Date of Award

January 2015

Document Type

Open Access Thesis

Degree Name

Master of Public Health (MPH)


School of Public Health

First Advisor

James Childs


Introduction: West Nile virus (WNV) is a mosquito-borne flavivirus that was first reported in North America in 1999. Human infections with West Nile are often mild or asymptomatic, but some infections progress to life-threatening encephalitis. The Connecticut Agricultural Experiment Station performs viral cell culture screening on mosquito pools collected throughout the state each summer to detect viruses circulating in mosquito populations. Three different real-time RT-PCR assays are used to identify WNV in positive cell cultures. Many surveillance programs do not use cell culture and only use real-time PCR to screen pools. Although this can provide for faster analyses, some positive pools may be missed if mutations or other factors cause the PCR reaction to fail. Objectives: My goal was to evaluate the performance of three primer/probe sets in comparison to cell culture to evaluate the feasibility of direct PCR analysis for surveillance. Methods: RNA was extracted from 90 WNV positive mosquito pools and 90 controls from the 2013 surveillance season. Real-time RT-PCR was performed once on all samples for each primer and probe set. Pools known to be positive in cell culture that failed to amplify were sequenced and aligned with primer and probe sequences to identify any mutations in the primer or probe binding regions. Results: Of the three assays, the Tang, et al. series published in 2006 had the highest sensitivity and specificity. Only one strain that failed to amplify had mutations in the critical primer/probe binding regions, so most false negatives are likely due to other factors. Conclusion: The three assays performed well, with one set performing better than the other two. Surveillance using only one real-time RT-PCR assay to test samples should use the assay designed by Tang, et al (2006).


This is an Open Access Thesis.