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 develop 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 Africa. The probes were able to detect AHSV isolates from recent field cases of AHS in South Africa, despite being derived from historical reference strains. With regard to sensitivity and time considerations: radioactive 32P-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 completed in 4 days, which is less than half the time required for conventional isolation and serotyping. This report on the use of a complete set of cloned AHSV VP2-gene probes is the first demonstration of typing for a whole specie (serogroup) in a genus of the family Reoviridae.
Koekemoer
J J
JJ
Onderstepoort Veterinary Institute, 0110, Onderstepoort, South Africa.
Potgieter
A C
AC
Paweska
J T
JT
Van Dijk
A A
AA
D000355:African Horse Sickness / Q000175:diagnosis* / Q000821:virology; D000356:African Horse Sickness Virus / Q000235:genetics* / Q000302:isolation & purification; D000818:Animals; D000833:Animals, Suckling; D001345:Autoradiography; D015152:Blotting, Northern; D002213:Capsid / Q000235:genetics*; D036022:Capsid Proteins; D002460:Cell Line; D002645:Chickens; D002522:Chlorocebus aethiops; D006224:Cricetinae; D015342:DNA Probes / Q000096:biosynthesis* / Q000235:genetics; D016679:Genome, Viral; D006736:Horses; D008163:Luminescent Measurements; D051379:Mice; D012367:RNA, Viral / Q000032:analysis; D011933:Reagent Kits, Diagnostic; D012680:Sensitivity and Specificity; D012703:Serotyping; D014709:Vero Cells
D036022:Capsid Proteins; D015342:DNA Probes; D012367:RNA, Viral; D011933:Reagent Kits, Diagnostic; C442519:VP2 protein, African horse sickness virus; C442520:VP2 protein, Bluetongue virus