African horsesickness virus serotyping and identification of multiple co-infecting serotypes with a single genome segment 2 RT-PCR amplification and reverse line blot hybridization.
Since protection against African horsesickness (AHS) is serotype-specific, rapid serotyping of AHSV is crucial to identify the correct vaccine serotype for efficient control of the spread of AHS outbreaks, especially when they occur in non-endemic regions. This paper describes the first one-day serotyping procedure that requires only a single RT-PCR and hybridization and which can identify multiple serotypes in mixed infections in one assay. The same region of genome segment 2 of all nine AHSV serotypes is amplified in a single RT-PCR. A universal primer set, designed to amplify the 5'-terminal 521-553bp of genome segment 2 of all of the nine AHSV serotypes with one reaction, was used to generate serotype-specific probes from dsRNA prepared from infected tissue cultures or organ samples. These probes hybridized serotype-specifically with immobilized genome segment 2 cDNA of the nine AHSV reference serotypes in a checkerboard reverse line blot format. All nine AHSV reference and the seven vaccine strains and field viruses isolated up to 28 years apart could be serotyped accurately within a day. The sensitivity of the method is 1pg dsRNA which is sufficient to serotype AHSV directly from lung and spleen specimens of infected horses.
Biochemistry Division, Onderstepoort Veterinary Institute, Onderstepoort 0110, South Africa. KoekemoerO@arc.agric.za
J J O
JJ
Koekemoer
A A van
AA
Dijk
D000355:African Horse Sickness / Q000175:diagnosis / Q000821:virology; D000356:African Horse Sickness Virus / Q000145:classification* / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D000818:Animals; D018076:DNA, Complementary; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism; D016679:Genome, Viral; D006736:Horses / Q000821:virology; D008168:Lung / Q000821:virology; D009693:Nucleic Acid Hybridization; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D012703:Serotyping; D013154:Spleen / Q000821:virology
D018076:DNA, Complementary; D004279:DNA, Viral; D012367:RNA, Viral