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Anthropogenic actions, including deforestation, disorganized urbanization and globalization contribute to emergence and re-emergence of arboviruses worldwide where Flavivirus is the most prevalent and its continuous monitoring can help in preventive control strategies. Thus, the aim of this study was to detect flaviviruses RNA in single hematophagous insects, used as sentinels. Total RNA was extracted from six Aedes aegypti stored since 2003 and from 100 Culicidae, collected through CDC trap in a public park of a Brazilian Northwest city of São Paulo State. Flavivirus was detected through RT/PCR targeting 230-250 bp of the RNA polymerase coding sequence (NS5). PCR amplicons were sequenced by Sanger method; used in comparative analysis over Basic Local Alignment Search Tool (BLAST) in GeneBank; and subjected to Neighbor-Joining phylogenetic analyses. Efficiency of Flavivirus diagnosis was confirmed by detection of Dengue virus serotype 2 in Aedes aegypti. From the 100 collected insects, 19 were positive for Culex flavivirus (CxFV). NS5 partial sequence phylogenetic analysis clustered all CxFV in one branch separated from vertebrate flaviviruses, being applicable to Flavivirus species identification. The dipteran RNA extraction methodology described in this work supports detection of flaviviruses in single insects maintained in 80% ethanol, which can be used to constant arboviruses surveillance.

Novel Single Hematophagous Insect RNA Detection Method Supports Its Use as Sentinels to Survey Flaviviruses Circulation