A Single-Tube Multiplex Reverse Transcription—Polymerase Chain Reaction for Detection and Differentiation of Vesicular Stomatitis Indiana 1 and New Jersey Viruses in Insects

Abstract

Abstract.A multiplex single-tube reverse transcription-polymerase chain reaction (RT-PCR) has been developed for the detection and differentiation of vesicular stomatitis viruses (VSV), Indiana 1 and New Jersey, from insect samples. Using this assay, detection of either or both viruses in as little as 20 fg of total RNA from tissue culture was achieved, along with detection of vesicular stomatitis (VS) RNA from macerates containing 2 infected mosquitoes in pools of 10-30 noninfected mosquitoes. Vesicular stomatitis virus was detected by RT-PCR in all culture-positive samples, and detection as low as 4 plaque forming units per milliliter was achieved. Comparison between RT-PCR and tissue culture revealed that RT-PCR was able to detect VSV in a volume of insect macerate averaging almost 100 times less than that required for detection by tissue culture. The reported RT-PCR is a potential valuable tool for rapid and sensitive detection and differentiation of VS in insects because intense work associated with viral isolation, the cytotoxicity of insect extracts, and separate virus identification steps can be avoided. Potential application to detection and differentiation of VSV serotypes from vertebrate hosts is addressed.Vesicular stomatitis virus (VSV) causes vesicular lesions on the epithelia of the tongue and mouth, as well as coronary bands of hooves of cattle, pigs, and horses. 9 Humans, rodents, and numerous other mammals and fowl can also be infected. 12,16 Vesicular stomatitis virus has been isolated on many occasions in several species of insects, including mosquitoes, during viral epidemics and in forested endemic foci. 2,21-23 At least 2 groups of insects, sand flies (Lutzomyia spp) and black flies (Diptera: Simuliidae), have been shown to carry the virus in nature and to replicate and transmit VSV to susceptible hosts after laboratory infection. 2,13,20 In addition, it has been shown that black flies can act as vectors to disseminate VSV by cofeeding on a nonviremic host. 14 However, the exact role played by insects in VSV's natural cycle has not been definitely proven. Vesicular stomatitis (VS) is of considerable economic importance as quarantines must be enforced until diagnosis is complete because its clinical symptoms are indistinguishable from those of foot and mouth disease (FMD).

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

“…The PriProET assay for VSV described in this paper is a further refinement of the method for multiplex detection and differentiation. So far only a few diagnostic RT-PCR assays for VSV have been reported (4,7,8,14). These are traditional gel-based assays, and none of these are designed for the quantitative detection of VSV.…”

“…These are traditional gel-based assays, and none of these are designed for the quantitative detection of VSV. Two of the assays (7,8) are multiplex assays that detect both VSV serotypes with a combination of serotype-specific primers. To our knowledge a VSV-specific primer set has not been described before, and the high degree of genetic variability in the VSV genomes complicates the design of such primers.…”

“…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 multiplex assay (7,8).In this study, we developed a quantitative RT-PCR assay for the detection and differentiation of the two main VSV serotypes based on the novel primer-probe energy transfer (PriProET) detection system (5, 12). This fluorescence resonance energy transfer detection system combines probe-based real-time monitoring of PCR amplification with confirmation of probe hybridization from the melting temperature (T m ) curve.…”

“…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 multiplex assay (7,8).…”

“…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 multiplex assay (7,8).…”