Caracterização genética e evolução dos vírus dengue no Estado do Rio Grande do Norte

Dengue is considered as the most important arthropod-borne viral disease throughout the world due to the high number of people at risk to be infected, mainly in tropical and subtropical regions of the planet. The etiologic agent is Dengue Virus (DENV), it is a single positive-stranded RNA virus of the family Flavivirus, genus Flaviviridae. Four serotypes are known, DENV-1, DENV-2, DENV-3 and DENV-4. One of the most important characteristic of these viruses is the genetic variability, which demands phylogenetic and evolutionary studies to understand key aspects like: epidemiology, virulence, migration patterns and antigenic characteristics. The objective of this study is the genetic characterization of dengue viruses circulating in the state of Rio Grande does Norte from January 2010 to December 2012. The complete E gene (1485 pb) of DENV1, 2 e 4 from Brazilian (Rio Grande do Norte) patients was sequenced. Phylogenetic analysis was performed using MEGA 5.2 software, Tamura-Nei model and Neighbor-Joining trees were inferred for the datasets. In Brazil, there is just one DENV-1 genotype (genotype V), one DENV-2 genotype (Asian/American) and two DENV-4 genotypes (genotypes I and II). Brazilian strains of DENV-1 are subdivided in two different lineages (BR-I and BR-II), the Brazilian strains of DENV-2 are subdivided in four lineages (BRI-IV) and genotype II of DENV-4 is subdivided in three Brazilian lineages (BRI-III). The viruses isolated in RN belong to lineage BR-II (DENV-1), BR-IV (DENV-2) and BR-III (DENV-4).The Caribbean and near Latin American countries are the main source of these viruses to Brazil. Amino acids substitutions were detected in three domains of E protein, this makes clear the necessity of studies that associate epidemiological and molecular data to better understand the effects of these mutations. This is the first study about genetic characterization and evolution of Dengue viruses in Rio Grande do Norte, Brazil

Caracterização da resposta imunológica de Aedes aegypti e Culex quinquefasciatus à infecção pelo sorotipo 1 do Dengue vírus

Os insetos podem atuar como pragas agrícolas e vetores de patógenos causadores de doenças ao homem e outros animais. Investigações a respeito do sistema imunológico de Ae. aegypti e Cx. quinquefasciatus poderão contribuir para o desenvolvimento de métodos de controle das doenças veiculadas por estes insetos, principalmente a dengue, enfermidade causadora de sério problema de saúde pública no mundo. Apesar de Ae. aegypti ser a única espécie vetora confirmada na transmissão do vírus Dengue no Brasil, considera-se também importante um melhor entendimento dos mecanismos imunológicos de Cx. quinquefasciatus tido como refratário ao vírus. Neste estudo foram utilizadas linhagens de Ae. aegypti e Cx. quinquefasciatus mantidas no Insetário do Departamento de Entomologia do CPqAM/FIOCRUZ. Três grupos experimentais de fêmeas com 10 dias de idade foram formados para cada espécie. Grupo I, composto por fêmeas alimentadas com solução sacarose (10 por cento); grupo II, fêmeas alimentadas com sangue limpo e grupo III, fêmeas alimentadas com sangue infectado com o sorotipo DENV-1. De cada grupo foram obtidos hemolinfa, glândula salivar, intestino médio e corpo gorduroso para avaliação da expressão dos antimicrobianos defensina e transferrina. Essa avaliação foi realizada através de PCR em Tempo Real utilizando o kit QuantiFast SYBR Green - One-Step qRT-PCR. A avaliação da hemodinâmica foi realizada utilizando 10 microlitros de hemolinfa de cada grupo, através da contagem das células em câmara de Neubauer. Nossos resultados demonstraram que o Cx. quinquefasciatus tem um maior aumento da expressão de defensina e um maior número total de hemócitos quando infectados com DENV-1 em relação ao Ae. aegypti e a transferrina teve sua expressão alterada somente no Ae. aegypti. Em ambas as espécies estudadas, apenas a alimentação sanguínea não interfere na produção de hemócitos ou quanto na indução de defensina e transferrina. Esses dados sugerem que fêmeas de Cx. quinquefasciatus parecem apresentar uma resposta imune celular e humoral mais intensa do que Ae. aegypti quando infectados com DENV-1

Indicadores entomológicos dos levantamentos rápidos de infestação de Aedes aegypti e ocorrência de dengue na cidade de Penápolis, São Paulo Brasil. 2006-2010

Pós-graduação em Ciência Animal - FMVA

Desenvolvimento de marcadores SNPs (Single Nucleotide Polymorphisms) para estudos populacionais do mosquito Aedes aegypti no Brasil

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Índices climáticos e infestação predial por larvas de Aedes aegypti (Linnaeus, 1762) e Aedes albopictus (Skuse, 1894) em Araçatuba-sp Brasil

Aedes is synanthropic; associated with climatic conditions and urban environment. This study performed a retrospective evaluation of the climatic indices and the A. aeypti and A. albopictus infestation larvae rates (IP), registered from the month of October, 2005 to 2009, in the Araçatuba city, São Paulo State. There was no variation in the average temperature in the period (26.3°C). The rain falls were high in 2005 (133.5 mm3 ) fell in 2006, 2007 and 2008 (55.0 mm3 , 78.5 mm3 and 79.0 mm3 , respectively) and increased in 2009 (104.0 mm3 ). The infestation IP was 1.10, 1.39, 0.36, 0.28 and 3.30, respectively in 2005, 2006, 2007, 2008 and 2009, none of them were A. albopictus larvae. There was no association between IP and temperature, but there was significant difference (P <0.001) between the IP of 2009, with the IP of 2007 and 2008. This study it was concluded that among the environmental factors just rain influenced the rate of larvae, "infestation", committed only by A. aegypti.

