies to the following virus of local interest: Hantaan, Rocio, Ilheus, Eastern, Western and Venezuelan equine encephalitis virus and st. Louis encephalitis virus using inhibition hemagglutination test, plaque reduction neutralization test, indirect immunofluorescence test and immunoglobulin M antibody capture enzyme immunoassay (MAC-ELISA). The associations were calculated using chi-square statistics and the odds ratio. Sensitivity, especificity, positive and negative predictive values of the individual and familiar characteristics were also analyzed to evaluate their capacity to discriminate between infected and non-infected people and to predict arbovirus and hantavirus infections. The prevalence of antibodies to arbovirus was 26.9% and 62.1% of the families had at least one member infected by these agents. Hantavirus antibodies were found in 1.6% of the sera analysed and 5.2% of the families had members infected by these agents. Antibodies to western equine encephalitis virus were not found. IgM antibodies were not observed sugesting no recent infection for those agents in that population. Age, ocupation, nativity and the habit to enter the forest were shown to be risk factors to arbovirus infections. The presence of annexes to the house, to breed chickens and the presence of rodents inside the house were considered predictive factors to arbovirus infections. Risk or predictive factors to hantavirus infections were not observed. The results suggest the use of some of the individual or family characteristics as a tool on the surveillance of arbovirus infections, to discriminate people with major probability of infections, specially in fie1d conditions, where human and laboratorial resources are scarce.

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.

Devido a existência de 2 alphavírus artritogênicos no Brasil, os vírus Mayaro (MAYV) e Chikungunya (CHIKV) tornou-se importante desenvolver testes diagnósticos eficazes para discriminar suas infecções. No presente trabalho, desenvolvemos ELISAs indiretos para diagnóstico de CHIKV e MAYV utilizando proteínas de envelope viral E2 recombinantes, produzidas em Escherichia coli, as rE2-CHIKV e rE2-MAYV ELISAs. As proteínas E2 recombinantes tiveram suas antigenicidades verificadas nos ensaios utilizando anticorpos policlonais oriundos de camundongos hiperimunizados com CHIKV, MAYV e outros alphavírus. O rE2-CHIKV ELISA detectou anticorpos murinos de forma homotípica e não produziu reações cruzadas evidenciáveis utilizando anticorpos murinos específicos contra outros Alphavírus. O rE2-MAYV ELISA detectou anticorpos murinos homotípicos e também, reagiu cruzadamente com anticorpos murinos anti-CHIKV, mas não para outros Alphavírus. Esses ELISAs, também, foram usados na detecção de anticorpos em soros de pacientes com suspeita de infecção arboviral. Pelo o rE2-CHIKV ELISA, testaram-se 59 soros, resultando em 26 amostras IgG positivas. Resultados desse ELISA, quando comparados aos obtidos por teste de neutralização, demonstraram sensibilidade de 89,66% e especificidade de 100%. Soros humanos IgG positivos foram detectados em altas diluições pelo rE2-CHIKV ELISA. Quanto a detecção de IgM, o rE2- CHIKV ELISA apresentou moderada concordância com outros ensaios sorológicos. Com rE2- MAYV ELISA, testaram-se 68 soros resultando em 23 amostras IgG positivas, das quais 11 também mostraram-se positivas em teste de neutralização, demonstrando sensibilidade de 100% e especificidade de 78,95%. Portanto, os rE2-CHIKV e rE2 MAYV ELISAs, particularmente para detecção de IgG, mostraram-se adequadamente sensíveis e específicos para serem validados em estudos com maiores números de amostras e serem aplicados ao diagnóstico de pacientes infectados com CHIKV e MAYV. / Due the existence of 2 arthritogenic alphaviruses in Brasil, the viruses Mayaro (MAYV) and Chikungunya (CHIKV), it became important the development of efficient diagnose tests to discriminate their infections. In the present work, we developed indirect ELISAs for CHIKV and MAYV diagnosis using viral recombinant envelope proteins E2, produced in Escherichia coli, the rE2-CHIKV and rE2-MAYV. The recombinant E2 proteins had their antigenicity confirmed in the assay by using polyclonal antibodies produced in hyperimmunized mice with CHIKV, MAYV and other alphaviruses. The rE2-CHIKV ELISA detected homotypic murine antibodies and did not produced detectable cross-reactivity signal when using murine antibodies from other alphaviruses. The rE2-MAYV ELISA detected homotypic antibodies and also cross-reacted with murine anti-CHIKV antibodies, but not to other alphaviruses. These ELISAs were also tested for the detection of human antibodies, using patient sera suspected of arboviral infection. For rE2- CHIKV ELISA, it were tested 59 sera, resulting in 26 positive IgG samples. These ELISA results, when compared to those of a neutralizing assay, demonstrated a sensibility of 89.66% and specificity of 100%. The IgG positive human sera were detected in high dilutions by rE2-CHIKV ELISA. Regarding the detection of IgM, the rE2-CHIKV ELISA showed a moderate samples detection agreement when compared to other serologic assays. For rE2-MAYV ELISA, it were tested 68 sera, resulting in 23 positive IgG samples, of which 11 demonstrated to be positive by the neutralization assay, demonstrating a sensibility of 100% and specificity of 78.95%. Therefore, the rE2-CHIKV and rE2-MAYV ELISAs, especially for IgG detection, demonstrated to be properly sensitive and specific to be validated in studies using a greater number of samples, and also to be applied in the diagnosis of infected CHIKV and MAYV patients.