Abstract
Collection of mosquitoes and testing for vector-borne viruses is a key surveillance activity that directly influences the vector control efforts of public health agencies, including determining when and where to apply insecticides. Vector control districts in California routinely monitor for three human pathogenic viruses including West Nile virus (WNV), Western equine encephalitis virus (WEEV), and St. Louis encephalitis virus (SLEV). Reverse transcription quantitative polymerase chain reaction (RT-qPCR) offers highly sensitive and specific detection of these three viruses in a single multiplex reaction, but this technique requires costly, specialized equipment that is generally only available in centralized public health laboratories. We report the use of reverse transcription loop-mediated isothermal amplification (RT-LAMP) to detect WNV, WEEV, and SLEV RNA extracted from pooled mosquito samples collected in California, including novel primer sets for specific detection of WEEV and SLEV, targeting the nonstructural protein 4 (nsP4) gene of WEEV and the 3’ untranslated region (3’-UTR) of SLEV. Our WEEV and SLEV RT-LAMP primers allowed detection of <0.1 PFU/reaction of their respective targets in <30 minutes, and exhibited high specificity without cross reactivity when tested against a panel of alphaviruses and flaviviruses. Furthermore, the SLEV primers do not cross-react with WNV, despite both viruses being closely related members of the Japanese encephalitis virus complex. The SLEV and WEEV primers can also be combined in a single RT-LAMP reaction, with discrimination between amplicons by melt curve analysis. Although RT-qPCR is approximately one order of magnitude more sensitive than RT-LAMP for all three targets, the RT-LAMP technique is less instrumentally intensive than RT-qPCR and provides a more cost-effective method of vector-borne virus surveillance.
Highlights
Testing mosquito vectors for human pathogenic viruses like West Nile virus is a central feature of successful surveillance approaches and an early predictor of human epidemics
We describe use of reverse transcription loop-mediated isothermal amplification (RT-Loop-mediated isothermal amplification (LAMP)) to detect West Nile virus (WNV), Western equine encephalitis virus (WEEV), and St. Louis encephalitis virus (SLEV), and apply the technique to RNA extracted from Culex mosquitoes collected in California
The WEEV nonstructural protein 4 (nsP4) and SLEV 3’ untranslated region (3’-UTR) primer sequences are shown in Table 2, and sequence alignments showing the positioning of the primers are presented in Figs 1 and 2
Summary
Testing mosquito vectors for human pathogenic viruses like West Nile virus is a central feature of successful surveillance approaches and an early predictor of human epidemics. Three medically important vector-borne viruses, West Nile virus (WNV), Western equine encephalitis virus (WEEV), and St. Louis encephalitis virus (SLEV) are endemic in California and cause sporadic seasonal outbreaks, primarily during the summer months. SLEV was detected in field-caught mosquitoes in California in 2015, for the first time since the arrival of WNV in 2003 [2, 3]. Despite the much higher prevalence of WNV in California since 2003, SLEV and WEEV are still included in routine mosquito surveillance since vector-borne viruses can exist in cryptic and sporadic transmission cycles without the detection of human or equine disease, as exemplified by the re-emergence of SLEV in 2015
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