O sequenciamento de alto desempenho, pela redução dos custos nos últimos anos, vem sendo cada vez mais utilizado para prospectar e identificar vírus. Estes métodos são extremamente mais sensíveis que outros métodos moleculares, e capazes de sequenciar genomas virais sem conhecimento prévio, clonagem ou isolamento. Neste estudo, utilizamos o sequenciamento de alto desempenho para conhecer, e caracterizar genomas completos de arbovírus isolados nas Américas, incluindo a prospecção de vírus em amostras de pequenos mamíferos do estado de São Paulo, Brasil. Assim, sequenciamos e caracterizamos 44 Bunyavirales, 35 no gênero Orthobunyavirus, família Peribunyaviridae, oito no gênero Phlebovirus, família Phenuiviridae, e um orthonairovírus, família Nairoviridae. Entre os Bunyavirales identificamos uma provável nova estratégia de codificação da proteína não estrutural do segmento pequeno, e ainda identificados sete vírus que são reassortants naturais. Caracterizamos o genoma completo do vesiculovírus Piry, determinando sua relação filogenética com arbovírus pertencentes ao gênero Vesiculovirus, família Rhabdoviridae. Prospectamos novos vírus, os quais incluímos em três famílias, Parvoviridae, Anelloviridae e Hepeviridae. Na família Parvoviridae, identificamos 20 chapparvovírus endógenos e exógenos, oriundos de grande diversidade de hospedeiros vertebrados e invertebrados, e que representam uma nova subfamília, a Chapparvovirinae. Também, descrevemos onze novas espécies de Anelloviridae em roedores silvestres e marsupiais, fornecendo importantes informações sobre a diversidade, a taxonomia, e ainda ampliamos a gama de hospedeiros de anellovírus conhecidos. Por fim, identificamos e caracterizamos uma nova espécie de Orthohepevirus de roedores Sigmodontinae, nomeada \"Orthohepevirus E\". Acreditamos que estamos a fornecer relevantes informações sobre genômica, epidemiologia molecular, evolução e taxonomia de 45 arbovírus americanos, bem como sobre 13 novas espécies virais encontradas em pequenos mamíferos. Tais informações deverão dar subsídios para múltiplos futuros estudos visando compreender a importância destes novos vírus e a desenvolver métodos diagnósticos. / In last years, high-throughput sequencing (HTS) has been cost-effective and increasingly used for prospection and identification of viruses. These methods are extremely more sensitive than other molecular methods and are capable of sequencing viral genomes without prior knowledge, cloning or isolation. In this study, we used HTS approach to identify and characterize complete genomes of arbovirus isolated in the Americas, as well as viral prospection in samples of small mammals from São Paulo State, Brazil. Thus, we sequenced and characterized 44 viruses from Bunyavirales order, including 35 in Orthobunyavirus genus, family Peribunyaviridae, eight in Phlebovirus genus, family Phenuiviridae, and one in Orthonairovirus genus, family Nairoviridae. Among the Bunyavirales we identified a novel putative strategy for encoding the non-structural protein of the small segment, as well as we identified seven viruses that are natural reassortants. Also, we characterized the complete genome of the Piry vesiculovirus, determining its phylogenetic relationship with arboviruses belonging to the Vesiculovirus genus, family Rhabdoviridae. On the other hand, we have prospected novel viruses, which included in three families, Parvoviridae, Anelloviridae, and Hepeviridae. In the Parvoviridae family, we identified 20 endogenous and exogenous chapparvoviruses from a broad diversity of vertebrate and invertebrate hosts, representing a new subfamily, the Chapparvovirinae. Also, we have described eleven new species of Anelloviridae in wild rodents and marsupials, providing important information on diversity, taxonomy and even broadening the range of known anelloviruses hosts. Finally, we identified and characterized a novel species of orthohepevirus in Sigmodontinae rodent, named \"Orthohepevirus E\". We believe that we are providing relevant relevant on genomics, molecular epidemiology, evolution and taxonomy of 45 American arboviruses, as well as on 13 new viral species found in small mammals. Thus, these informations should provide support for multiple future studies to understand the importance of these new viruses, as well as to develop diagnostic methods.

