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Title: Surveillance for Western equine encephalitis St. Louis encephalitis and West Nile viruses using reverse transcription loop-mediated isothermal amplification

In this study, 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, despitemore » 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.« less
 [1] ;  [2] ;  [3] ;  [1] ;  [4] ;  [1]
  1. Univ. of California, Davis, CA (United States)
  2. Sandia National Lab. (SNL-CA), Livermore, CA (United States)
  3. Sandia National Lab. (SNL-CA), Livermore, CA (United States); Univ. of Washington, Seattle, WA (United States)
  4. Univ. of California, Davis, CA (United States); Sacramento-Yolo Mosquito and Vector Control District, Elk Grove, CA (United States)
Publication Date:
OSTI Identifier:
Report Number(s):
Journal ID: ISSN 1932-6203; 607270
Grant/Contract Number:
Accepted Manuscript
Journal Name:
Additional Journal Information:
Journal Volume: 11; Journal Issue: 1; Journal ID: ISSN 1932-6203
Public Library of Science
Research Org:
Sandia National Laboratories (SNL-CA), Livermore, CA (United States)
Sponsoring Org:
Country of Publication:
United States
59 BASIC BIOLOGICAL SCIENCES West Nile virus; Western equine encephalitis virus; RNA extraction; Eastern equine encephalitis virus; Japanese encephalitis virus; polymerase chain reaction; sequence alignment; RNA viruses