Le virus Zika (ZIKV), virus découvert pour la première fois à la fin des années quarante, est un arbovirus émergent récemment arrivé sous le feu des projecteurs à l’occasion d’une pandémie rapide à l’échelle mondiale. Appartenant à la famille des Flaviviridae, ce flavivirus est transmis par les moustiques du genre Aedes. Alors qu’on le croyait relativement peu pathogène, ce virus se révèle être la cause probable d’une vague de complications neurologiques, incluant l’apparition de microcéphalies et de syndromes de Guillain-Barré. De plus, il n’existe à l’heure actuelle ni vaccins ni traitements spécifiques, la lutte contre le virus se résumant largement à la mise en place de mesures de prévention contre la piqûre de moustiques et la lutte anti-vectorielle.Une meilleure connaissance de l’ensemble de la biologie du virus, depuis les modalités d’entrée dans l’organisme, en particulier au niveau cutanée, jusqu’aux mécanismes moléculaires intimes de la réplication du virus s’avère nécessaire. Par des approches moléculaires et cellulaires, nous avons mis en évidence le tropisme du virus, identifié ses récepteurs et déterminé les réponses cellulaires induites par ce dernier. Nos travaux ont également identifié un potentiel mécanisme d’évasion mise en place par le ZIKV. Nous avons également entrepris un travail original sur un mécanisme moléculaire favorisant la pathogénicité des flavivirus. Une meilleure connaissance de ce mécanisme pourrait déboucher sur l’identification de potentiels cibles thérapeutiques. Enfin, le tropisme neuronal avéré du ZIKV nous a amené à travailler sur la réponse immune des astrocytes humain. En effet, les astrocytes forment une population cellulaire très importante dans le système nerveux central qui est fortement impliquée dans les mécanismes de neurogénèse dans le cerveau des fœtus. / The Zika virus (ZIKV) was first isolated from non-human primates the late 1940s. This emerging arbovirus has recently been under the spotlight due to a rapid world pandemic. Belonging to the Flaviviridae family, this flavivirus is transmitted by Aedes’ genus mosquitoes. Historically low pathogenic, a new major concern is the possible association of ZIKV with diverse of neurological complications, including the development of microcephaly and Guillain-Barré syndrome, particularly in newborns of infected mothers. In addition, there is currently no vaccine or specific treatment to cure the disease, so the main preventive measures to fight the spreading of the virus are to prevent mosquitoes’ bites and to plan an effective vector control. A better understanding of the biology of the virus, from the entry in the body, especially at the skin level, to the molecular mechanisms of viral replication, is therefore necessary.Using different molecular and cellular strategies, we investigated the tropism of the virus, identified cell surface receptors and determined the cell’s responses to the infection. Our work also permitted to identify a potential mechanism by which ZIKV evades the host immune system to facilitated his own replication. We also have undertaken original work on a molecular mechanism increasing the pathogenicity of flavivirus. A better knowledge of this mechanism may lead to the identification of potential therapeutic targets. Finally, considering the neuronal tropism of the ZIKV, we studied the immune response of human astrocytes, a very important cell population in the central nervous system, playing a major role in the mechanisms of neurogenesis during the fetus’ brain development.

