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Title: Validation of a reverse transcriptase multiplex PCR test for the serotype determination of U.S. isolated of bluetongue virus

Author
item JOHNSON, DONNA - USDA, APHIS, NVSL
item Wilson, William
item PAUL, P - IOWA STATE UNIV.

Submitted to: Veterinary Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/27/2000
Publication Date: 11/1/2000
Citation: Johnson, D.J., Wilson, W.C., Paul, P.S. 2000. Validation of a reverse transcriptase multiplex PCR test for the serotype determination of U.S. isolated of bluetongue virus. Veterinary Microbiology. 76:105-115. 2000.

Interpretive Summary: Bluetongue virus is the causative agent of disease in sheep and cattle. The presence of the virus in the USA has been used to impose trade restriction of livestock and germplasm. This study validates the recently developed genetic test the differentiate the US types of this virus. This rapid test should expedite the movement of US livestock.

Technical Abstract: Bluetongue (BT) is an arthropod-borne viral disease affecting ruminants primarily in tropical and temperate regions of the world. Of the 24 serotypes of BT virus (BTV) identified worldwide, five have been found in the United States. Serotype identification of BTV isolates is important to the epidemiology of the virus, but current methods are cumbersome. A single-tube multiplex reverse transcriptase polymerase chain reaction (mRT-PCR) assay, previously developed for the serotype determination of U.S. BTV isolates, was evaluated. The determination of serotype was based on the size of the resultant amplified product. The procedure was evaluated using all 24 serotypes of BTV and nine serotypes of epizootic hemorrhagic disease virus (EHDV), a closely related orbivirus. Only the five U.S. serotypes of BTV were detected by the mRT-PCR. The assay was further tested using 132 BTV isolates originating from 24 western and southern states of the United States, from several different host species, spanning a period of 24 years. The serotypes of the isolates were determined by both a virus neutralization (VN) procedure and the mRT-PCR. Comparison of the mRT- PCR to the standard VN showed that the mRT-PCR successfully identified the serotypes of 130 of the isolates and was shown to be more reliable and specific than the VN assay.