PCR DETECTION OF NORTH-AMERICAN AND CENTRAL AFRICAN ISOLATES OF EPIZOOTIC HEMORRHAGIC DISEASE-VIRUS (EHDV) BASED ON GENOME SEGMENT-10 OF EHDV SEROTYPE-1

PCR amplification technology for the detection of epizootic hemorrhagi c disease virus (EHDV) ribonucleic acid in cell culture and clinical s pecimens was developed. With oligoribonucleotide primers selected from genome segment 10 of EHDV serotype 1 (EHDV-1), which codes for tyro n onstructural proteins (NS3 and NS3a), the PCR-based assay resulted in a 535-bp PCR product. RNAs from North American EHDV-1 prototype, EHDV- 2 prototype, and a number of EHDV field isolates, including the Centra l African isolates of ENDV-5 and EBDV-318 propagated in cell cultures, mere detected by this PCR-based assay. The specific 535-bp PCR produc ts mere visualized onto agarose gels, and the identity of the PCR prod ucts was confirmed by chemiluminescent hybridization with a 352-bp int ernal probe. The sensitivity of the EHDV PCR assay was increased by ch emiluminescent hybridization; by this ENDV-NS3 PCR, 10 fg of EHDV RNA was detected (equivalent to 600 viral particles). Amplification produc t was not detected when the PCR-based assay was applied to RNAs from N orth American bluetongue virus prototype serotypes 2, 10, 11, 13, and 17; total nucleic acid extracts from uninfected BHK-21 cells; or unfra ctionated blood from calves and deer that were EHDV seronegative and v irus isolation negative. The described EHDV PCR-based assay with prime rs derived from segment 10 of EHDV-1 resulted in detection of EHDV RNA from blood and tissues collected from calves and deer with natural an d experimental EHDV infections and provides a valuable tool to study t he epidemiology of EHDV infection in susceptible ruminants.