Quantitative Multiplex Assay for Simultaneous Detection and Identification of Indiana and New Jersey Serotypes of Vesicular Stomatitis Virus

Abstract

In order to establish a rapid and reliable system for the detection of vesicular stomatitis virus (VSV), we developed a quantitative reverse transcription-PCR assay for the detection, quantification, and differentiation of the major serotypes, VSV Indiana and VSV New Jersey, using a closed-tube multiplex format. The detection system is based on the recently invented primer-probe energy transfer (PriProET) system. A region of the gene encoding the RNA-dependent RNA polymerase was amplified by using VSV-specific primers in the presence of two serotype-specific fluorescent probes. By incorporating nucleotide analogues in the primers, both serotypes were amplified with similar efficiencies. The generation of specific amplicons resulted in fluorescent signals for either of the two serotypes, and the specificities of the reactions were confirmed from the melting temperature profiles of the fluorescent probes. The limits of detection were found to be less than 10 50% tissue culture infective doses/ml for both serotypes. The diagnostic value of the new method was tested with clinical materials from experimentally infected pigs, and it is concluded that the method is a powerful tool for the rapid identification of VSV.Vesicular stomatitis (VS) is a zoonotic vesicular disease caused by a negative-sense single-stranded RNA virus member of the Rhabdoviridae family, genus Vesiculovirus. VS virus (VSV) contains two main serotypes, Indiana-1 (Ind-1) and New Jersey (NJ), and multiple subtypes, including Ind-2 and Ind-3 (13). The infection is limited to the Western Hemisphere, but it has been described in France and South Africa (6). The NJ serotype accounts for more than 80% of the clinical cases reported, and the Ind-1 serotype accounts for the remaining clinical cases in areas of endemicity (13). The Office International des Epizooties classifies VS as a list A disease (9), as the clinical signs in domestic livestock, such as cattle and pigs, are indistinguishable from those of foot-and-mouth disease (FMD) (6). The host spectrum of VSV is wide; cattle, pigs, horses, and numerous other species can be infected, which severely complicates efforts for the eradication of VSV. Even humans can occasionally be infected with VSV (6).The appearance of typical clinical signs of vesicular disease in domestic livestock should always cause a suspicion of FMD. When clinical samples are negative for FMD virus (FMDV), rapid laboratory tests that reliably identify VSV are of urgent need. The diagnostic tools presently available for the diagnosis of VSV infection include virus isolation (by inoculation of cell cultures) and viral antigen detection (enzyme-linked immunosorbent assay and complement fixation and neutralization tests) (9). Detection of VSV by amplification of viral RNA by reverse transcription-PCR (RT-PCR) has been reported previously (4,7,8,14). These assays are exclusively based on gel-based identification of PCR products and are designed to detect NJ (14), Ind-1 and/or NJ by a heminested assay (4), and Ind-1 and NJ by a multiple...

Detection and Serotype-Specific Differentiation of Vesicular Stomatitis Virus Using a Multiplex, Real-Time, Reverse Transcription-Polymerase Chain Reaction Assay

“…7,10,12 Thus far, there are a minimal number of real-time assays in literature for comparison. 14 This real-time RT-PCR assay proved to be sensitive and specific for the detection of VSV-NJ and VSV-IN (1-3) in a single reaction. More extensive validation, including testing of field samples of distinct genetic lineages and broad geographic origin will be needed to evaluate the full potential of this assay.…”

“…Other published assays only amplify VSV-IN1 and VSV-NJ, and are potentially more complicated and/or have not been tested on field samples. 7,10,12,14,17 …”

N-Glycans on Nipah Virus Fusion Protein Protect against Neutralization but Reduce Membrane Fusion and Viral Entry

“…For the quantitation of viral genome copies, viral RNA extracted from the viral preparations using an RNA extraction kit (QIAGEN, Valencia, CA) was used for a reverse transcriptase reaction using an iScript cDNA synthesis kit (Bio-Rad, Hercules, CA). The number of VSV genomes was then quantified using quantitative PCR according to a protocol which can quantify the number of VSV genomes from cell culture material (29). This assay uses primers and probes targeted to the L gene of the Indiana serotype, the parental virus from which the reporter vector was derived.…”

Development of Primer-Probe Energy Transfer real-time PCR for the detection and quantification of porcine circovirus type 2

“…In general, these systems require perfect hybridisation between the probe and the viral target region for comparative quantification of viral load, since the results might be influenced by point mutations in the PCV2 genomes. During our work with variable viral genomes it has turned out that the robust and sensitive primerprobe energy transfer (PriProET) method is able to solve this problem, since the results of this test are less influenced by point mutations in the targeted viral genomes (Rasmussen et al, 2003;Rasmussen et al, 2005;Hakhverdyan et al, 2006).…”