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Title: A SINGLE-TUBE MULTIPLEX RT-PCR FOR DETECTION AND DIFFERENTIATION OF VESICULAR STOMATITIS INDIANA 1 AND NEW JERSEY VIRUSES IN INSECTS

Author
item MAGNUSON, ROBERTA - COLORADO STATE UNIV.
item TRIANTIS, JONI - COLORADO STATE UNIV.
item Rodriguez, Luis
item PERKINS, ALISHA - COLORADO STATE UNIV.
item MEREDITH, CYNTHIA - COLORADO STATE UNIV
item BEATY, BARRY - COLORADO STATE UNIV
item MCCLUSKEY, BRIAN - USDA APHIS FTCOLLINS CO
item SALMAN, MO - COLORADO STATE UNIV.

Submitted to: Journal of Veterinary Diagnostic Investigation
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/1/2003
Publication Date: 11/15/2003
Citation: MAGNUSON, R.J., TRIANTIS, J., RODRIGUEZ, L.L., PERKINS, A., MEREDITH, C.O., BEATY, B., MCCLUSKEY, B., SALMAN, M. 2003. A SINGLE-TUBE MULTIPLEX RT-PCR FOR DETECTION AND DIFFERENTIATION OF VESICULAR STOMATITIS INDIANA 1 AND NEW JERSEY VIRUSES IN INSECTS. JOURNAL OF VETERINARY DIAGNOSTIC INVESTIGATION. 15(6):561-567.

Interpretive Summary: Vesicular stomatitis is a vesicular disease that clinically is indistinguishable from foot and mouth disease. Unlike foot and mouth disease, vesicular stomatitis is caused by a virus (VSV) that is carried and transmitted by insects particularly sand flies and black flies. Detection of VSV infected insects can be difficult due to the intense work involved, problems with the toxicity of insect extracts to cells and the time consuming task of confirming the identity of any virus isolated in cells. Here we describe a single-tube test based in reverse transcription-PCR that is capable of detecting and at the same time differentiating the two major types of VS: vesicular stomatitis Indiana 1 and New Jersey viruses, not only in insect samples but also from infected cells. Using this assay we could detect either or both viruses in the same samples with very litle amount of total RNA. We could also detect vesicular stomatitis RNA in macerates containing two infected mosquitoes in pools of 10-30 non-infected mosquitoes. This test is a potential valuable tool not only for rapid detection and differentiation of vesicular stomatitis in insects but also for rapid identification of VSV in cells inoculated with clinical samples.

Technical Abstract: Vesicular stomatitis virus causes vesicular disease that clinically is indistinguishable from foot and mouth disease. Unlike foot and mouth disease, vesicular stomatitis is an arthropod-borne virus and has been isolated on many occasions from several species of insects. Two groups of insects, sand flies and black flies, have been shown to cary the virus in nature and also can replicate and transmit the virus to susceptible hosts after laboratory infection. Virus isolation from insects can be difficult due to the intense work involved, problems with cytotoxicity of insect extracts and the fact that any cytopathic effect must be confirmed by virus identification. Here we describe a multiplex single-tube reverse transcription-PCR for the detection and differentiation of vesicular stomatitis Indiana 1 and New Jersey viruses, from insect samples. Using this assay we could detect either or both viruses, in as little as 20 femtograms of total RNA. We could also detect vesicular stomatitis RNA in macerates containing two infected mosquitoes in pools of 10-30 non-infected mosquitoes. The reported reverse transcription PCR is a potential valuable tool for rapid detection and differentiation of vesicular stomatitis in insects.