Dengue Fever and Dengue Hemorrhagic Fever are diseases affecting approximately 100 million people/year and are a major concern in developing countries. In the present study, the phylogenetic relationship of six strains of the first autochthonous cases of DENV-4 infection occurred in Sao Paulo State, Parana State and Rio Grande do Sul State, Brazil, 2011 were studied. Nucleotide sequences of the envelope gene were determined and compared with sequences representative of the genotypes I, II, III and Sylvatic for DEN4 retrieved from GenBank. We employed a Bayesian phylogenetic approach to reconstruct the phylogenetic relationships of Brazilian DENV-4 and we estimated evolutionary rates and dates of divergence for DENV-4 found in Brazil in 2011. All samples sequenced in this study were located in Genotype II. The studied strains are monophyletic and our data suggest that they have been evolving separately for at least 4 to 6 years. Our data suggest that the virus might have been present in the region for some time, without being noticed by Health Surveillance Services due to a low level of circulation and a higher prevalence of DENV-1 and DENV- 2.

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The increasing population of Aedes aegypti mosquitoes on Madeira Island (Portugal) resulted in the first autochthonous dengue outbreak, which occurred in October 2012. Our study establishes the first genetic evaluation based on the mitochondrial DNA (mtDNA) genes [cytochrome oxidase subunit I (COI) and NADH dehydrogenase subunit 4 (ND4)] and knockdown resistance ( kdr ) mutations exploring the colonisation history and the genetic diversity of this insular vector population. We included mosquito populations from Brazil and Venezuela in the analysis as putative geographic sources. The Ae. aegypti population from Madeira showed extremely low mtDNA genetic variability, with a single haplotype for COI and ND4. We also detected the presence of two important kdr mutations and the quasi-fixation of one of these mutations (F1534C). These results are consistent with a unique recent founder event that occurred on the island of Ae. aegypti mosquitoes that carry kdr mutations associated with insecticide resistance. Finally, we also report the presence of the F1534C kdr mutation in the Brazil and Venezuela populations. To our knowledge, this is the first time this mutation has been found in South American Ae. aegypti mosquitoes. Given the present risk of Ae. aegypti re-invading continental Europe from Madeira and the recent dengue outbreaks on the island, this information is important to plan surveillance and control measures.

Population genetic studies of insect vectors can generate knowledge to improve epidemiological studies focused on the decrease of pathogen transmission. In this study, we used nine SNPs across the Aedes aegypti genome to characterize seasonal population variations of this important dengue vector. Mosquito samples were obtained by ovitraps placed over Botucatu SP from 2005 to 2010. Our data show that, regardless of the large variation in mosquito abundance (deduced from the number of eggs obtained from ovitraps), the effective population size remained stable over the years. These results suggest that Ae. aegypti is able to maintain a sufficiently large active breeding population during the dry season to keep genetic frequencies stable. These results open new perspectives on mosquito survey and control methods.

SUMMARYWe report the first isolation of Dengue virus 4 (DENV-4) in the state of São Paulo, from two patients -one living in São José do Rio Preto and the other one in Paulo de Faria, both cities located in the Northwest region of the state. The virus isolations were accomplished in the clone C6/36 Aedes albopictus cell line, followed by indirect immunofluorescence assays, performed with typespecific monoclonal antibodies that showed positive reactions for DENV-4. The results were confirmed by Nested RT-PCR and RealTime RT-PCR assays. The introduction of DENV-4 in a country that already has to deal with the transmission of three other serotypes increases the possibility of the occurrence of more severe cases of the disease. The importance of early detection of dengue cases, before the virus spreads and major outbreaks occur, should be emphasized.