La dengue, une des maladies les plus largement émergents actuellement, avec 390 millions d'infections chaque année (OMS), est causée par le virus de la dengue contre lequel il n’existe pas de traitements. La protéine NS5 a un rôle important dans le cycle de réplication. Cette protéine se compose d'une méthionine S-transférase d’adénosyl en N-terminal et une ARN polymérase dépendante de l'ARN (RdRp) en C-terminal. Cette NS5 RdRp peut catalyser non seulement la synthèse du brin négatif de l'ARN, utilisé comme matrice pour synthétiser l'ARN brin plus-supplémentaire, mais aussi pour la synthèse d'un ARN complémentaire à partir d'une matrice court e d'ARN sans amorce (de novo). Dans ce travail de thèse, nous présentons la production et le test de l'activité de la protéine NS5, ainsi que du domaine polymérase RdRp pour les quatre sérotypes du virus de la dengue en développant un nouveau test enzymatique, en utilisant comme un réactif fluorescent. L'utilisation de ce réactif fluorescent a également contribué à la détermination des conditions optimisées pour développer un essai de criblage de l'activité polymérase pour identifier des inhibiteurs contre le virus de la dengue. En outre, quatre flavonoïdes, Hinokiflavone, apigénine, la quercétine et Amentoflavone ont montré des valeurs d’IC50 équivalentes contre toutes les constructions NS5 et les domaines polymérase des quatre sérotypes. / Dengue fever, one of the most widely emerging diseases nowadays with 390 million infections each year (WHO), is caused by Dengue virus in which no official antiviral reagent or vaccine is available. The NS5 protein has an important role in the replication cycle. This protein consists of a S-adenosyl methionine transferase at N-terminal and a RNA dependent RNA polymerase (RdRp) at C-terminal. This NS5 RdRp can catalyse for not only synthesis of minus-strand RNA to be used as the template to synthesize additional plus-strand RNA but also synthesizing a complement RNA from a short RNA template without primer (de novo). In this research we present the production and activity test for NS5 protein and N-terminal extended sequence 266-900 from NS5 RdRp of all first four serotypes of Dengue virus and a construct of sequence 273-900 using a new enzymatic assay, using Picogreen as fluorescent reagent. Using this fluorescent reagent also helped determining the optimised conditions to develop a screening assay for inhibitors against dengue polymerase activity. In addition, four flavonoids, Hinokiflavone, Apigenin, Quercetin and Amentoflavone showed approximate IC50 values when testing on all NS5 and polymerase protein constructs of all four serotypes.

La dengue et le chikungunya sont deux arboviroses émergentes qui sont transmises à l'homme par la piqûre de moustiques du genre Aedes. Il n'existe ni vaccin ni traitements commercialisés pour ces arboviroses. Il apparaît donc nécessaire de développer de nouvelles stratégies pour isoler les virus circulants et bloquer leur transmission. La compréhension des mécanismes mis en jeu dans les cellules des vecteurs Aedes lors d'une infection par le virus de la dengue (DENV) sont encore très peu étudiés, notamment pour les sérotypes 1 et 3. Par des analyses protéomiques de l'infection d'une lignée cellulaire du moustique Aedes albopictus par ces séroytypes, nous avons démontré qu'en réponse à l'infection, les cellules de moustiques utilisent les mécanismes antioxydants combinés à la production d'énergie pour faire face au virus. Les résultats de notre étude devraient permettre de mieux comprendre l'interaction DENV-vecteur Aedes au niveau cellulaire dans le but de concevoir des stratégies efficaces pour le contrôle du DENV. Nous avons également regroupé dans une revue les connaissances acquises sur les études protéomiques des