Modification and validation of a reference real-time RT-PCR method for the detection of a new African horse sickness virus variant

African horse sickness (AHS) is a disease affecting equids caused by the AHS virus (AHSV). The World Organization for Animal Health (WOAH) includes AHS as a notifiable disease and, upon detection within the European Union, immediate control and eradication measures are mandated. Thus, validated diagnostic methods for rapid AHSV detection are essential. The Agüero 2008 and Guthrie 2013 rRT-PCR methods have been widely validated for detection of any AHSV strain and are included as reference rRT-PCRs in the WOAH manual. However, the WOAH reference laboratory for AHS in the Republic of South Africa (RSA), reported an AHSV variant undetected by the Agüero 2008 rRT-PCR. Therefore, a set of modified primers and probe, containing degenerate positions to avoid mismatches with the sequence of the new RSA strain, was developed. The modified-Agüero method was validated by the WOAH reference laboratories in Spain and the UK employing a broad collection of AHSV strains, clinical samples as well as a synthetic RNA mimicking the target sequence of the new RSA AHSV variant (AHSV-sRNA-RSA). Comparative assessment of the modified-Agüero versus the WOAH reference rRT-PCRs, showed that the modified method exhibited a good diagnostic performance and enabled the detection of the new RSA AHSV variant nucleic acid.