Validation of a real-time RT-PCR for vesicular stomatitis virus

Authors: Letchworth, Geoffrey; JIMENEZ, CARLOS - UNIV NACIONAL, COSTA RICA; HERRERO, MARCO - UNIV NACIONAL, COSTA RICA; CORNISH, TODD - UNIVERSITY OF WYOMING; Wilson, William; Smoliga, George; Pauszek, Steven; DORNAK, CARRIE - UNIVERSITY OF WYOMING; GEORGE, MARCOS - LADIVES PANAMA CITY PANAM; Rodriguez, Luis

Submitted to: American Association of Veterinary Laboratory Diagnosticians
Publication Type: Abstract Only
Publication Acceptance Date: 9/13/2004
Publication Date: 10/21/2004
Citation: Letchworth, G., Jimenez, C., Herrero, M., Cornish, T., Wilson, W.C., Smoliga, G.R., Pauszek, S.J., Dornak, C., George, M., Rodriguez, L.L. 2004. Validation of a real-time RT-PCR for vesicular stomatitis virus. Proc. Am. Assoc. of Vet. Lab. Diagn. 47:121. 2004.

Interpretive Summary: Sporadic outbreaks of vesicular stomatitis (VS) in the United States result in significant economic losses for the US livestock industries because VS clinically mimics foot-and-mouth disease. Control strategies for the two diseases differ radically and thus mandate rapid and accurate differentiation. The objective of this study was to validate a rapid and accurate diagnostic polymerase chain reaction (PCR) test for VS. Because VS apprears to spread to the U.S. from Mexico and Central America, where many different lineages of virus are present, we obtained samples from cases of VS throughout Central America. We compared the classical diagnostic methods to PCR. The PCR identified virtually all cases detected by classical diagnostic tests. The few samples missed by PCR are driving improvements in the PCR assay. The PCR also detected some cases not identified by classical tests, suggesting that PCR may be more sensitive than other diagnostic methods under some conditions.

Technical Abstract: Sporadic outbreaks of vesicular stomatitis (VS) in the United States result in significant economic losses for the US livestock industries because VS clinically mimics foot-and-mouth disease. Control strategies for the two diseases differ radically and thus mandate rapid and accurate differentiation. 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 from North America to northern South America. This test has undergone bench validation at the Plum Island Animal Disease Center as is described in a separate report. Vesicular lesion samples obtained from cattle, horses, or swine from throughout Central America were tested at the Laboratorio de Diagnóstico de Enfermedades Vesiculares (LADIVES) in Panama; and samples from Costa Rica were tested at the School of Veterinary Medicine in Heredia Costa Rica. A portion of each sample was prepared for virus isolation in Vero cells and VSNJV and VSINV specific antibodies were used to confirm the presence of each virus. A second portion of each lesion sample was prepared for RT-PCR by RNA extraction using a commercial extraction kit and tested by rRT-PCR. Of approximately 400 samples tested, half yielded a cytopathogenic agent, identified in most cases as VSNJV and occasionally as VSINV by reaction to serotype specific antibodies, reaction with serotype-specific primers and probes, and genomic sequence. Most samples (90%) from which VSV was isolated also reacted in the rRT-PCR but a few specific strains originating from a specific region of Costa Rica were not detected by rRT-PCR. The molecular basis of this lack of detection is being investigated. VSV was not isolated from many RT-PCR positive samples, these samples were confirmed as positive by sequence analysis of N and other viral genes. We conclude that although rRT-PCR detected the largest number of positives from field samples, there were few specific strains that were not recognized by the current primer/probe combination. Additional primers and probes might be required to identify all of the Central American VSV's. An important secondary result of this research was the collection of hundreds of new VSV isolates. This benchmark collection constitutes a foundation from which many additional studies can arise. Please reference the following: (1) HQ issued Clearance/Sensitivity Responsibility memo (attached), and also found by clicking on the Help button when in the ARIS Project Info tab for ARS-115 (2) NPA Policy Memorandum PM-03-006, dated 06/25/2003 Subject: Submission of the ARS-115/Publication in ARIS, Northern Plains Area