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.

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

Effects of Plasmodium gallinaceum on hemolymph physiology of Aedes aegypti during parasite development

Insect disease vectors show diminished fecundity when infected with Plasmodium. This phenomenon has already been demonstrated in laboratory models such as Aedes aegypti, Anopheles gambiae and Anopheles stephensi. This study demonstrates several changes in physiological processes of A. aegypti occurring upon infection with Plasmodium gallinaceum, such as reduced ecdysteroid levels in hemolymph as well as altered expression patterns for genes involved in vitellogenesis, lipid transport and immune response. Furthermore, we could show that P. gallinaceum infected A. aegypti presented a reduction in reproductive fitness, accompanied by an activated innate immune response and increase in lipophorin expression, with the latter possibly representing a nutritional resource for Plasmodium sporozoites. (C) 2010 Elsevier Ltd. All rights reserved.

A novel trypsin Kazal-type inhibitor from Aedes aegypti with thrombin coagulant inhibitory activity

Kazal-type inhibitors play several important roles in invertebrates, such as anticoagulant, vasodilator and antimicrobial activities. Putative Kazal-type inhibitors were described in several insect transcriptomes. In this paper we characterized for the first time a Kazal unique domain trypsin inhibitor from the Aedes aegypti mosquito. Previously, analyses of sialotranscriptome of A. aegypti showed the potential presence of a Kazal-type serine protease inhibitor, in female salivary glands, carcass and also in whole male, which we named AaTI (A. aegypti trypsin inhibitor). AaTI sequence showed amino acid sequence similarity with insect thrombin inhibitors, serine protease inhibitor from Litopenaeus vannamei hemocytes and tryptase inhibitor from leech Hirudo medicinalis (LDTI). In this work we expressed, purified and characterized the recombinant AaTI (rAaTI). Molecular weight of purified rAaTI was 7 kDa rAaTI presented dissociation constant (K(i)) of 0.15 and 3.8 nM toward trypsin and plasmin, respectively, and it weakly inhibited thrombin amidolytic activity. The rAaTI was also able to prolong prothrombin time, activated partial thromboplastin time and thrombin time. AaTI transcription was confirmed in A. aegypti female salivary gland and gut 3 h and 24 h after blood feeding, suggesting that this molecule can act as anticoagulant during the feeding and digestive processes. Its transcription in larvae and pupae suggested that AaTI may also play other functions during the mosquito`s development. (C) 2010 Elsevier Masson SAS. All rights reserved.

The Aedes aegypti larval transcriptome: a comparative perspective with emphasis on trypsins and the domain structure of peritrophins

The genome sequence of Aedes aegypti was recently reported. A significant amount of Expressed Sequence Tags (ESTs) were sequenced to aid in the gene prediction process. In the present work we describe an integrated analysis of the genomic and EST data, focusing on genes with preferential expression in larvae (LG), adults (AG) and in both stages (SG). A total of 913 genes (5.4% of the transcript complement) are LG, including ion transporters and cuticle proteins that are important for ion homeostasis and defense. From a starting set of 245 genes encoding the trypsin domain, we identified 66 putative LG, AG, and SG trypsins by manual curation. Phylogenetic analyses showed that AG trypsins are divergent from their larval counterparts (LG), grouping with blood-induced trypsins from Anopheles gambiae and Simulium vittatum. These results support the hypothesis that blood-feeding arose only once, in the ancestral Culicomorpha. Peritrophins are proteins that interlock chitin fibrils to form the peritrophic membrane (PM) that compartmentalizes the food in the midgut. These proteins are recognized by having chitin-binding domains with 6 conserved Cys and may also present mucin-like domains (regions expected to be highly O-glycosylated). PM may be formed by a ring of cells (type 2, seen in Ae. aegypti larvae and Drosophila melanogaster) or by most midgut cells (type 1, found in Ae. aegypti adult and Tribolium castaneum). LG and D. melanogaster peritrophins have more complex domain structures than AG and T. castaneum peritrophins. Furthermore, mucin-like domains of peritrophins from T. castaneum (feeding on rough food) are lengthier than those of adult Ae. aegypti (blood-feeding). This suggests, for the first time, that type 1 and type 2 PM may have variable molecular architectures determined by different peritrophins and/or ancillary proteins, which may be partly modulated by diet.