principaux compartiments des arthropodes vecteurs de maladies humaines. Dans un second volet, nous avons mis en évidence une méthode rapide d'isolement et de concentration des DENV et du chikungunya. Cette technique d'isolement basée sur la capture de virus sur des billes magnétiques enrobées de polymères anioniques permet d'obtenir des particules virales infectieuses. Cette méthode combinée à des approches classiques de détection de virus pourrait non seulement permettre l'identification des échantillons infectés ayant une faible charge virale, mais aussi l'isolement simultanée de particules infectieuses de dengue et de chikungunya à partir d'un seul échantillon. / Dengue (DENV) and Chikungunya (CHIKV) viruses are two emerging arboviruses that are transmitted to humans by the bite of Aedes sp. mosquito vectors. Neither vaccines, nor medical treatments, are commercially available for these infections. It is, therefore, necessary to elaborate novel strategies to isolate the circulating viruses and block their transmission.Our understanding of the molecular mechanisms involved, during the infection of the Aedes vector by dengue virus (DENV), especially serotypes 1 and 3, remains very scant. We, therefore, performed a proteomics analysis of an Aedes albopictus cell line, infected by these two DENV serotypes, and showed that the cells use both anti-oxidant and energy-production mechanisms in the fight against the virus. These results should help to improve our knowledge of the interaction of the DENV virus and the Aedes mosquito vector, at the cellular level, with the aim of designing efficient strategies for the control of this virus. We have, in addition, developed a rapid and sensitive isolation technique, based on viral particle adsorption to magnetic beads coated with an anionic polymer. The use of this technique is of great interest, as it permits the rapid and simultaneous detection and isolation of CHIKV and DENV from samples with reduced viral loads.

Dans cette thèse, nous présentons deux modèles mathèmatiques qui simulent la dynamique d'une épidémie de dengue à Cuba. Le premier modèle est décrit par un système d'équations différentielles ordinaires dans le but d'estimer certains paramètres essentiels dans l'évolution de l'èpidérnie. Le deuxième modèle utilise une inclusion différentielle afin de simuler la dynamique sous incertitudes. Nous passons aussi en revue une famille de modèles d'équations différentielles pour l'épidémie de VIH/SIDA à Cuba et nous reformulons l'un de ces modèles en une inclusion différentielle. En outre, pour analyser le comportement de ces deux épidémies et l'effet de l'intervention nous développons un algorithme pratique pour le calcul des ensembles atteignables des inclusions différentielles, qui fournit un cadre mathématique nous permettant de faire des prédictions appropriées de toutes les variables d'état des modèles / Ln this work we present two mathematical models that simulate the dynamics of an epidemic outbreak of dengue disease in Cuba. The i first model is described by a system of ordinary differential equations with the purpose of estimaling some essential parameters in the evolution of the epidemie. The second model uses a differential inclusion in order to simulate the dynamic under uncertainties. Also, we review a family of ordinary differential equations models of HIV/AIDS epidemic in Cuba and one of these models is reformulated as a differential inclusion. In addition, to analyze the behavior of these two epidemies and the effect of intervention, we develop a practical algorithm for the computation of the reachable sets of the differential inclusions, providing a mathematical framework allowing us to make suitable predictions of aII state variables of the models at a future time.