A synthesis of all results of researches done by the author on arboviruses cycles and bioecology of their vectors in French Guiana, Central African Republic and Brazil, from 1975 to 1996. Emphasis is put on the ecoepidemiology of yellow fever and dengue viruses. Studies are also reported on the impacts of environmental changes on the transmission of arboviruses in Brazilian Amazonia.<br />An exhaustive list of publications and communications of the author is presented.

L'épidémie de West-Nile en Amérique du Nord en 2002, qui a touché plus de quarante états aux Etats-Unis, a conduit les Agences de santé à s'interroger sur le risque d'émergence, à l'extérieur de la zone intertropicale, de zoonoses vectorielles. Cette épidémie associée au changement climatique, a bien mis en évidence le rôle central de l'avifaune migratrice dans la diffusion du virus. La biologie des oiseaux, tout particulièrement le phénomène migratoire, permet un transport des virus sur de longues distances et entre espèces très diversifiées. Le Moyen-Orient, qui est situé au carrefour de différents continents, est extrêmement propice à la propagation des virus émergents dans les pays du Nord. La circulation du virus West Nile a été rapportée dans différents pays de la région, tels que l'Egypte, Israël, Liban, Irak, Emirats Arabes Unis et Iran. Saidi et al. (1970) ont montré la présence d'anticorps anti-virus du Nil occidental au sein de la population de la côte caspienne (Nord de l'Iran), des provinces du Khorassan (Nord-Est) et du Khuzestan (Sud-Ouest). Notre étude, conduite dans le cadre d'un programme associant TIMC-IMAG UMR 5525 UJF CNRS VetAgroSup, le Réseau International des Instituts Pasteurs et le Centre National d'Etudes Spatiales, vise: (i) à caractériser la circulation du virus de West-Nile au sein des populations équines d'Iran ; et (ii) et à modéliser l'impact sanitaire de l'environnement et du climat sur la transmission. Les résultats acquis permettent d'apprécier le risque associé à la dissémination spatio-temporelle du virus par les oiseaux migrateurs. Une attention toute particulière est portée à l'étude des déterminants environnementaux et climatiques susceptibles d'accroitre le potentiel de transmission du virus.

Thesis (Ph.D.) - University of Queensland, 2004. / Program shared between two schools. Includes bibliography.

Mestre -- Universidade de Sao Paulo. Faculdade de Saude Publica. Departamento de Epidemiologia, Sao Paulo, 2001.

Identifica las fuentes de alimentación en mosquitos colectados en áreas endémicas de arbovirosis en el Departamento de Loreto utilizando herramientas moleculares, y evalúa la presencia de arbovirus en tales mosquitos. De enero a marzo del 2009, los mosquitos son recolectados en las provincias de Alto Amazonas y Datem del Marañón, utilizando trampas de luz CDC, cebo humano y mochilas aspiradoras. Un total de 125 mosquitos colectados con evidencia de sangre son identificados usando claves dicotómicas, homogenizados individualmente, sometidos a extracción de DNA y amplificación de PCR utilizando 3 sets de primers consenso dirigidos al gen del citocromo b de mamíferos, aves y reptiles. El rendimiento del ensayo molecular es previamente validado usando DNA extraído de sangre de vertebrados conocidos. Se obtienen amplicones de PCR en 115 (92%) mosquitos, y son secuenciados para identificar al potencial hospedero. Psorophora albigenu, Ochlerotatus. serratus y Och. fulvus tienen comportamiento de alimentación ecléctico, Culex (Melanoconiom) vomerifer y Cx. (Aedinus) amazonensis se alimentan de ratas espinosas. Cx. (Mel.) dunni y Cx. (Mel.) occosa se alimentan de un gato y un oso perezoso respectivamente. Ps. cingulata, Mansonia humeralis, Anopheles oswaldoi s.l, An. benarrochi tienen comportamientos antropofílicos. Éste es el primer estudio que identifica fuentes de alimentación en Cx. (Mel.) spp., Ps.albigenu, Och. serratus, Och. fulvus, y que soporta sus roles como vectores en el ciclo de transmisión de alfavirus en la Cuenca Amazónica del Perú. / Tesis