The wMel Wolbachia strain blocks dengue and invades caged Aedes aegypti populations

Dengue fever is the most important mosquito-borne viral disease of humans with more than 50 million cases estimated annually in more than 100 countries. Disturbingly, the geographic range of dengue is currently expanding and the severity of outbreaks is increasing. Control options for dengue are very limited and currently focus on reducing population abundance of the major mosquito vector, Aedes aegypti. These strategies are failing to reduce dengue incidence in tropical communities and there is an urgent need for effective alternatives. It has been proposed that endosymbiotic bacterial Wolbachia infections of insects might be used in novel strategies for dengue control. For example, the wMelPop-CLA Wolbachia strain reduces the lifespan of adult A. aegypti mosquitoes in stably transinfected lines. This life-shortening phenotype was predicted to reduce the potential for dengue transmission. The recent discovery that several Wolbachia infections, including wMelPop-CLA, can also directly influence the susceptibility of insects to infection with a range of insect and human pathogens has markedly changed the potential for Wolbachia infections to control human diseases. Here we describe the successful transinfection of A. aegypti with the avirulent wMel strain of Wolbachia, which induces the reproductive phenotype cytoplasmic incompatibility with minimal apparent fitness costs and high maternal transmission, providing optimal phenotypic effects for invasion. Under semi-field conditions, the wMel strain increased from an initial starting frequency of 0.65 to near fixation within a few generations, invading A. aegypti populations at an accelerated rate relative to trials with the wMelPop-CLA strain. We also show that wMel and wMelPop-CLA strains block transmission of dengue serotype 2 (DENV-2) in A. aegypti, forming the basis of a practical approach to dengue suppression.

Impact of Wolbachia on infection with chikungunya and yellow fever viruses in the mosquito vector Aedes aegypti

Incidence of disease due to dengue (DENV), chikungunya (CHIKV) and yellow fever (YFV) viruses is increasing in many parts of the world. The viruses are primarily transmitted by Aedes aegypti, a highly domesticated mosquito species that is notoriously difficult to control. When transinfected into Ae. aegypti, the intracellular bacterium Wolbachia has recently been shown to inhibit replication of DENVs, CHIKV, malaria parasites and filarial nematodes, providing a potentially powerful biocontrol strategy for human pathogens. Because the extent of pathogen reduction can be influenced by the strain of bacterium, we examined whether the wMel strain of Wolbachia influenced CHIKV and YFV infection in Ae. aegypti. Following exposure to viremic blood meals, CHIKV infection and dissemination rates were significantly reduced in mosquitoes with the wMel strain of Wolbachia compared to Wolbachia-uninfected controls. However, similar rates of infection and dissemination were observed in wMel infected and non-infected Ae. aegypti when intrathoracic inoculation was used to deliver virus. YFV infection, dissemination and replication were similar in wMel-infected and control mosquitoes following intrathoracic inoculations. In contrast, mosquitoes with the wMelPop strain of Wolbachia showed at least a 10(4) times reduction in YFV RNA copies compared to controls. The extent of reduction in virus infection depended on Wolbachia strain, titer and strain of the virus, and mode of exposure. Although originally proposed for dengue biocontrol, our results indicate a Wolbachia-based strategy also holds considerable promise for YFV and CHIKV suppression.

Limited dengue virus replication in field-collected Aedes aegypti mosquitoes infected with Wolbachia

Introduction Dengue is one of the most widespread mosquito-borne diseases in the world. The causative agent, dengue virus (DENV), is primarily transmitted by the mosquito Aedes aegypti, a species that has proved difficult to control using conventional methods. The discovery that A. aegypti transinfected with the wMel strain of Wolbachia showed limited DENV replication led to trial field releases of these mosquitoes in Cairns, Australia as a biocontrol strategy for the virus. Methodology/Principal Findings Field collected wMel mosquitoes that were challenged with three DENV serotypes displayed limited rates of body infection, viral replication and dissemination to the head compared to uninfected controls. Rates of dengue infection, replication and dissemination in field wMel mosquitoes were similar to those observed in the original transinfected wMel line that had been maintained in the laboratory. We found that wMel was distributed in similar body tissues in field mosquitoes as in laboratory ones, but, at seven days following blood-feeding, wMel densities increased to a greater extent in field mosquitoes. Conclusions/Significance Our results indicate that virus-blocking is likely to persist in Wolbachia-infected mosquitoes after their release and establishment in wild populations, suggesting that Wolbachia biocontrol may be a successful strategy for reducing dengue transmission in the field.