La dengue est une préoccupation mondiale majeure en santé publique, touchant principalement les jeunes enfants en Asie du sud est. Les vaccins contre la dengue sont actuellement en développement. Des études coût-efficacité seront nécessaires en cas d'introduction d'un nouveau vaccin. Pour cela, il est indispensable d’obtenir des informations sur le coût économique lié à la dengue (qui sont relativement rares) et des données précises sur l'incidence de la maladie, ceci afin de fournir des estimations solides quant à l'importance de la dengue dans les régions endémiques.Ce travail de thèse propose une approche méthodologique par laquelle le Ministère de la Santé devrait envisager lors de l'évaluation de la pertinence et de la faisabilité de l'adoption d’un vaccin contre la dengue par les programmes de nationaux de vaccination, particulièrement dans les pays en voie de développement ; le Cambodge par exemple. Nous avons mis en place des études prospectives d'incidence de la dengue sur plusieurs années, réalisées dans la population générale, incluant un large éventail de groupes d'âge. Les résultats de ces études ont été comparés avec les données de surveillance nationale réunis dans la même région en calculant des facteurs de multiplication afin d’estimer la sous-déclaration. D'autres études de coûts de la maladie ont été menées pour estimer le poids financier la dengue sur plusieurs années et d'identifier les groupes vulnérables. Cette approche a également permis de mieux comprendre l'épidémiologie de la dengue dans le pays, dont certaines caractéristiques sont nécessaires pour évaluer la faisabilité de l'introduction d’un nouveau vaccin contre la dengue. Les résultats rapportés dans cette thèse vont permettre (1) d’initier à partir des données cambodgiennes des simulations de coût-efficacité à partir des caractéristiques attendues du vaccin et (2) de développer un modèle dynamique afin de permettre aux autorités nationales d'analyser les études de coût-efficacité en tenant compte des spécificités immunologiques et épidémiologiques des pays concernés. Si un vaccin efficace et fiable est disponible à un prix abordable, son adoption par le programme national de vaccination serait probablement rentable pour le Cambodge mais répondrait surtout à un problème d'inéquité sociale. / Dengue is a major public health concern worldwide, particularly in young children in Southeast Asia. Dengue vaccines are currently in development and policymakers need appropriate economic studies to determine their potential financial and public health impact. Alongside economic assessment, accurate disease incidence data are required to provide robust estimates of disease burden across the regions where dengue is endemic. This thesis proposes a methodological approach by which the Ministry of Health should consider when assessing feasibility of adoption dengue vaccines, with specific reference to developing country settings, e.g. Cambodia. We implemented prospective dengue incidence studies over several years, performed in a general population and include a wide range of ages. The results of these studies were compared with national surveillance data gathered in the same region by calculating multiplication factors to estimate underreporting on several years. Additional prospective cost of illness study was conducted to generate accurate economic burden of disease estimates over several years and identify vulnerable groups. This strategy also allowed better understanding of the epidemiology of dengue in Cambodia, of which some characteristics are needed to evaluate the feasibility of introducing dengue vaccine. Designing cost-effectiveness studies before a vaccine has been fully evaluated requires assumptions about variables such as efficacy and effectiveness, dosage and costs. Our next step will be to determine the threshold price – sensitivity analyses - for a vaccine to be cost-effective rather than assigning a specific cost, because it is difficult to predict cost for a vaccine that has not been marketed. Should this vaccine be safe and affordable enough, the adoption of the new vaccine into a National Immunization Programme would probably be cost-effective but, above all, a matter of social equity.