O sequenciamento de alto desempenho, pela redução dos custos nos últimos anos, vem sendo cada vez mais utilizado para prospectar e identificar vírus. Estes métodos são extremamente mais sensíveis que outros métodos moleculares, e capazes de sequenciar genomas virais sem conhecimento prévio, clonagem ou isolamento. Neste estudo, utilizamos o sequenciamento de alto desempenho para conhecer, e caracterizar genomas completos de arbovírus isolados nas Américas, incluindo a prospecção de vírus em amostras de pequenos mamíferos do estado de São Paulo, Brasil. Assim, sequenciamos e caracterizamos 44 Bunyavirales, 35 no gênero Orthobunyavirus, família Peribunyaviridae, oito no gênero Phlebovirus, família Phenuiviridae, e um orthonairovírus, família Nairoviridae. Entre os Bunyavirales identificamos uma provável nova estratégia de codificação da proteína não estrutural do segmento pequeno, e ainda identificados sete vírus que são reassortants naturais. Caracterizamos o genoma completo do vesiculovírus Piry, determinando sua relação filogenética com arbovírus pertencentes ao gênero Vesiculovirus, família Rhabdoviridae. Prospectamos novos vírus, os quais incluímos em três famílias, Parvoviridae, Anelloviridae e Hepeviridae. Na família Parvoviridae, identificamos 20 chapparvovírus endógenos e exógenos, oriundos de grande diversidade de hospedeiros vertebrados e invertebrados, e que representam uma nova subfamília, a Chapparvovirinae. Também, descrevemos onze novas espécies de Anelloviridae em roedores silvestres e marsupiais, fornecendo importantes informações sobre a diversidade, a taxonomia, e ainda ampliamos a gama de hospedeiros de anellovírus conhecidos. Por fim, identificamos e caracterizamos uma nova espécie de Orthohepevirus de roedores Sigmodontinae, nomeada \"Orthohepevirus E\". Acreditamos que estamos a fornecer relevantes informações sobre genômica, epidemiologia molecular, evolução e taxonomia de 45 arbovírus americanos, bem como sobre 13 novas espécies virais encontradas em pequenos mamíferos. Tais informações deverão dar subsídios para múltiplos futuros estudos visando compreender a importância destes novos vírus e a desenvolver métodos diagnósticos. / In last years, high-throughput sequencing (HTS) has been cost-effective and increasingly used for prospection and identification of viruses. These methods are extremely more sensitive than other molecular methods and are capable of sequencing viral genomes without prior knowledge, cloning or isolation. In this study, we used HTS approach to identify and characterize complete genomes of arbovirus isolated in the Americas, as well as viral prospection in samples of small mammals from São Paulo State, Brazil. Thus, we sequenced and characterized 44 viruses from Bunyavirales order, including 35 in Orthobunyavirus genus, family Peribunyaviridae, eight in Phlebovirus genus, family Phenuiviridae, and one in Orthonairovirus genus, family Nairoviridae. Among the Bunyavirales we identified a novel putative strategy for encoding the non-structural protein of the small segment, as well as we identified seven viruses that are natural reassortants. Also, we characterized the complete genome of the Piry vesiculovirus, determining its phylogenetic relationship with arboviruses belonging to the Vesiculovirus genus, family Rhabdoviridae. On the other hand, we have prospected novel viruses, which included in three families, Parvoviridae, Anelloviridae, and Hepeviridae. In the Parvoviridae family, we identified 20 endogenous and exogenous chapparvoviruses from a broad diversity of vertebrate and invertebrate hosts, representing a new subfamily, the Chapparvovirinae. Also, we have described eleven new species of Anelloviridae in wild rodents and marsupials, providing important information on diversity, taxonomy and even broadening the range of known anelloviruses hosts. Finally, we identified and characterized a novel species of orthohepevirus in Sigmodontinae rodent, named \"Orthohepevirus E\". We believe that we are providing relevant relevant on genomics, molecular epidemiology, evolution and taxonomy of 45 American arboviruses, as well as on 13 new viral species found in small mammals. Thus, these informations should provide support for multiple future studies to understand the importance of these new viruses, as well as to develop diagnostic methods.