Wolbachia reduces the transmission potential of Dengue-infected Aedes aegypti

BACKGROUND: Dengue viruses (DENV) are the causative agents of dengue, the world's most prevalent arthropod-borne disease with around 40% of the world's population at risk of infection annually. Wolbachia pipientis, an obligate intracellular bacterium, is being developed as a biocontrol strategy against dengue because it limits replication of the virus in the mosquito. The Wolbachia strain wMel, which has been introduced into the mosquito vector, Aedes aegypti, has been shown to invade and spread to near fixation in field releases. Standard measures of Wolbachia's efficacy for blocking virus replication focus on the detection and quantification of virus in mosquito tissues. Examining the saliva provides a more accurate measure of transmission potential and can reveal the extrinsic incubation period (EIP), that is, the time it takes virus to arrive in the saliva following the consumption of DENV viremic blood. EIP is a key determinant of a mosquito's ability to transmit DENVs, as the earlier the virus appears in the saliva the more opportunities the mosquito will have to infect humans on subsequent bites. METHODOLOGY/PRINCIPAL FINDINGS: We used a non-destructive assay to repeatedly quantify DENV in saliva from wMel-infected and Wolbachia-free wild-type control mosquitoes following the consumption of a DENV-infected blood meal. We show that wMel lengthens the EIP, reduces the frequency at which the virus is expectorated and decreases the dengue copy number in mosquito saliva as compared to wild-type mosquitoes. These observations can at least be partially explained by an overall reduction in saliva produced by wMel mosquitoes. More generally, we found that the concentration of DENV in a blood meal is a determinant of the length of EIP, saliva virus titer and mosquito survival. CONCLUSIONS/SIGNIFICANCE: The saliva-based traits reported here offer more disease-relevant measures of Wolbachia's effects on the vector and the virus. The lengthening of EIP highlights another means, in addition to the reduction of infection frequencies and DENV titers in mosquitoes, by which Wolbachia should operate to reduce DENV transmission in the field.

Otimização da RT-PCR Multiplex para detecção de vírus dengue em amostras de Aedes aegypti infectados artificialmente

A dengue é a mais importante doença viral transmitida por mosquitos, no que diz respeito à morbidade e mortalidade, que afeta os seres humanos. Este vírus é transmitido pelos vetores Aedes albopictus e Aedes aegypti, este último é o principal vetor nas Américas. O controle da doença se baseia na vigilância laboratorial e vigilância entomológica. A vigilância laboratorial visa aprimorar a capacidade do diagnóstico, detectando precocemente a circulação viral e monitorando os sorotipos circulantes. Dentro deste tipo de vigilância, a RT-PCR é um método bastante usado no diagnóstico da doença em humanos e mosquitos, porém, a má conservação do material pode comprometer a integridade do RNA e trazer resultados falso-negativos. O desenvolvimento de melhores métodos de vigilância do vírus dengue (DENV) em mosquitos é de grande valor para os programas de controle. Desta maneira, o presente projeto visou otimizar a técnica de RT-PCR Multiplex para detecção de DENV em amostras de Ae. aegypti infectadas artificialmente pelo vírus. Primers que amplificam uma região de 80 pb do gene rpL8 de mosquito foram desenhados no site Primer3 e avaliados na ferramenta online Multiple Primer Analyzer, junto com primers que amplificam os sorotipos DENV. Não houve competição de primers e foi observado bandas distintas no gel de agarose. Foi avaliado o efeito de diferentes formas de preservação do material genético das amostras (RNAlater®, freezer -80°C e nitrogênio líquido) por 7 dias, onde não houve diferenças significativas em relação à integridade do RNA. O efeito de diferentes formas de extração de RNA (Kit da QIAGEN® , TRIzol® e Chomczymski-Sacchi) também foi avaliado e o método ChomczymskiSacchi obteve o melhor desempenho. A otimização desta técnica permitirá uma maior confiabilidade nos resultados, já que além da detecção dos sorotipos, haverá uma confirmação da qualidade do RNA, aprimorando a capacidade do diagnóstico e auxiliando a prevenção e controle da transmissão da dengue.

Atividade tóxica de isolados de Bacillus Thuringiensis a larvas de Aedes Aegypti (l.) (diptera: culicidae)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Estudo da variabilidade genética de populações de Aedes aegypti (Diptera,Culicidae), resistente e suscetíveis a inseticidas

Pós-graduação em Genética - IBILCE

Predição in vitro da atividade tóxica de isolados de Bacillus thuringiensis Berliner e efeito sinergístico no controle de larvas de Aedes aegypti (L.) (Diptera: Culicidae)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Estratégias adaptativas de Aedes aegypti e Aedes albopictus em resposta a temperatura e qualidade de criadouros

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Atividade larvicida dos óleos essenciais de Syzygium aromaticum e Citrus sinensis em populações de Aedes aegypti

Pós-graduação em Biociências - FCLAS