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.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2015-12-09T14:33:14Z No. of bitstreams: 1 tatianeribeirodesiqueira.pdf: 1746220 bytes, checksum: 01a239ee1ea9876e3d413b78f8f1446b (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2015-12-09T14:56:33Z (GMT) No. of bitstreams: 1 tatianeribeirodesiqueira.pdf: 1746220 bytes, checksum: 01a239ee1ea9876e3d413b78f8f1446b (MD5) / Made available in DSpace on 2015-12-09T14:56:33Z (GMT). No. of bitstreams: 1 tatianeribeirodesiqueira.pdf: 1746220 bytes, checksum: 01a239ee1ea9876e3d413b78f8f1446b (MD5) Previous issue date: 2015-02-26 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A infecção por dengue vírus (DENV) tem sido considerada atualmente a mais importante arbovirose no mundo. Até o presente momento foram descritos quatro diferentes sorotipos do DENV: DENV-1, DENV-2, DENV-3 e DENV-4. A infecção pelo DENV pode produzir uma ampla variedade de distúrbios a febre do dengue ou formas graves da doença, como a febre hemorrágica do dengue (FHD) e síndrome do choque do dengue (SCD), onde os quatro sorotipos de DENV podem causar desde uma infecção assintomática até FHD e SCD. A patogênese da FHD/SCD é multifatorial e vários estudos mostram algumas hipóteses para explicar as manifestações mais graves na infecção por DENV: (i) fatores virais, (ii) associação entre FHD/SCD em casos de infecção heterotípica pelo DENV e (iii) fatores do próprio hospedeiro, que poderiam estar relacionados à resposta imune. Estudos que buscam entender o porquê de pacientes com dengue apresentarem diferentes prognósticos são de grande importância para a Saúde Pública. Embora a doença causada por DENV seja considerada um grande problema de saúde pública, ainda não estão disponíveis drogas antivirais e vacinas a fim de tratar ou prevenir a infecção. O combate do vetor tem sido ineficiente, permitindo o aparecimento de novas epidemias. A cidade de Juiz de Fora vem passando por diversas epidemias de dengue nos últimos anos, com o registro de casos graves e óbitos. Diante deste contexto, este trabalho visou investigar os fatores sorológicos e genéticos relacionados com a predisposição ao desenvolvimento das formas graves de dengue em Juiz de Fora. Em setembro e outubro de 2013 e fevereiro e maio de 2014, amostras de sangue total foram coletadas. As amostras foram estudas para estudos de SNPs, pequisa de anticorpos e do DENV. No grupo estudado, foi observada uma soroprevalência de 16,1%. Foram detectados genótipos predisponentes e genótipos protetores de FHD, nos genes FCRIIa, JAK-1 e DCSIGN, em moradores de Juiz de Fora, entretanto, não foi observada associação desses genótipos individualmente e/ou em combinação com a distribuição de gênero, diferentes regiões de Juiz de Fora onde os participantes residiam e relato de apresentação de sintomas de dengue pelos pacientes. Foram detectados três pacientes que apresentaram material genético de DENV. O conhecimento de áreas e pessoas predispostas à FHD constituem informações valiosas do ponto de vista epidemiológico e na estruturação de políticas públicas que visem o controle da dengue. / Dengue virus infection (DENV) is considered the most important arbovirose in the world and the greatest impact on public health. Four different serotypes of DENV: DENV-1, DENV-2, DENV-3 and DENV-4 have been described. DENV infection may produce a wide variety of disorders, dengue fever or severe disease, such as dengue hemorrhagic fever (DHF) and dengue shock syndrome (SCD). Any of the four DENV can cause asymptomatic infection from one to DHF and DSS. The pathogenesis of DHF / DSS is multifactorial and several studies showed some hypotheses to explain the most serious manifestations of infection by DENV: (i) viral factors (ii) secondary infection DENV and (iii) the host factors could be related to exaggerated immune response. Studies trying to understand why dengue patients have different prognoses are of great importance for public health. Although dengue is considered one major public health problem, there are not available antiviral drugs and vaccines to treat or prevent the infection. The vector control has been inefficient, allowing the emergence of new outbreaks. The city of Juiz de Fora has experienced several dengue epidemics in recent years, with the record of serious