L’infection par le virus du Chikungunya est devenu en quelques années un problème de santé publique. D’abord limitée dans les régions tropicales et subtropicales, la diffusion mondiale du vecteur de l’infection couplée à la migration humaine et aux adaptations du virus contribue à la survenue de phénomènes épidémiques fréquents et touchant de nombreux territoires jusqu’aux pays tempérés. Dans les prochaines décades, la population des 65 ans ou plus sera probablement largement impactée lors des épidémies. Les conséquences de la maladie dans cette population est méconnu du fait de l’absence de données d’observation disponibles. Or, les spécificités de cette population sont connues (risque de comorbidité associée, immunosenescence, modifications physiologiques affectant le système rénal, cardiaque, pulmonaire…) et influencent fortement son mode de réponse et sa capacité à supporter un épisode infectieux. A travers l’observation et le suivi de la cohorte ChikOld constituée de 687 sujets âgés de 65 ans ou plus ayant contracté ou non la maladie en 2014, nous avons mis en évidence que les outils d’aide au diagnostic élaborés dans une population d’adultes jeunes avaient des performances médiocres dans la population âgée. Nous avons également pu mettre en évidence que les tableaux cliniques habituellement décrits de la maladie ne sont pas ceux observés le plus fréquemment dans une population de sujets de 65 ans ou plus. Ces observations ont abouti à l’élaboration un outil d’aide au diagnostic (score) spécifique à cette population. De nombreuses questions subsistent concernant cette infection, notamment sur les conséquences à moyen et long terme, sachant les conséquences en phase aiguë et l’existence de formes chroniques. Une étude préliminaire que nous avons conduit suggère l’absence de surmortalité à moyen terme (un an) et la majoration de la dépendance dans les suites d’une infection par le virus du Chikungunya. D’autres travaux seront nécessaires pour caractériser la forme habituelle de la maladie chez les sujets âgés ainsi que pour mieux appréhender les conséquences à moyen et long terme concernant la mortalité et la dépendance. / Chikungunya virus infection is an emergent arthropod-borne alpha-virus transmitted by mosquito bites, and causes fever with debilitating arthritic illness. Chikungunya virus infection is still considered as an emerging public health problem in both tropical and temperate regions. The presence of favourable conditions in temperate regions has enabled propagation of the vector, leading to the emergence of the first autochthonous cases of CHIKV in Europe and the USA. Older people may be particularly concerned about infection during an outbreak. CVhikungunya virus infection prevalence rates are not fully known, and vary from 18% to 48% The use of predictive scores would thus be very helpful in this situation. We have showed that predictive scores developed in young population have poor diagnostic performances in elderly population. In fact, the populations described in observational studies of chikungunya virus infection were predominantly young subjects. Clinical and epidemiological data in older subjects (aged 65 and over) are sparse. The mortality and morbidity related to infection in elderly people is poorly documented. We showed that the usual clinical expression of CHIKV infection is different in elderly subjects (absence of fever, arthralgia or both). We have developed and validated a new Chikungunya virus infection screening score specifically for use in the aged population.Some questions remain, in particular concerning mid- or long-term consequences of infection in elderly people. In a preliminary study, we have showed that the mid-term mortality rate of aged people infected by Chikungunya was lower than that of uninfected aged people.We need to continue our work on this thematic to explore more precisely the consequences of chikungunya virus infection in elderly people (mid- and long-term mortality, loss of autonomy, chronic form…).