La Guadeloupe fait partie des pays où la Dengue est endémique avec des épidémies tous les 2 à 3 ans. Depuis 3 ans, d'autres arboviroses sont apparues sur le continent américain avec le Chikungunya en 2013 puis le Zika en 2015, causant d'importantes épidémies notamment en Guadeloupe. Le seul vecteur reconnu de ces maladies en Guadeloupe est le moustique Aedes aegypti. Il n'y a pas de vaccin, ni de traitements spécifiques contre ces infections et les moyens de prévention contre ces maladies passent par la surveillance et le contrôle des populations de moustiques sur le terrain. Les méthodes de surveillance sont basées le plus souvent sur l'analyse d'indices larvaires, parfois controversés. De plus, les moyens de contrôle des vecteurs ont longtemps été basés sur l'utilisation massive d'insecticides chimiques entraînant la résistance des moustiques à ces produits. Ce travail de thèse s'est donc articulé autour de deux grands axes devant permettre d'améliorer la prévention et le contrôle de ces arboviroses: i) la recherche d'un nouvel outil de surveillance des populations vectrices, basé sur la physiologie des femelles adultes et ii) l'évaluation des niveaux et l'étude de certains mécanismes de résistance à trois insecticides chimiques, le Téméphos, le Malathion (utilisés dans le passé) et la Deltaméthrine (utilisée actuellement). Un modèle de surveillance des populations vectrices basé sur les taux de parité en lien avec l'espérance de vie des femelles, en fonction des températures a été développé, et des pistes sur les situations entomologiques les plus à risques se sont dessinées. Les épreuves de résistance effectuées sur des larves de moustiques de Guadeloupe ont globalement révélé de forts niveaux de résistance au Téméphos et de faibles niveaux de résistance au Malathion. Les tests adulticides ont mis en évidence une résistance modérée des femelles à la Deltaméthrine. Les investigations moléculaires ont démontré des fréquences alléliques très élevées pour les mutations Kdr V1016I et F1534C connues pour être liées à la résistance aux pyréthrinoïdes. De plus, l'évaluation des niveaux d'expression constitutifs de certains gènes de détoxification a révélé des surexpressions significatives des populations testées par rapport à la souche sensible Bora-Bora, pour la carboxy-choline-estérase CCEAE3A, quatre cytochromes P450 à mono-oxygénases (014614, CYP6M11, CYP6BB2 et CYP9J23) et la glutathione-S-transférase GSTE2. / Guadeloupe is an endemic country for Dengue with epidemics every 2 to 3 years. In the past 3 years, other arboviruses have reached the Americas with Chikungunya virus in 2013 and Zika virus in 2015, causing major epidemics including in Guadeloupe. The only known vector of these diseases in Guadeloupe is the mosquito Aedes aegypti. As there is no vaccine nor specific treatment against these infections, prevention against these diseases is achieved through the monitoring and control of mosquito populations. Monitoring methods are based mostly on larval indices, with sometimes controversial results. In addition, vector control methods are based since a very long time on the massive use of chemical insecticides, causing mosquito resistance to these products. This work has therefore focused on two main areas to improve the prevention and control of these arboviruses: i) the search of a new vector population monitoring tool, based on the physiology of adult females and ii) the assessment of the resistance levels and mechanisms regarding three chemical insecticides, Temephos, Malathion (used in the past) and Deltamethrin (currently used). A vector population monitoring model based on females life expectancy as a function of parity rates and according to temperatures has being developed, and tracks on the entomological situations most at risk have emerged. Insecticide resistance tests performed on mosquito larvae have generally found strong Temephos resistance levels and low resistance to Malathion. Adulticide tests showed a moderate resistance of females to Deltamethrin. Molecular investigations have shown very high allelic frequencies for kdr mutations V1016I and F1534C, known to be associated with pyrethroid resistance. Moreover, the evaluation of constitutive expression levels of some detoxification genes revealed significant overexpression in tested Aedes aegypti populations compared to the susceptible Bora-Bora strain, for the carboxy-choline-esterase CCEAE3A, four cytochrome P450 mono-oxygenases (014614, CYP6M11, CYP6BB2 and CYP9J23) and the glutathione-S-transferase GSTE2.

