VECTOR-BORNE DISEASES, SURVEILLANCE, PREVENTION West Nile Virus Surveillance: A Simple Method for Verifying the Integrity of RNA in Mosquito (Diptera: Culicidae) Pools

In aWestNile virus (WNV)-free ecosystem, it is essential to verify the integrity ofRNA before concluding thatRNAextracted frommosquito specimens is negative forWNVgene sequences. Theprimaryobjectiveofour studywas todevelopa rapidmolecular assay to rapidly screenmosquitoes for the presence of 18S RNA andWNV gene sequences. Mosquitoes, collected frommultiple sites on the island of OÔahu, were pooled into groups of 1Ð50 mosquitoes according to capture site, date, and species.Usingprimerdesign software and theGenBankdatabase, generic oligonucleotideprimerpairs were designed to amplify mosquito18S rRNA gene sequences from different species. RNA was extracted from mosquito pools, and reverse transcriptase-polymerase chain reaction (RT-PCR) was performed for the presence of mosquito18S rRNA and WNV gene sequences. Three of the seven primer pairs successfully detected 18S rRNA sequences for bothAedes andCulex byRT-PCR, and one primer pair successfully ampliÞed 18S rRNA sequences for 15 different mosquito species. All 64 mosquito pools from 10 different sites on the island of Oahu, Hawaii, were negative for WNV nonstructural protein-5 gene sequences. This simple, one-step RT-PCR method for screening mosquito pools for arboviruses will become an increasingly valuable tool as WNV becomes endemic throughout the Americas.