A Multiplex Real-Time Reverse Transcription Polymerase Chain Reaction Assay With Enhanced Capacity to Detect Vesicular Stomatitis Viral Lineages of Central American Origin.

Kate Hole,Lauro Velazquez-Salinas,Charles Nfon,Luis L Rodriguez

doi:10.3389/fvets.2021.783198

Abstract

Vesicular stomatitis virus (VSV) causes a disease in susceptible livestock that is clinically indistinguishable from foot-and-mouth disease. Rapid testing is therefore critical to identify VSV and rule out FMD. We previously developed and validated a multiplex real-time reverse transcription polymerase chain reaction assay (mRRT-PCR) for detection of both VS New Jersey virus (VSNJV) and VS Indiana virus (VSIV). However, it was subsequently apparent that this assay failed to detect some VSNJV isolates in Mexico, especially in genetic group II, lineage 2.1. In order to enhance the sensitivity of the mRRT-PCR for VSNJV, parts of the assay were redesigned and revalidated using new and improved PCR chemistries. The redesign markedly improved the assay by increasing the VSNJV detection sensitivity of lineage 2.1 and thereby allowing detection of all VSNJV clades. The new assay showed an increased capability to detect VSNJV. Specifically, the new mRRT-PCR detected VSNJV in 100% (87/87) of samples from Mexico in 2006-2007 compared to 74% for the previous mRRT-PCR. Furthermore, the analytical sensitivity of the new mRRT-PCR was enhanced for VSNJV. Importantly, the modified assay had the same sensitivity and specificity for VSIV as the previously published assay. Our results highlight the challenges the large genetic variability of VSV pose for virus detection by mRRT-PCR and show the importance of frequent re-evaluation and validation of diagnostic assays for VSV to ensure high sensitivity and specificity.

Highlights

Vesicular stomatitis virus (VSV) is an arbovirus and prototype of the Rhabdovirus viral family and vesiculovirus genus from which vesicular stomatitis Indiana virus (VSIV) and vesicular stomatitis New Jersey virus (VSNJV) constitute the main serotypes [1]
We aimed to evaluate the performance of our mRRT-PCR assay to detect samples from both the Central American lineage 2.1 recently introduced in Mexico and the epidemic lineage 1.1
When PCR products were visualized by agarose gel electrophoresis, amplification products matching the expected size of VSNJV amplicon were observed in most of the mRRT-PCR negative reactions indicating failure of VSNJV detection in these samples by mRRT-PCR might be associated with mismatches in the probes

Summary

Introduction

Vesicular stomatitis virus (VSV) is an arbovirus and prototype of the Rhabdovirus viral family and vesiculovirus genus from which vesicular stomatitis Indiana virus (VSIV) and vesicular stomatitis New Jersey virus (VSNJV) constitute the main serotypes [1]. The genetic diversity of VSNJV has been associated with at least six different phylogenetic clades, which are directly linked to the geographical regions where these viruses are typically circulating [3]. In southern Mexico, where VSV is endemic, clinical cases are recorded on an annual basis [4]. These VSV endemic zones are colonized by multiple lineages belonging to North American Clade I [3]. Most VSV outbreaks recorded in the United States have been linked to endemic ancestors from these regions [4,5,6]

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Methods

Results