Depuis 2007, le Gabon est régulièrement confronté à des infections par les virus Chikungunya (CHIKV) et Dengue (DENV). Au total, près de 4300 prélèvements provenant de patients se présentant avec un syndrome fébrile algique, en phase clinique aiguë, ont pu être collectés et analysés pour la période de 2007 à 2010. En effet, deux importantes épidémies concomitantes de CHIKV et de DENV ont sévi au Gabon (i.e. Libreville en 2007 et Franceville en 2010). Entre ces deux flambées épidémiques, de nombreux cas sporadiques d'infection à CHIKV ou à DENV ont continué à être enregistrés à travers le pays. Des cas de co-infection à CHIKV/DENV ont également été diagnostiqués lors des deux flambées épidémiques. Ces deux arbovirus se sont ainsi propagés en quelques années dans un mouvement de nord-ouest à sud-est à travers le pays. L’étude plus avancée des cas de co-infection à CHIKV/DENV a pu démontrer que ce phénomène pouvait survenir soit de manière simultanée soit séquentielle au cours du repas sanguin d'Aedes albopictus, principal vecteur du CHIKV et du DENV au Gabon.(…)En conclusion, ce travail de thèse décrit précisément la survenue brutale d’épidémies imputables à plusieurs arbovirus circulant simultanément au Gabon et responsables de nombreux cas cliniques se présentant sous la forme d'un syndrome fébrile algique. La co-circulation de ces virus suggère l’apparition d’une dynamique de type épidémique/endémique et implique un problème de santé publique latent dans cette région d’Afrique, voire dans l’ensemble de la sous-région d’Afrique Centrale. / Following to the first simultaneous Chikungunya (CHIKV) and Dengue (DENV) viruses outbreak in 2007, an active surveillance of febrile syndromes was set up in Gabon, a central African country. During a three-year period, we observed a rapid spread of CHIKV and DENV in a southward movement from north-west to south-east of the country. Indeed, CHIKV and DENV have disseminated within a non-immune population, widely favored by the extraordinary capacity of Aedes albopictus vector to colonize diverse environments and to replace local mosquito’s species. In 2010, a second outbreak occurred in Gabon with further CHIKV/DENV co-infections in both human and mosquito. This is the first documented evidence of co-infection in a wild-caught Aedes albopictus. Additionally, an underlying Zika (ZIKV) virus epidemic transmission by the same invasive vector was retrospectively recorded during the outbreak in 2007. These data reveal an unusual ZIKV natural life cycle, occurring in an urban environment and potentially representing a new arboviral emerging threat from Aedes albopictus.(…)In conclusion, these data highlighted the recent introduction and rapid dissemination of CHIKV and DENV in Gabon. The Aedes albopictus vector has shown its extraordinary capacity to sustain epidemic transmissions, leading to arboviral co-circulations (i.e. CHIKV, DENV-2, DENV-1, DENV-3, ZIKV) and notably to some CHIKV/DENV-2 co-infection cases. This multiple arboviral circulation suggests an epidemic/endemic dynamic in Gabon, involving a latent public health problem in this region of Africa.

Les arbovirus transmis par les moustiques Aedes représentent un problème majeur de santé publique dans les pays du Sud et certains comme la dengue et le chikungunya risquent d'émerger dans les pays du Nord. La lutte contre ces maladies repose essentiellement sur le contrôle des populations de vecteurs. Pour un meilleur contrôle de ces arbovirus, beaucoup d'efforts sont déployés pour développer de nouveaux outils. La mesure de la réponse anticorps (Ac) de l'homme contre les protéines salivaires des arthropodes a été utilisée pour évaluer son exposition aux piqûres des vecteurs et estimer le risque de transmission des pathogènes. L'objectif de notre travail a été de valider par une approche immuno-épidémiologique le concept « réponse Ac anti salive comme bio-marqueur d'exposition aux Aedes ». Nous avons également évalué la spécificité de cette réponse par rapport aux populations exposées uniquement à Ae. aegypti ou Ae. albopictus. Dans un second volet, nous avons identifié les protéines salivaires d'Ae. albopictus impliquées dans cette réponse. Et enfin, nous avons évalué la potentialité d'utiliser ce bio-marqueur comme critère d'efficacité de la lutte anti vectorielle contre Ae. albopictus. Nos résultats montrent une corrélation entre la réponse Ac anti salive et l'intensité d'exposition aux vecteurs indiquant ainsi la pertinence de son utilisation comme bio-marqueur d'exposition aux piqûres des Aedes. Nous notons une faible réaction croisée de cette réponse