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.

Abstract

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.