Development of multiplex real-time reverse transcriptase PCR assays for detecting eight medically important flaviviruses in mosquitoes

A multiplex real-time reverse transcriptase PCR has been developed for the rapid detection and identification of eight medically important flaviviruses from laboratory-reared, virus-infected mosquito pools. The method used involves the gene-specific amplification of yellow fever virus (YFV), Japanese encephalitis virus (JEV), West Nile virus (WNV), St. Louis encephalitis virus (SLEV), and dengue virus (DENV) serotypes 1 to 4 (DENV-1 to DENV-4, respectively) by use of the Havivirus consensus amplimers located at the RNA-dependent RNA polymerase domain of nonstructural protein 5. Virus-specific amplicons were detected by four newly characterized TaqMan fluorogenic probes (probes specific for YFV, JEV, V*`NV, and SLEV) and four previously published probes specific for DENV-1 to -4 (L. J. Chien, T. L. Liao, P. Y. Shu, J. H. Huang, D. J. Gubler, and G. J. Chang, J. Clin. Microbiol. 44:1295-1304, 2006). This assay had a specificity of 100% and various sensitivities of at least 3.5 PFU/ml for YFV, 2.0 PFU/ml for JEV, 10.0 PFU/ml for WNV, and 10.0 PFU/ml for SLEV. Additionally, we have developed an in vitro transcription system to generate RNase-resistant RNA templates for each of these eight viruses. These templates can be incorporated into the assay as RNA copy number controls and/or as external controls for RNA-spiked mosquito pools for quality assurance purposes. Although further study with mosquitoes collected in the field is needed, the incorporation of this assay into mosquito surveillance could be used as an early-warning system for the detection of medically important flaviviruses, particularly when the cocirculation of multiple viruses in the same region is suspected.