Ac entre la salive d'Ae. aegypti et d'Ae. albopictus. Les protéines salivaires antigéniques d'Ae. albopictus identifiées sont essentiellement impliquées dans la prise du repas sanguin. L'utilisation de ce bio-marqueur a permis de détecter la baisse de la densité vectorielle après les mesures de lutte contre Ae. albopictus, suggérant son utilité pour mesurer l'efficacité des stratégies de contrôle. L'ensemble de ces travaux contribuent à une meilleure connaissance de l'interaction de l'homme avec les Aedes. L'identification de protéines/peptides spécifiques d'espèce permettrait d'améliorer l'utilisation de ce bio-marqueur. / Aedes borne virus are considered to be public health problems in Southern countries while several such diseases like dengue and chikungunya threaten to emerge in the developed world. The control of these diseases is currently based on vector population control. Much effort is being devoted to develop new tools to control such arbovirus. Recent findings suggest that the evaluation of human antibody (Ab) response to arthropod salivary proteins is relevant to measure the level of human exposure to mosquito bites and to evaluate the risk of pathogen transmission. Using an immuno-epidemiological approach, the present study aimed to validate the concept “anti saliva Ab response, biomarker of Aedes exposure”. We evaluated the specificity of this Ab response according to populations only exposed to Ae. aegypti and Ae. albopictus, respectively. Ae. albopictus salivary proteins involved in this Ab response were also identified. We evaluated the usefulness of this biomarker for measuring the efficacy of Ae. albopictus control strategies. Our results showed a significant correlation between anti saliva Ab response and exposure level to vectors bites, thus indicating its usefulness as biomarker of Aedes exposure. We observed low Ab cross reactivity between Ae. aegypti and Ae. albopictus salivary gland extracts. The Ae. albopictus antigenic salivary proteins which were identified are mostly involved in blood feeding. The decrease of Ae. albopictus density after control measures has been detected by this biomarker, suggesting its usefulness for evaluating control strategies.This work contributes significantly to the study of human antibody response to Aedes salivary proteins which remains so far poorly documented. The identification of species specific salivary proteins/peptides should improve the use of this biomarker.

Mosquitos transgênicos incapazes de transmitir malária podem ser um controle alternativo, mas atualmente não estão disponíveis. O estudo da interação mosquitopatógeno é importante para melhorar o desenho de genes. A proteína circumsporozoita (CSP) tem dois domínios conservados que podem estar envolvidos na penetração dos esporozoítos na glândula salivar. Nosso objetivo foi expressar peptídeos contendo essas regiões na hemolinfa do mosquito usando o sistema de expressão transiente vírus Sindbis e a tecnologia de transgênese. Se a CSP está envolvida neste processo, os peptídeos competirão com spz impedindo a penetração. Cinco vírus sindbis e quatro linhagens transgênicas foram construídos e desafiados por P. gallinaceum. Nossos resultados mostram que os peptídeos não impediram a penetração de spz na glândula salivar, principalmente porque os peptídeos recombinantes não foram produzidos ou detectados. Aprimorar o desenho de genes, usando a otimização de códons e outras tecnologias, será essencial para a expressão de proteínas exógenas em mosquitos transgênicos. / Transgenic mosquitoes that impair malaria transmission can be an alternative control but currently an effective line is not available. A better understanding of mosquito interaction with pathogens is very important to improve refractory transgene design. Circumsporozoite protein (CSP) has two conserved domains that could be involved in spz penetration into mosquito salivary glands. Our aim was to express peptides encompassing these conserved regions in the mosquito hemolymph using Sindbis virus transient expression system and transgenesis technology. If CSP is involved in this process these peptides will compete with sporozoites impairing its penetration. Five Sindbis virus and four transgenic lines were constructed and challenged with P. gallinaceum. Our results showed these peptides could not impair sporozoites penetration in salivary glands, mainly because the recombinant proteins could not be produced or detected. Improving transgene design using codon usage and other technologies will be essential for expressing foreign proteins in transgenic mosquitoes.