LARVICIDAL ACTIVITY OF GRANDISIN AGAINST AEDES AEGYPTI

Dengue is a tropical disease caused by an arbovirus transmitted by the mosquito Aedes aegypti. Because no effective vaccine is available for the disease, the strategy for its prevention has focused on vector control by the use of natural insecticides. The aim of this study was to evaluate the larvicidal activity of the lignan grandisin, a leaf extract from Piper solmsianum, against Ae. aegypti.

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

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Larvicidal activity of the water extract of Moringa oleifera seeds against Aedes aegypti and its toxicity upon laboratory animals

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Estudo teórico-bioclimático da potencialidade de desenvolvimento do Aedes aegypti no estado do Rio Grande do Norte

In the current climate of global public health there is the emergence of urban dengue, a disease regarded as acute infectious fever. The disease annually, has affected millions of people worldwide, mostly in the range of the intertropical globe. The disease's main vector in urban areas, the Aedes aegypti mosquito. Recent studies indicate that the distribution of the insect vector of dengue in the geographical area is directly tied to the behavior of environmental restrictions that area, especially among those, the air temperature and relative humidity. From that context, the work aims to estimate and spatializing, monthly, for each municipality in the state of Rio Grande do Norte, the potential of biophysical conditions conducive to the development of Aedes aegypti. Yet, made use of the following methodology: collection of epidemiological data and climatological, Normal climatological, descriptive statistics (measures of central tendency and scatter), uniform distribution, estimation geostatistics and sufer program, version 8.0. The results flagged for a behavior very heterogeneous, both in space and in time, in the case of the potential of biophysical conditions conducive to the development of Aedes aegypti in the state of Rio Grande do Norte. Still, he noted that there is a tendency for lifting the potential of development for the entire state, from the month of January, ending in the month of April mainly in central and western portions of the state. By contrast, there is the permanence of increased potential for development in the eastern portion of the state. The latter record maximum potential in the month of July, resulting probability of greater than 70% have been favorable conditions for the development of Aedes aegypti in that area. In the period between the months of August to December, it is small potential for development of Aedes aegypti in all parts of the state

Association of insecticide use and alteration on Aedes aegypti susceptibility status

Dengue and dengue hemorrhagic fever, vector-borne diseases transmitted by the mosquito Aedes aegypti, are presently important public health problems in Brazil. As the strategy for disease control is based on vector control through the use of insecticides, the development of resistance is a threat to programs efficacy. The objective of this study was to compare the Aedes aegypti susceptibility in nine vector populations from the state of São Paulo and seven from Northeast region of Brazil, since there was a difference on group of insecticide used between the areas. Bioassays with larvae and adult were performed according to the World Health Organization methods.The results showed higher resistance levels to organophosphates group in populations from the Northeast region where this group was used for both larvae and adult control than in São Paulo where organophosphates were used for larvae and pyretroids for adult control. Resistance to pyretroids in adults was widespread in São Paulo after ten years of use of cypermethrin while in vector populations from the Northeast region it was punctual. The difference in resistance profile between the areas is in accordance to the group of insecticide used.

Controlling dispersal dynamics of Aedes aegypti

The dengue virus is transmitted in regions previously infested with the mosquito Aedes aegypti. To assess the spreading and establishment of the dengue disease vector, a mathematical model is developed that takes into account the diffusion and advection phenomena. A discrete model based on the cellular automata approach, which is a good framework to deal with small populations, is also developed to be compared with the continuous modeling.

ASSESSING THE SUITABILITY of STERILE INSECT TECHNIQUE APPLIED TO AEDES AEGYPTI

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Mitochondrial DNA polymorphism and heteroplasmy in populations of Aedes aegypti in Brazil

The tropical mosquito Aedes aegypti (Diptera: Culicidae) is the most important domestic vector of urban yellow fever and dengue viruses. Ae. aegypti originated from Africa and was probably introduced into Brazil during the colonial period through embarkations, and dengue epidemics soon followed. Genetic analysis of 12 Ae. aegypti populations from five states in Brazil was conducted based on two mitochondrial DNA fragments: cytochrome oxidase I and NADH dehydrogenase subunit 4. Analyses comparing individual haplotypes indicated the existence of two well-defined clades, probably representing two mitochondrial lineages. Analysis of molecular variance showed significant variability in genetic structure among collections within groups. Mantel regression analysis showed a correlation between genetic and geographic distances, mainly because of northern and northeastern populations, in comparison with those in the southeast. The population from Santos, the largest port in Brazil, showed the greatest diversity, with 10 unique haplotypes, an indication of recent introductions that have not yet spread to other Brazilian cities. Different mitochondrial DNA sequences were found in three specimens, indicating the presence of heteroplasmy.