USE OF REVERSE-TRANSCRIPTASE POLYMERASE-CHAIN-REACTION (RT-PCR) AND DOT-BLOT HYBRIDIZATION FOR THE DETECTION AND IDENTIFICATION OF AFRICAN HORSE SICKNESS VIRUS NUCLEIC-ACIDS

A coupled reverse transcriptase-polymerase chain reaction assay (RT-PC R) for the detection of African horse sickness virus (AHSV) dsRNA, has been developed using genome segment 7 as the target template for prim ers. RNA from isolates of all nine AHSV serotypes were readily detecte d. The potential inhibitory effects of either ethylene diamine tetra a cetic acid (EDTA) or heparin on the RT-PCR were eliminated by washing blood samples before lysis of the red blood cells and storage. There w as a close agreement in the sensitivity and the specificity of the RT- PCR and an indirect sandwich ELISA. Confirmation of the presence of AH SV using RT-PCR and dot-blot hybridization on blood samples collected from horses experimentally infected with AHSV serotype 4 (AHSV 4) and AHSV serotype 9 (AHSV 9), was achieved within 24 hours, compared to th e period of several days required for virus isolation. The RT-PCR and virus isolation methods showed similar levels of sensitivity when used for the detection of AHSV in 3 horses infected with AHSV 4, and in 2 out of 3 horses infected with a less virulent isolate of AHSV 9. Altho ugh viraemia was detected in the third horse by virus isolation, from 6 to 14 days after infection, this animal remained consistently negati ve by RT-PCR. Conversely, AHSV viral RNA was detected by RT-PCR in the blood of 4 donkeys and 4 mules up to 55 days after their experimental infection despite the absence of any detectable infectious virus. RT- PCR is a sensitive and rapid method for detecting AHSV nucleic acids d uring either the incubation period at the start of an African horse si ckness (AHS) epizootic, or for epidemiological investigations in speci es where clinical signs may be inapparent.