A espécie Aedes scapularis é um dos culicídeos de grande importância médica. Está distribuída nas Américas e tem grande competência vetora para diversos arbovírus. No estado de São Paulo, há ocorrência de Ae. scapularis em vários municípios, como em Pariquera-Açu e São Paulo. O objetivo desse trabalho foi testar se há diferenciação genética - morfológica entre essas populações, podendo diagnosticar existência de espécies crípticas em Aedes scapularis. As populações estudadas foram Pariquera-Açu (PAR), Parque Ecológico do Tietê em São Paulo (PET) e Butantã (BUT). Os parâmetros utilizados foram: Morfometria geométrica da asa (forma e tamanho), estudo do gene mitocondrial COI, análise dos espaçadores internos transcritos ITS2 e análise morfológica de ovos. Com os resultados obtidos, podemos concluir que a divergência populacional é atestada por padrões geográficos de forma alar, gene mitocondrial COI e razão comprimento e largura dos ovos, mas extensões de estudos em outras áreas precisam ser feitos para poder atestar espécies crípticas em Aedes scapularis. / The species Aedes scapularis is a culicidae of medical importance. It is distributed in the Americas and has a high vector competence for many arboviruses. In state of São Paulo, have occurrence of Ae. scapularis in many cities, such as Pariquera-Acu and the city of São Paulo. The aim of this study was to determine differentiation genetic- morphology among these populations, being able to diagnose the existence of cryptic species in Aedes scapularis. The populations studied were Pariquera-Acu (PAR), the Tietê Ecological Park in Sao Paulo (PET) and Butantã (BUT). The parameters used were: wing geometric morphometry (shape and size), study of mitochondrial gene COI, analysis of internal transcribed spacers ITS2 and morphological analysis of eggs. With these results, we conclude that divergence population is attested by the geographical patterns of wing shape, and COI mitochondrial gene length and width ratio of eggs, but extensions of studies in other areas need to be made in order to attest cryptic species in Aedes scapularis.

Estudos populacionais de Aedes aegypti são de grande importância para o desenvolvimento de estratégias de controle, mas infelizmente o Estado de São Paulo carece desse tipo de estudo. Devido a esse contexto, o objetivo do trabalho foi avaliar a variabilidade genética e fenotípica de 4 populações de Ae. aegypti na área metropolitana de São Paulo. As análises interpopulacionais de forma em ambos os sexos mostraram variabilidade morfológica alar que, no entanto não indica estruturação populacional. Não foi observada correlação entre valores de distância fenética alar e distância geográfica para machos (r =-0,03) e fêmeas (r=0,34). Estruturação populacional foi detectada (Fst global=0,062) e baixo fluxo gênico (Nm=0,47) foi presumido nas quatro localidades. Foi observada correlação entre as distâncias genética e geográfica (r=0,76). Os dois marcadores populacionais aqui empregados aparentemente têm graus de resolução distintos para eventos microevolutivos, tendo sido DNA microssatélite ligeiramente mais sensível para acusar estruturação populacional. / Populational studies of Ae. aegypti are important to develop control strategies, but unfortunately the State of São Paulo lacks such studies. Despite this context, the objective of this work was to evaluate genetic and phenotypic variability of population samples of Ae. aegypti from four collecting sites in the metropolitan area of São Paulo city. The interpopulational analysis of shape for females and males showed morphological variability in wings, but did not indicate population structure. Pairwise fenetic distances among populations were not correlated to the geographic distances for males (r=-0.03) and females (r =0.34). Populational structure was detected (global Fst= 0.062) and low gene flow (Nm= 0.41) was presumed among four locations. Pairwise genetic distances among populations were correlated to the geographic distances (r = 0.76). The two populational markers used here apparently have different degrees of resolution for microevolutionary events, being that microsatellite DNA were slightly more sensitive in revealing populational structure.

Com o aumento progressivo do número de casos de infecção por diferentes arbovírus, por exemplo, dengue, zica e chikungunya, faz-se necessário o desenvolvimento de novas técnicas para o combate a esses arbovírus. A manipulação genética possibilitou a obtenção de mosquitos geneticamente modificados que sejam capazes de suprimir a população selvagem ou impedir a transmissão de agentes etiológicos gerando doenças. O estudo teve como objetivo o estabelecimento de linhagens para supressão populacional de Aedes aegypti. Esta construção apresenta esterilidade condicionada à presença ou ausência de antibiótico no meio em que esses mosquitos se desenvolvem durante a fase larval. Dessa forma, sem a necessidade de se utilizar radiação para obter insetos estéreis, é possível melhorar a qualidade dos machos adultos liberados e aumentar a competitividade dos mesmos em competir por fêmeas selvagens e adicionalmente gerar o desejado quadro de supressão populacional. / The increasing number of infection cases by arboviruses, for instance Dengue, Zika and Chikungunya, it is necessary to develop new techniques to fight against these arboviruses. Genetic manipulation allowed the production of genetically modified mosquitoes that are capable of suppressing the wild population or prevent pathogens transmission and avoid disease development. The project proposes to establish lines for population suppression of Aedes aegypti, one of the main vectors of those diseases. This construction presents the conditional sterility due to the presence or absence of antibiotics in the environment where these mosquitoes are developed during the larval stage. Thus, without the need to use radiation for sterile insects, it is possible to improve the quality of the released adult males and enhance the competitiveness thereof in competing with wild females and additionally generate population suppression.