cases and deaths. Given this context, this study aimed to investigate the serological and genetic factors related to the predisposition to severe forms of dengue in Juiz de Fora. In September/ October 2013 and February to May 14 342 samples of whole blood were collected were collected. Samples were used to investigate the immune response do dengue, SNPs and DENV. In the study group, a seroprevalence of 16.1% was observed. Predisponent and protector genotypes were detected genes FCyRIIa, JAK-1 and DCSIGN in Juiz de Fora residents, however, there was no association of these genotypes individually and / or in combination with the gender distribution, different Juiz de Fora regions where the participants lived and report presentation of dengue symptoms by patients. Three patients were detected with DENV infection, by the time of sample collection.The knowledge of areas and persons who are more prone to have FHD is a valuable information from the epidemiological point of view and the structuring of public policies aimed at controlling dengue.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2016-01-29T12:34:38Z No. of bitstreams: 1 jerusabotelhosouza.pdf: 1803631 bytes, checksum: e5453b5a95de704649e79eb74037206d (MD5) / Rejected by Adriana Oliveira (adriana.oliveira@ufjf.edu.br), reason: Adicionar instituição on 2016-02-01T15:45:28Z (GMT) / Submitted by Renata Lopes (renatasil82@gmail.com) on 2016-02-01T16:11:51Z No. of bitstreams: 1 jerusabotelhosouza.pdf: 1803631 bytes, checksum: e5453b5a95de704649e79eb74037206d (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2016-02-01T20:10:36Z (GMT) No. of bitstreams: 1 jerusabotelhosouza.pdf: 1803631 bytes, checksum: e5453b5a95de704649e79eb74037206d (MD5) / Made available in DSpace on 2016-02-01T20:10:36Z (GMT). No. of bitstreams: 1 jerusabotelhosouza.pdf: 1803631 bytes, checksum: e5453b5a95de704649e79eb74037206d (MD5) Previous issue date: 2014-02-14 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A dengue é a arbovirose de maior importância para a saúde pública no Brasil. A cidade de Juiz de Fora vem passando por diversas epidemias de dengue nos últimos anos, com o registro de casos graves e óbitos. Diante deste contexto, este trabalho visou investigar a circulação de Dengue virus (DENV) em Aedes aegypti coletados do ambiente e em amostras clínicas de pacientes com suspeita de dengue por meio de técnicas moleculares e sorológicas. Amostras de Aedes aegypti (larvas e mosquitos) e soro de pacientes passaram pelo processo de extração de RNA total e este material foi utilizado para detecção de DENV por RT-PCR. Amostras clínicas de pacientes que apresentavam até seis dias de febre foram testadas para a proteína NS1 para Dengue virus. Além disso, foi realizada a análise das fichas dos pacientes nas quais foi possível verificar o dia de início da febre, os dados hematológicos (hematócrito, leucometria global e contagem de plaquetas) e sorológicos (IgM e/ou IgG). Aedes aegypti foi coletado em todas as regiões da cidade e dos 163 pools de larvas analisados, o DENV foi detectado em seis (cinco pools positivos para DENV-1 e um pool positivo para DENV-2). Estes pools haviam sido coletados nas regiões Norte, Centro e Sul. Das 166 amostras clínicas analisadas na detecção molecular, seis foram positivas (uma amostra positiva para DENV-1 e cinco amostras positivas para DENV-2). Sessenta amostras clínicas foram testadas para NS1 e 11 (18,33%) foram positivas. A partir da análise das fichas dos pacientes foi possível observar que 132 apresentavam resultados de sorologia. Destes, 39/132 (29,54%) apresentaram IgM, 9/132 (6,81%) apresentaram IgG e 15/132 (11,36%) apresentaram IgM e IgG. Além disso, 109 pacientes mostraram dados hematológicos. Entre estes, 18 (16,51%) pacientes apresentaram hematócrito inferior ao valor de referência, 32 (29,35%) desenvolveram leucometria abaixo de 3.500/mm3 e 24 (22,02%) tiveram contagem de plaquetas inferior ao valor de referência. Este é o primeiro estudo de investigação molecular de DENV em Juiz de Fora e os resultados indicaram a cocirculação dos sorotipos 1 e 2 em Juiz de Fora e a ocorrência da transmissão transovariana de DENV no Aedes aegypti. A combinação dos testes moleculares e sorológicos permitiu a identificação de 50 pacientes na fase aguda da