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

Abstract

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 flavivirus 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, WNV, 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.

FIG. 1.

Multiple-nucleotide-sequence alignment of the YFV group, the DENV group, and the JEV group. The aligned region corresponds to nucleotides 8997 to 9258 of YFV. The 50% majority consensus sequence is listed as the reference to highlight the conservation of this region. In addition to the eight viruses used in this study, viruses in the YFV group (Sepik virus [SEPV], Yokose virus [YOKV] group, and Entebbe bat virus [ENTV] group) and the JEV group (Murray Valley encephalitis virus [MVEV] and Usutu virus [USUV]) are aligned with the respective virus group. The flavivirus consensus amplimers (amplimers mFU1 and CFD2) and eight virus-specific probes are highlighted in boldface and italic.

FIG. 2.

Performance of the TaqMan RT-PCR assay for detection of four different flaviviruses in a single-dye or four-dye multiplex format. The detection thresholds, expressed as C_T values representative of the two experiments, are plotted against the log of a known infectivity (PFU per reaction) of YFV, JEV, WNV, and SLEV in four-tube, single-dye probe assays (A) or in a one-tube, four-color multiplex probe assay (B).