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Title: Preliminary development of a real-time PCR for all serotypes of Epizootic Hemorrhagic Disease Virus

Author
item Wilson, William
item O Hearn, Emily

Submitted to: American Association of Veterinary Laboratory Diagnosticians
Publication Type: Abstract Only
Publication Acceptance Date: 8/8/2006
Publication Date: 10/15/2006
Citation: Wilson, W.C., O Hearn, E.S. 2006. Preliminary development of a real-time PCR for all serotypes of Epizootic Hemorrhagic Disease Virus. American Association of Veterinary Laboratory Diagnosticians.

Interpretive Summary: Epizootic hemorrhagic diseases virus (EHDV) has been associated with bluetongue-like disease in cattle. Although US EHDV strains have not been experimentally proven to cause disease in cattle there is serologic evidence of infection in cattle. Bluetongue virus (BTV) causes an estimated $125,000,000 annual loss to the U.S. livestock industry and about $3,000,000,000 annual losses worldwide. Therefore rapid diagnosis and differentiation of BTV and EHDV is required. Our laboratory has developed a genetic test that detects all EHDV serotypes based on DNA sequence analysis. The EHDV detection assay does not cross-react with BTV serotypes; however, this assay is less sensitive than double amplification protocols. The sensitivity for all eight serotypes is sufficient for diagnostic applications without the contamination problems associated with standard amplification and especially double amplification tests.

Technical Abstract: Epizootic hemorrhagic diseases virus (EHDV) has been associated with bluetongue-like disease in cattle. Although US EHDV strains have not been experimentally proven to cause disease in cattle there is serologic evidence of infection in cattle. Bluetongue virus (BTV) causes an estimated $125,000,000 annual loss to the U.S. livestock industry and about $3,000,000,000 annual losses worldwide. Therefore rapid diagnosis and differentiation of BTV and EHDV is required. Our laboratory has been developing the molecular basis for early detection of indigenous and exotic BTV and EHDV disease outbreaks. The foundation for these tests was accomplished by phylogenetic analysis of two conserved target genes; one that is highly expressed in infected mammalian cells, the other is highly expressed in infected insect cells. The analysis of all BTV and EHDV prototype strains indicated that a complex primer design will be necessary for a comprehensive gene amplification diagnostic test. This information has been used as the basis for the development of rapid EHDV real-time PCR that detects all EHDV serotypes. The EHDV detection assay does not cross-react with BTV serotypes; however, this assay is less sensitive than nested-PCR protocols. The sensitivity of 1 pg double-stranded RNA for all eight serotypes is sufficient for diagnostic applications without the contamination problems associated with standard PCR and especially nested-PCR tests.