Development of probes for typing African horsesickness virus isolates using a complete set of cloned VP2-genes

A set of cloned full-length VP2-genes from the reference strain of each of the nine serotypes of African horsesickness virus (AHSV) was used to develo p probes for typing AHSV isolates. The VP2-gene probes hybridised serotype- specific to purified viral dsRNA from its corresponding serotype. No cross- hybridisation was observed between the different AHSV serotypes or with RNA from equine encephalosis virus or bluetongue virus (BTV) which are related viruses within the genus Orbivirus that co-circulate with AHSV in South Af rica. The probes were able to detect ANSV isolates from recent field cases of AHSV in South Africa, despite being derived from historical reference st rains. With reguard to sensitivity and time considerations: radioactive P-3 2-labelling resulted in a marginal increase in sensitivity over digoxigenin -labelled probes. By infecting cell cultures at different multiplicities of infection (m.o.i.) and harvesting at various times post infection, it was established that AHSV RNA could be detected 16 h post infection (p.i.) at a m.o.i. of 1.00 pfu per cell and 48 h p.i. at a m.o.i. of 0.01 pfu per cell . Typing of AHSV isolates by means of VP2-gene probe hybridisation can be c ompleted in ii days. which is less than half the time required for conventi onal isolation and serotyping. This report on the use of a complete set of cloned AHSV VP2-gene probes is the first demonstration of typings for a who le specie (serogroup) in a genus of the family Reoviridae. () 2000 Elsevier Science B.V. All rights reserved.