The influence of forest canopy disturbance (FCD) on La Crosse virus (LACV), leading cause of US pediatric arboviral encephalitis, is critical to understand in landscapes where forests are periodically harvested. Southwestern Virginia is part of an emerging focus of this interior forest bunyavirus. I investigated how the temperate forest mosquito community, LACV vectors, and the LACV amplifying vertebrate host (chipmunks) were impacted by logging. This research was conducted across an experimental FCD gradient (from least to most disturbed: contiguous control, fragmented control, clearcut, and high-leave shelterwood (SW)). Using gravid traps, I found that the mosquito community was resilient to logging with no significant difference in diversity or community composition across treatments. Mean number of female mosquitoes caught per trap-night declined with disturbance. FCD significantly affected the abundance of vector species in different ways. The primary LACV vector, Aedes triseriatus, and the recent invasive Ae. japonicus declined with logging. Other vectors (Ae. albopictus, Ae. canadensis, and Ae. vexans) thrived with logging. Culex pipiens/restuans was affected by disturbance but had no treatment preference. A mark-recapture study revealed that chipmunk abundance and LACV seroprevalence were greatest on the SW. In sync with Ae. triseriatus abundance but in contrast to the chipmunk results, mosquito LACV detection was significantly greater on unlogged sites. Surprisingly, LACV was detected in Ae. japonicus and Cx. pipiens/restuans. In a follow-up study, I isolated LACV from field-collected Ae. japonicus. Although LACV was previously isolated from Cx. pipiens, the vector competence was unknown. Therefore, I examined the vector competence of Cx. pipiens and Cx. restuans. Although poor vectors, I did detect LACV in the saliva of both species. An additional experiment found that nutritionally-stressed Cx. restuans were better vectors than those in the control group, indicating that environmental stressors (e.g., FCD) may alter the ability of accessory vectors to spread LACV. The influence of FCD on LACV is complex. Because logging decreases Ae. triseriatus abundance, human LACV risk is likely lowered by decreased transovarial vertical transmission. However, high chipmunk seroprevalence on disturbed sites suggest horizontal transmission with accessory vectors plays a larger role in LACV risk on recently logged sites. / Ph. D.

La Fièvre catarrhale ovine (FCO) est une arbovirose émergente en Europe depuis la fin desannées 90. Elle affecte principalement les ruminants par la piqûre de petits moucheronshématophages, les Culicoides (Diptera : Ceratopogonidae). Pendant l’été 2006, l’introductiondu sérotype 8 de la FCO, dans la région de Maastricht (Pays-Bas) a rapidement diffusé dansles Ardennes, générant de lourdes pertes pour les éleveurs de bovins et d’ovins. Cesévènements interrogent sur la capacité des Culicoides de la région paléarctique à transmettrela FCO. Ils révèlent la nécessité de mieux connaître la biologie de ces diptères.Nous avons développé successivement dans ce travail, trois axes de recherche qui se sontappuyés sur un travail de terrain réalisé principalement au sein de deux élevages situés dansles Ardennes françaises.Dans un premier temps, nous avons réalisé une expérimentation de gorgement de Culicoidesde captures et d’émergences, provenant des Ardennes, sur petits ruminants virémiques pour leBTV8. A l’issue des expérimentations, une femelle gorgée de l’espèce Culicoides obsoletus apondu et a été retrouvée faiblement positive lors de la recherche du génome du virus de laFCO. Les résultats obtenus ainsi que les difficultés rencontrées lors de la réalisation de cetype d’expérimentation sont discutés.Le deuxième travail exposé s’est intéressé au comportement trophique des Culicoides parl’étude de l’origine du repas sanguin de femelles de Culicoides piégées dans des biotopesvariés. A cette fin, nous avons utilisé des marqueurs moléculaires pour amplifier l’ADN devertébré présent dans les estomacs de femelles gorgées. Ces analyses ont permis de mettre enévidence que des espèces appartenant aux complexes Obsoletus, Pulicaris, ou encore,Culicoides dewulfi, avaient un spectre d’hôte large. Certaines