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Title: Detection of all eight serotypes of epizootic hemorrhagic disease virus (EHDV) by real-time RT-PCR

Author
item Wilson, William - Bill
item O Hearn, Emily
item TELLGREN-ROTH, CHRISTIAN - UNIVERSITY OF WYOMING
item STALLKNECHT, DAVID - UNIVERSITY OF GEORGIA
item MEAD, DANIEL - UNIVERSITY OF GEORGIA
item Mecham, James

Submitted to: Journal of Veterinary Diagnostic Investigation
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/10/2008
Publication Date: 3/21/2009
Citation: Wilson, W.C., O Hearn, E.S., Tellgren-Roth, C., Stallknecht, D., Mead, D., Mecham, J.O. 2009. Detection of all eight serotypes of epizootic hemorrhagic disease virus (EHDV) by real-time RT-PCR. Journal of Veterinary Diagnostic Investigation. Vol 21:220-225

Interpretive Summary: This manuscript assay describes a new genetic test for Epizootic hemorrhagic disease virus (EHDV) that does not cross-react with bluetongue virus BTV strains and performed identically to a previous assay that was more labor intensive and prone to contamination problems. Differentiation of BTV and EHDV is necessary, since diagnosis of infection caused by these viruses is often confused and EHDV has been associated with bluetongue-like disease in cattle. Although U.S. EHDV strains have not been experimentally proven to cause disease in cattle, there is serologic evidence of infection in cattle.

Technical Abstract: Epizootic hemorrhagic disease virus (EHDV) has been associated with bluetongue-like disease in cattle. Although U.S. EHDV strains have not been experimentally proven to cause disease in cattle, there is serologic evidence of infection in cattle. Differentiation of bluetongue virus (BTV) and EHDV is necessary, since diagnosis of infection caused by these viruses is often confused. Our laboratory has previously developed nested RT-PCR tests for early detection of indigenous EHDV disease outbreaks. These tests, although sensitive and specific, are prone to contamination problem, because of multiple sample handling steps. Additionally, the EHDV nested RT-PCR only detects seven of the eight serotypes. To develop improved diagnostic tests, sequence analysis was performed on two conserved target genes; one is highly expressed in infected mammalian cells, while the other is highly expressed in infected insect cells. Analysis of all EHDV prototype strains indicated that a complex primer design is necessary for a comprehensive gene amplification diagnostic test. This information was used to develop a rapid EHDV real-time PCR that detects all eight EHDV serotypes. The EHDV assay did not cross-react with BTV strains and performed identically to the nested RT-PCR tests with archived clinical samples. In addition, it is superior to the nested RT-PCR, since it is a closed system with fewer cross-contamination problems, and is also less time-consuming.