DEVELOPMENT OF PCR-BASED TESTS FOR THE IDENTIFICATION OF NORTH-AMERICAN ISOLATES OF EPIZOOTIC HEMORRHAGIC-DISEASE VIRUS

A serogroup-specific polymerase chain reaction (PCR) assay and isolate identification strategies (restriction endonuclease analysis (REA) an d nucleotide sequencing) were developed for the detection of North Ame rican isolates of epizootic haemorrhagic disease virus (EHDV). PCR pri mers (EHDV-pr4, EHDV-pr5) were designed to hybridize to the L3 gene of a North American isolate of EHDV serotype 1, Total nucleic acid was e xtracted from preparations of infected tissue culture and PCR was perf ormed using a CDNA-PCR kit, according to the manufacturer's specificat ions, The PCR assay generated a 459 base pair product from North Ameri can isolates of EHDV serotypes 1 and 2, while bluetongue virus (BTV) s erotypes 10, 11, 13, and 17, and cell controls, failed to demonstrate PCR products, Slight modifications allowed for the PCR detection of EH DV-1 and -2 in white-tailed deer blood (Odocoileus virginiatus); PCR f ragments were not amplified from uninfected deer blood, A number of re striction endonucleases and sequencing primers were evaluated for thei r utility in isolate identification experiments, Specifically, REA emp loying HincII and cycle sequencing with an internal primer (EHDV-1-pr3 ) proved most successful for identifying isolate-specific genome marke rs, The techniques presented are expected to prove valuable for rapid and specific detection of possible future EHDV incursions in wild and domestic animal species.