d’entre elles peuvent se gorger àla fois sur les ruminants domestiques et sur la faune sauvage. De plus, ce type d’étuderenseigne sur l’écologie des différentes espèces de Culicoides.Enfin, nous présentons les résultats d’une étude faunistique fondée sur des captures avec despièges lumineux, mais aussi, des prélèvements de boue pour la recherche des gîtes larvaires.Les résultats de piégeages entre les deux exploitations ont été comparés, notamment en termesde biodiversité, et sont discutés en regard des différences de pratiques d’élevage entre lesdeux exploitations choisies d’une part, et la mise en évidence des gîtes larvaires d’autre part.De nombreuses espèces de Culicoides ont émergé au laboratoire à partir des prélèvements deboues, qui ont été caractérisés macroscopiquement. Les gîtes larvaires de C. obsoletus, peuconnus jusqu’alors, ont été mis en évidence dans les deux fermes. Ils ont fait l’objet d’un suivisur plusieurs mois.L’ensemble de ces études contribue à la meilleure connaissance des Culicoides présents dansles Ardennes et de leur biologie, elles permettent de rendre compte des espèces qui semblenttrès inféodées à l’élevage de bovins, et celles qui sont plus ubiquistes. Certains travauxprésentés pourraient être poursuivis pour mettre en évidence les espèces ou populations deCulicoides plutôt sylvatiques, et pour mettre en place de nouvelles expérimentations sur lacompétence et la capacité vectorielle des Culicoides. / Since the late 90’s, Bluetongue disease (BT) can be considered as an emerging arbovirose inEurope. This disease is mainly transmitted to ruminants by the bites of minute size midges,the Culicoides (Diptera: Ceratopogonidae), also known as biting midges. An outbreak of BTserotype 8 occurred during summer 2006, in the region of Maastricht (Netherlands) andspread quickly to the Ardennes region. The epizooty lead to severe losses in cattle and sheepholdings. These events highlighted the lack of knowledge on the vectorial capacity ofpaleartic Culicoides species, and more generally on their biology.Three approaches are successively treated in this document. They are all based on field workconducted mainly in two holdings located in the Ardennes region.First, an experiment to assess oral susceptibility of Culicoides to Bluetongue virus (BTV) 8was undertaken. Field collected and emerging Culicoides coming from the Ardennes wereengorged on viremic small ruminants. At the end of the experiments, one Culicoides obsoletusfemale was found bloodfed and laid eggs. She was tested for BTV and was found weaklypositive for BTV genome. This result and the difficulties met during the experiment havebeen discussed.The second study focused on the bloodmeal origin of engorged females of Culicoides. Thesewere collected by light traps set in different kinds of environment. Molecular markers wereused in order to amplify the DNA of vertebrates present in the stomach of bloodfed females.Some of the species processed belonging to the Obsoletus or the Pulicaris complex, andCulicoides dewulfi fed on a wide variety of hosts, including domestic ruminants and wildanimals. Moreover, this kind of study brings information on the ecology of different speciesof Culicoides.Finally, a faunistic survey is presented. It was achieved through light trap collections ofmidges and also thanks to the sampling of potential breeding sites. Biodiversity in thecollection of midges captured by light traps between the two holdings were compared.Differences observed are discussed taking into account the differences in breeding practicesbetween the two holdings and the breeding sites investigations. Numerous species ofCulicoides emerged in the laboratory from soil samples which were macroscopicallydescribed. Breeding sites of C. obsoletus, which were not well documentated in the literature,were found in both farms. These were monitored over some months.This work contributes to a better knowledge of the Culicoides present in the Ardennes andtheir biology. It highlights the species which are closely related to the cattle holdingenvironment, and those which are ubiquist. Some of these studies could be continued in orderto highlight the species more related to the forested areas, and to set new experiments onvectorial competence and capacity.