Enhancing African horse sickness virus detection: comparing and adapting PCR assays

African horse sickness (AHS) is the only equine disease for which the World Organisation for Animal Health (WOAH) gives official disease-free status, given that it poses a major threat to the equine industry. The disease is caused by AHS virus (AHSV; family Sedoreoviridae, taxon species Orbivirus alphaequi), which is endemic in sub-Saharan Africa. Reverse-transcription quantitative real-time PCR (RT-qPCR) is a rapid, sensitive detection method used in the diagnosis of AHS and the certification of animals as negative for AHSV for the purpose of movement. Genetic variability of AHSV may influence the accuracy of RT-qPCR detection methods because of possible mispriming and/or probe binding failures. We evaluated the diagnostic accuracy of the current WOAH-recommended RT-qPCR assays for the detection of AHSV, namely the Agüero et al. and Guthrie et al. methods. Utilizing 150 AHSV-positive diagnostic samples, we performed in vitro analysis using both assays. The Agüero assay failed to detect AHSV in 13 samples (8.7% false-negative rate). The AHSV VP7 genes of the 13 negative samples, and publicly archived sequences were used to perform in silico analysis, and we incorporated minor changes into the primers and probes of modified Guthrie and modified Agüero assays. A second in vitro analysis yielded 100% sensitivity for both assays. Differences in both the in silico and in vitro analyses highlight the need for continuous monitoring of the efficacy of molecular protocols used for the detection of AHSV.