doença e 22 na fase de convalescença. O conhecimento dos tipos virais circulantes no município e o diagnóstico da dengue em pacientes por mais de um teste e/ou parâmetro laboratorial constituem informações valiosas do ponto de vista epidemiológico e na estruturação de políticas públicas que visem o controle da dengue. / Dengue is the most important arboviral disease to public health in Brazil. The city of Juiz de Fora has undergone several dengue epidemics in recent years, with the record of severe cases and deaths. Given that, this work aimed to investigate the circulation of dengue virus (DENV) in Aedes aegypti collected from the natural environment and in clinical samples from patients with clinical symptoms of dengue, using molecular and serological approaches. Samples of Aedes aegypti (larvae and mosquitoes) and serum of patients were used for total RNA extraction, and the total RNA was used for detection of DENV by RT-PCR. Clinical samples from patients who had fever for up to six days were tested for Dengue virus NS1 protein. Furthermore, analysis of patient records was performed in which we could check the day the fever began the hematological data (hematocrit, total leukocyte count, and platelet count) and serologic response (IgM and/or IgG). Aedes aegypti was collected in all regions of the city and from the 163 larvae pools that were analyzed, DENV was detected in six (five pools DENV-1 positive and one pool DENV-2 positive). These pools were collected in the North, Centre and South regions. A total of 166 serum samples were analyzed for molecular detection, being six DENV positive (one serum sample was DENV-1 positive and five serum samples were DENV-2 positive). Sixty clinical samples were tested for NS1 and 11 (18.33%) were positive. From the analysis of patient records was possible to observe that 132 had serology results. Of these, 39/132 (29.54%) were IgM positive, 9/132 (6.81%) were IgG positive and 15/132 (11.36%) were IgM and IgG positive. In addition, from 109 patients hematological data were available. Among these, 18 (16.51%) patients had less than the reference value, 32 (29.35%) developed leukocyte hematocrit below 3.500/mm3 and 24 (22.02%) had platelet counts below the reference value. This is the first study of molecular investigation of DENV in Juiz de Fora, and the results indicated the co-circulation of serotypes 1 and 2 in Juiz de Fora and the occurrence of DENV transovarial transmission in Aedes aegypti. The combination of molecular and serological tests allowed the identification of 50 patients in the acute phase of the disease and 22 in the convalescent phase. The knowledge of viral types circulating in the municipality and the diagnosis of dengue in patients using different techniques constitute valuable information from an epidemiological point of view and help to structure public policies aiming the control of dengue.

Dengue é uma doença infecciosa não contagiosa causada pelo vírus da dengue (gênero Flavivirus, família Flaviviridae), transmitida pela picada de artrópodes do gênero Aedes, principalmente Aedes aegypti, e sendo atualmente um importante problema de saúde pública em todo o mundo. Segundo a Organização Mundial de Saúde, cerca de 50 a 100 milhões de pessoas se infectam anualmente em mais de 100 países de todos os continentes. A dengue apresenta-se em três formas clínicas principais; doença febril indiferenciada, febre clássica do dengue (DF) e dengue hemorrágico com ou sem choque (DHF/DSS). Recentemente, viu-se um dramático aumento do número de casos de DHF/DSS nas Américas, e este aumento coincidiu com a introdução do dengue tipo 3, genótipo III. Neste trabalho, caracterizamos completamente o genoma de um vírus brasileiro de dengue tipo 3 (D3BR/RP1/2003) isolado de um paciente com dengue clássica. O genoma viral possui um tamanho de 10707 nucleotídeos e contém uma única região codificadora (posição 95 a 10264) flanqueada por duas regiões não codificadoras (RNC5, 1 a 94; RNC3, 10268 a 10707). A comparação do genoma viral com outros vírus isolados de pacientes com DF e DHF não mostrou diferenças significativas que sugerissem a presença de um fator genético associado à virulência. A analise filogenética baseada no genoma completo, mostra que a cepa D3BR/RP1/2003 pertence ao genótipo