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.
Koekemoer
J J O
JJ
Biochemistry Division, Onderstepoort Veterinary Institute, Onderstepoort 0110, South Africa. KoekemoerO@arc.agric.za
Dijk
A A van
AA
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