O mosquito Aedes aegypti é vetor de doenças como a febre amarela, dengue, chinkungunya e zika. O sistema digestório é responsável pela digestão e absorção de nutrientes, é também uma interface com o ambiente externo sendo a porta de entrada de organismos infecciosos. A presença de duas lipases digestivas foi confirmada por qPCR, uma na fase larval (L-Aa7051) e outra na fase adulta (L-Aa7055). Estas enzimas foram agrupadas na família das lipases neutras e apresentam alterações em resíduos envolvidos na especificidade, domínio tampa e alça β9. A L-Aa7055 recombinante foi expressa heterologamente em Escherichia coli na porção insolúvel, com atividade após a renaturação. Observamos que a expressão da lipase L-Aa7055 sofre uma redução de 30% na infecção Plasmodium gallinaceum, não sendo afetada pelo vírus dengue sorotipo 2 (DENV2). A digestão de lipídeos é importante na fase larval, com altos níveis de transcrito. Um estudo mais aprofundado ainda será necessário para compreender completamente o papel das lipases no processo infeccioso. / The Aedes aegypti borne diseases yellow fever, dengue fever, chinkungunya and zica are important public healthy problems. The digestion and absorption of nutrients are performed in the digestive system, which is also an external environment interface that allows the infection by pathogenic microorganisms. The presence of two digestive lipases were identified by qPCR, L-Aa7051 in the larval phase and L-Aa7055 in the adult female. The lipase sequences were grouped in the neutral family, and exhibit alterations in residues involved in specificity, lid domain and β9 loop. The recombinant L-Aa7055 was expressed in the insoluble fraction, and show activity after a renaturation process. We notice that the expression levels of L-Aa7055 are reduced by 30% in the Plasmodium gallinaceum infection and were not affected by serotype 2 dengue virus (DENV2). The lipid digestion is important in the larval phase, with higher transcript levels. New studies will be necessary to the complete understanding of lipase contribution in the infectious process.

O mosquito Aedes aegypti é vetor de doenças como a febre amarela, dengue, chinkungunya e zika. O sistema digestório é responsável pela digestão e absorção de nutrientes, é também uma interface com o ambiente externo sendo a porta de entrada de organismos infecciosos. A presença de duas lipases digestivas foi confirmada por qPCR, uma na fase larval (L-Aa7051) e outra na fase adulta (L-Aa7055). Estas enzimas foram agrupadas na família das lipases neutras e apresentam alterações em resíduos envolvidos na especificidade, domínio tampa e alça β9. A L-Aa7055 recombinante foi expressa heterologamente em Escherichia coli na porção insolúvel, com atividade após a renaturação. Observamos que a expressão da lipase L-Aa7055 sofre uma redução de 30% na infecção Plasmodium gallinaceum, não sendo afetada pelo vírus dengue sorotipo 2 (DENV2). A digestão de lipídeos é importante na fase larval, com altos níveis de transcrito. Um estudo mais aprofundado ainda será necessário para compreender completamente o papel das lipases no processo infeccioso. / The Aedes aegypti borne diseases yellow fever, dengue fever, chinkungunya and zica are important public healthy problems. The digestion and absorption of nutrients are performed in the digestive system, which is also an external environment interface that allows the infection by pathogenic microorganisms. The presence of two digestive lipases were identified by qPCR, L-Aa7051 in the larval phase and L-Aa7055 in the adult female. The lipase sequences were grouped in the neutral family, and exhibit alterations in residues involved in specificity, lid domain and β9 loop. The recombinant L-Aa7055 was expressed in the insoluble fraction, and show activity after a renaturation process. We notice that the expression levels of L-Aa7055 are reduced by 30% in the Plasmodium gallinaceum infection and were not affected by serotype 2 dengue virus (DENV2). The lipid digestion is important in the larval phase, with higher transcript levels. New studies will be necessary to the complete understanding of lipase contribution in the infectious process.