III e encontra-se proximamente relacionada a outro vírus isolado no Rio de Janeiro em 2002. Entretanto, quando essa análise filogenética é realizada com as regiões codificadoras das proteínas E e NS5 individualmente, a cepa D3BR/RP1/2003 encontra-se mais proximamente relacionada a um vírus isolado na Ilha de Martinica no Caribe. Este achado sugere que o isolado D3BR/RP1/2003 pode ter sido originado através de um evento de recombinação entre duas cepas virais distintas antes ou após sua introdução no Brasil. Dados sugestivos de recombinação foram observados também quando analisada a relação filogenética entre vírus isolados na Ásia. / Dengue is an infectious disease caused by dengue virus (genus Flavivirus, family Flaviviridae), transmitted by the bite of arthropods of the Aedes genus, mainly Aedes aegypti, and being currently an important problem of public health worldwide. According to the World Health Organization, about 50 the 100 million people are infected annually in more than 100 countries of all continents. Dengue can be presented in three main clinical forms; undifferentiated febrile illness, classic dengue fever (DF) and hemorrhagic dengue fever with or without shock (DHF/DSS). Recently, a dramatically increase of DHF/DSS cases in the Americas have been seen, and this increase coincided with the introduction of the dengue type 3, genotype III. In this work, we have completely characterized the genome of a Brazilian dengue type 3 (D3BR/RP1/2003 strain) isolated from a DF patient. The viral genome possesses a size of 10707 nucleotides and has a unique open reading frame (position 95 to 10264), flanked by two untranslated regions (UTR5\', 1 to 94; UTR3\', 10268 to 10707). The comparison of the viral genome with other viruses isolated from patients with DF and DHF did not show significant differences to suggest the presence of a genetic factor associate with virulence. Phylogenetic analysis based on the complete genome showed that D3BR/RP1/2003 strain belongs to genotype III and is close related to another virus isolated in Rio de Janeiro in 2002. However, when the phylogenetic analysis was based on the individual coding regions of E and NS5 proteins, D3BR/RP1/2003 strain was closely related to a virus isolated in the Island of Martinique in the Caribbean. This finding suggests that D3BR/RP1/2003 strain could have been originated through an event of recombination between different virus strains before or after its introduction to Brazil. Data supporting recombination events between viruses isolated in Asia have also been observed.

A dengue é uma doença infecciosa causada pelo vírus da dengue (DENV) e transmitida principalmente pela picada de mosquitos Aedes aegypti. A dengue é a doença viral transmitida por artrópodes de maior importância em saúde pública, afetando principalmente a países tropicais e subtropicais do mundo. As epidemias de dengue têm aumentado consideravelmente nos últimos anos em todo o mundo e as fronteiras de circulação do vírus vem se expandido constantemente. Assim, estudos de diferentes aspectos da doença e do vírus são de grande importância para aperfeiçoar os conhecimentos sobre esta ameaça. Neste sentido, é importante que algumas análises, como as filogenéticas e evolutivas dos vírus sejam realizadas para identificação dos genótipos circulantes, a origem dos mesmos, o relacionamento com outros subtipos e a evolução sofrida ao longo do tempo. Este estudo teve por objetivo analisar o relacionamento filogenético e evolutivo dos DENV isolados em Ribeirão Preto entre 2010 e 2011. Amostras de soro (n=79) de pacientes com dengue estocadas a -80ºC foram inoculadas em células C6/36 para tentativa de isolamento viral, o qual foi confirmado a partir de 39 amostras por imunofluorescência indireta e/ou RT-PCR em tempo real. Sequenciamento de parte do gene da proteína viral NS5 ou do gene da proteína E mostrou que 25 pertenciam ao DENV-1, seis ao DENV-2 e oito ao DENV-3. Para as análises filogenéticas e evolutivas, o gene da proteína E de 15 DENV-1, quatro DENV-2 e um DENV-3 foi sequenciado. Estas análises foram realizadas também utilizando toda a região codificadora de dois DENV-1, um DENV-2 e um DENV-3. As análises mostraram que todos os vírus introduzidos em Ribeirão Preto foram provenientes de vírus