Field Evaluation of a Multiplex Real-Time Reverse Transcription Polymerase Chain Reaction Assay for Detection of Vesicular Stomatitis Virus

Authors: Wilson, William - Bill; LETCHWORTH, GEOFFREY - FORMER ARS EMPLOYEE; JIMENEZ, C. - UNIV NACIONAL HEREDIA CR; HERRARO, M. - UNIV NACIONAL HEREDIA CR; NAVARRO, R. - CPA, MEXICO; PAZ, P. - CPA, MEXICO; CORNISH, T. - UNIV WYOMING, LARAMIE; Smoliga, George; Pauszek, Steven; DORNAK, C. - UNIV WYOMING, LARAMIE; GEORGE, M. - INFECTIOUS DISEASE LAB PN; Rodriguez, Luis

Submitted to: Journal of Veterinary Diagnostic Investigation
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/5/2009
Publication Date: 3/1/2009
Citation: Wilson, W.C., Letchworth, G., Jimenez, C., Herraro, M., Navarro, R., Paz, P., Cornish, T.E., Smoliga, G.R., Pauszek, S.J., Dornak, C., George, M., Rodriguez, L.L. 2009. Field Evaluation of a Multiplex Real-Time Reverse Transcription Polymerase Chain Reaction Assay for Detection of Vesicular Stomatitis Virus. Journal of Veterinary Diagnostic Investigation. 21(2):179-186.

Interpretive Summary: Vesicular stomatitis (VS) is an insect transmitted viral disease that occurs in horses, cattle and pigs throughout the Americas. The disease resembles foot-and-mouth disease, a devastating disease of cloven hooved animals. Rapid and accurate differentiation of these two diseases is critical because their consequences and control strategies differ radically. We describe field testing of a rapid diagnostic test based on real-time reverse transcriptase-polymerase chain reaction (rRT-PCR). This test was able to rapidly detect viruses from a comprehensive collection of 622 vesicular lesion samples obtained from cattle, horses, and swine from throughout Mexico and Central America with high sensitivity and specificity. Interestingly VSV isolates originating from a specific region of Costa Rica were not detected by rRT-PCR. Sequence comparisons of these viruses with the rRT-PCR showed important differences in sequence that required the adjustment of the rRT-PCR test by addition of lineage-specific primers and a probe capable of detecting the missing genetic lineage. Thus this assay reliably identified existing Mexican and Central American VSVs and proved readily adaptable as new VS viruses are encountered. An important secondary result of this research was the collection of hundreds of new VSV isolates that provide a foundation from which many additional studies can arise. This rRT-PCR test is an important addition to the countermeasure arsenal against foreign animal diseases.

Technical Abstract: Sporadic outbreaks of vesicular stomatitis (VS) in the United States result in significant economic losses for the US livestock industries because VS is an OIE reportable disease and also clinically mimics foot-and-mouth disease. Rapid and accurate differentiation of these two diseases is critical because their consequences and control strategies differ radically. The objective of this study was to field-validate a one-tube multiplexed real-time reverse transcriptase-polymerase chain reaction (rRT-PCR) assay for the rapid detection of VS-New Jersey virus (VSNJV) and VS-Indiana virus (VSINV) strains occurring in Mexico and North and Central America. A comprehensive collection of 622 vesicular lesion samples obtained from cattle, horses, and swine from throughout Mexico and Central America were tested by the rRT-PCR assay and virus isolation. Overall clinical sensitivity and specificity of the rRT-PCR were 83% and 99% respectively. Interestingly VSV isolates originating from a specific region of Costa Rica were not detected by rRT-PCR. Sequence comparisons of these viruses with the rRT-PCR showed mismatches with the rRT-PCR system in the probe, forward and reverse primer regions. Addition lineage-specific primers and a probe corrected the lack of detection of the missing genetic lineage. Thus this assay reliably identified existing Mexican and Central American VSVs and proved readily adaptable as new VS viruses are encountered. An important secondary result of this research was the collection of hundreds of new VSV isolates that provide a foundation from which many additional studies can arise.