A NESTED PCR FOR DETECTION OF NORTH-AMERICAN ISOLATES OF BLUETONGUE VIRUS-BASED ON NS1 GENOME SEQUENCE-ANALYSIS OF BTV-17

A nested polymerase chain reaction (PCR)-based assay, for detection of bluetongue virus (BTV) ribonucleic acid in cell culture and tissue sa mples, was developed. Two pairs of oligonucleotide primers (BTV1 and B TV4 and BTV2 and BTV3), selected from non-structural protein 1 (NS 1) gene of BTV-17, were used for the nested PCR in two amplification step s. First a 826-bp product was amplified using an outer primer pair BTV 1 and BTV4, The second amplification, using nested or internal primer pair BTV2 and BTV3, produced a 517-bp PCR product. RNA from North Amer ican prototype serotypes 2, 10, 11, 13 and 17, propagated in cell cult ures, were detected by this nested PCR-based assay. The nested primers BTV2 and BTV3 increased the sensitivity of the BTV PCR assay, and as little as 0.1 fg of BTV RNA (equivalent to 5 viral particles) could be detected. Amplification products were not detected when the PCR-based assay was applied to RNA from a closely related orbivirus, epizootic hemorrhagic disease virus (EHDV) prototype serotypes 1 and 2; total nu cleic acid extracts from uninfected BHK-21 cells; or whole blood from calves and deer that were BTV-seronegative and virus isolation negativ e, Application of this nested BTV PCR-based assay to clinical samples resulted in detection of BTV RNA from a variety of tissues collected f rom calves and deer with natural and experimental BTV infections. The described BTV PCR-based assay provides a valuable tool to study the ep idemiology of BTV infection in susceptible wild ruminants and domestic livestock. (C) 1998 Elsevier Science B.V.