originados no Estado do Rio de Janeiro. Duas linhagens de DENV-1, uma de DENV-2 e uma de DENV-3 circularam em Ribeirão Preto entre 2010 e 2011. O relacionamento filogenético dos vírus foi similar independentemente do uso da sequência do gene da proteína E ou de toda a região codificadora. / Dengue is an infectious disease caused by dengue virus (DENV) and transmitted mainly by Aedes aegypti mosquitoes. Dengue is the viral disease transmitted by arthropods of greater importance in public health, particularly affecting the tropical and subtropical countries of the world. Dengue epidemics have increased considerably in recent years throughout the world and the borders of virus circulation have been expanding constantly. Thus studies of different aspects of the disease and the virus are of great importance to improve knowledge about this threat. In this sense, it is important that some analyzes such as phylogeny and evolution be carried out to identify the circulating genotypes, their origin, their relationship with other subtypes and evolution along the time. This study aimed to analyze the phylogenetic and evolutionary relationships of DENV isolated in Ribeirão Preto between 2010 and 2011. Serum samples (n = 79) of dengue patients stored at -80°C were inoculated into C6/36 cells for virus isolation attempts, which was confirmed from 39 samples by indirect immunofluorescence and/or real-time RT-PCR. Sequencing of part of the viral NS5 gene protein or the E gene protein showed that 25 belonged to DENV-1, six to DENV-2 and eight to DENV-3. For the phylogenetic and evolutionary analyzes, the E gene protein of 15 DENV-1, four DENV-2 and one DENV-3 was sequenced. These analyzes were also carried out with the entire coding region of two DENV-1, one DENV-2 and one DENV-3. The analysis showed that all viruses were introduced in Ribeirão Preto from viruses originated in the state of Rio de Janeiro. Two lineages of DENV-1, one of DENV-2 and one of DENV-3 circulated between 2010 and 2011. The phylogenetic relationship of these viruses was similar regardless of the use of the E gene protein or the entire coding region sequences.

Células dendríticas são importantes na interação dos sistemas imune inato e adaptativo, apresentando antígenos para linfócitos T CD4+ e CD8+ e induzindo respostas adaptativas contra estes. Elas são alvo de interesse no desenvolvimento de novas estratégias vacinais que visam o direcionamento de antígenos para as diferentes populações de DCs in vivo. Consiste na administração de anticorpos contra receptores da superfície da DC em fusão com o antígeno. Nós comparamos o direcionamento da proteína NS1 de DENV2 para as duas principais populações de DCs no baço, na presença de diferentes agonistas. Poly I:C é capaz de induzir as respostas de Linfócitos T CD8+ mais robustas, mas somente quando o antígeno é direcionado para a população de DCs CD8+DEC205+. Com CpG observamos resposta de células T CD8+ equivalente quando a proteína foi direcionada para qualquer uma das populações estudadas. Direcionamento com Poly I:C gerou proteção contra desafio por DENV2 quando NS1 é direcionada para a população de DCs DEC205+, sendo esta proteção dependente de Linfócitos T CD8+ e CD4+. / Dendritic Cells are critical in the interaction between innate and the adaptive immune system, presenting antigens to T lymphocytes and inducing adaptive immune responses against these. DCs are target for development of new vaccine strategies based on targeting antigens to different DCs populations in vivo. This consists on the administration of antibodies specific to DC surface endocytic receptor fused to an antigen. In this study, we evaluated the differences in adaptive responses when the NS1 protein from the DENV2 was directed to the two main populations of DCs in the spleen along with different agonistic molecules. Highest CD8+ T cell responses were seen when Poly I:C was used, but only when directed to the CD8+DEC205+ DC population. In the presence of agonist CpG we observed similar responses when the protein was targeted to either CD8+DEC205+ or CD8-DCIR2+ DCs. In the presence of Poly I:C, only targeting to the DEC205+ DCs was able to protect mice against a challenge with DENV2. This protection was dependent on both CD8+ and CD4+ T cell responses.