Zika and dengue viruses infecting wild-caught mosquitoes in an environmental protection area in Brazil

Species of the genus Flavivirus are widespread in Brazil and are a major public health concern. The city of São Paulo is in a highly urbanized area with some green spaces which are used for recreation and where potential vertebrate hosts and mosquito vectors of these arboviruses can be found, a scenario that can contribute to the transmission of flaviviruses to humans. This study therefore sought to investigate natural flavivirus infection in mosquitoes collected in the Capivari-Monos Environmental Protection Area (EPA) in the south of the city. Monthly mosquito collections were carried out from March 2016 to April 2017 with CO 2 -baited CDC light traps. Specimens were identified morphologically and grouped in pools. A total of 260 pools of non-engorged females were inoculated into the C6/36 cell lineage after analysis by indirect immunofluorescence assay (IFA). IFA-positive specimens were tested by qRT-PCR with genus-specific primers targeting a region of ~260 nucleotides in the flavivirus NS5 gene, and the PCR products were sequenced to confirm and identify the flavivirus species. Anopheles ( Kerteszia ) cruzii and Wyeomyia ( Prosopolepis ) confusa were the most frequent species collected. Zika virus (ZIKV) nucleotide sequences were detected in three mosquito species, An. cruzii , Limatus durhami and Wy. confusa , and dengue virus 2 (DENV-2) sequences in Culex . spp. and Culex . ( Mel .) vaxus . To our knowledge, this is the first report of natural isolation of DENV-2 and ZIKV in sylvatic species of mosquitoes in the Capivari-Monos EPA. Our findings suggest that DENV-2 is present in Culex mosquitoes, and ZIKV in Anopheles , Wyeomyia and Limatus . The flavivirus species identified here are of medical importance; surveillance is therefore recommended in this EPA, where vertebrates and mosquitoes can act as flavivirus hosts and vectors.