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

Abstract

Abstract. Sporadic outbreaks of vesicular stomatitis (VS) in the United States result in significant economic losses for the U.S. livestock industries because VS is a reportable disease that clinically mimics footand-mouth disease. Rapid and accurate differentiation of these 2 diseases is critical because their consequences and control strategies differ radically. The objective of the current study was to field validate a 1-tube multiplexed real-time reverse transcription polymerase chain reaction (real-time RT-PCR) assay for the rapid detection of Vesicular stomatitis New Jersey virus and Vesicular stomatitis Indiana virus 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 was tested by the real-time RT-PCR assay and virus isolation. Overall, clinical sensitivity and specificity of the real-time RT-PCR were 83% and 99%, respectively. Interestingly, VS virus isolates originating from a specific region of Costa Rica were not detected by real-time RT-PCR. Sequence comparisons of these viruses with the real-time RT-PCR probe and primers showed mismatches in the probe and forward and reverse primer regions. Additional 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 VS viruses and proved readily adaptable as new VS viruses were encountered. An important secondary result of this research was the collection of hundreds of new VS virus isolates that provide a foundation from which many additional studies can arise.

Domestic cattle as a non-conventional amplifying host of vesicular stomatitis New Jersey virus

“…Wells with cytopathic effect were confirmed as positive for VSNJV using rRT-PCR. Total RNA was extracted using the Qiagen RNeasy kit (Qiagen, Inc., Valencia, CA, U.S.A.) following the manufacturer's protocol, and stored at −20 • C. Viral RNA was detected by semi-quantitative rRT-PCR specific for the nucleocapsid gene of VSNJV, as described previously (Wilson et al, 2009). …”

Improvement and Optimization of a Multiplex Real-Time Reverse Transcription Polymerase Chain Reaction Assay for the Detection and Typing of Vesicular Stomatitis Virus

“…7,20 In recent years, a number of rapid diagnostic tests for VSV have been developed, 1,[4][5][6]17 including different real-time reverse transcription polymerase chain reaction (real-time RT-PCR) assays. 11,12,19 These assays, however, either lack diagnostic sensitivity and may miss some current circulating strains of the virus 11,12 or use a cumbersome combination of a large number of primers to ensure accurate and sensitive detection of strains of VSV by targeting the N gene of the virus. 19 A previous study 5 reported the development of a multiplex real-time PCR that targets the L gene of VSV.…”

“…11,12,19 These assays, however, either lack diagnostic sensitivity and may miss some current circulating strains of the virus 11,12 or use a cumbersome combination of a large number of primers to ensure accurate and sensitive detection of strains of VSV by targeting the N gene of the virus. 19 A previous study 5 reported the development of a multiplex real-time PCR that targets the L gene of VSV. As part of the validation protocol of the assay, it was determined that the assay was not sensitive enough and failed to detect some of the current strains of the virus.…”

“…This is particularly important because of the potential genetic variation observed both within each serotype and between the serotypes. 2,3,9,10,16,18 To address this, a collection of phylogenetically characterized reference strains of VSV, representing current genetic lineages of the virus, including a prototype strain of the Tilarán lineage, 19 were evaluated. In addition, Mexican and Colombian VSV field isolates were also evaluated.…”

Detection of All Eight Serotypes ofEpizootic Hemorrhagic Disease Virusby Real-Time Reverse Transcription Polymerase Chain Reaction

“…The cycling parameters were based on the previously reported real-time RT-PCR for Vesicular stomatitis virus. 30 The cycle temperatures were 55uC for 25 min, 95uC for 2 min, 40 cycles of 95uC for 10 sec, and 55uC for 1 min. The samples were analyzed using 3 real-time PCR instruments.…”