DEVELOPMENT OF POLYMERASE CHAIN-REACTION FOR SPECIFIC IDENTIFICATION OF EPIZOOTIC HEMORRHAGIC-DISEASE VIRUS SEROTYPE-1

The diagnostic potential of the polymerase chain reaction (PCR) for sp ecific identification of epizootic hemorrhagic disease virus serotype 1 (EHDV-1) in cell culture and clinical specimens was evaluated. Using oligonucleotide primers, selected from genome segment 2 of EHDV-1 (Ne w Jersey strain), the PCR-based assay resulted in a 862 base pair (bp) PCR product. EHDV-1 RNA from United States prototype serotype 1 and a number of EHDV-1 held isolates, propagated in cell cultures, were det ected by this PCR based assay. The specific 862 bp PCR products were v isualized on ethidium bromide-stained agarose gel. Identity of the PCR product was confirmed by chemiluminescent hybridization with Iron rad iolabelled internal probe. Using chemiluminescent hybridization, the s ensitivity of the PCR assay was 1.0 fg of virus RNA (equivalent to 60 virus particles). Amplification product was not detected when the PCR- based assay was applied to RNA from EHDV serotype 2 (EHDV-2); the Unit ed States bluetongue virus (BLU) prototypes serotypes 2, 10, 11, 13, a nd 17; total nucleic acid extracts from uninfected BHK-21 cell; or blo od cells from calves and deer that were EHDV-seronegative and virus is olation negative. Application of this EHDV-1 PCR-based assay to clinic al samples resulted in detection of EHDV-1 RNA from blood samples, col lected from a calf experimentally infected with EHDV-1. The described PCR-based assay provides a simple, rapid, sensitive, specific and inex pensive method for specific identification of EHDV-1 infection in susc eptible ruminants.