Comparative analysis of 3 qPCR primer–probe sets for the detection of equid alphaherpesvirus 1

With the revision of the World Organisation for Animal Health (WOAH) Terrestrial Manual on equine rhinopneumonitis in 2024, 3 recommended qPCR primer-probe sets were added for the detection of equid alphaherpesvirus 1 (EqAHV1; formerly equine herpesvirus 1 [EHV1]; family <i>Orthoherpesviridae</i>, taxon species <i>Varicellovirus equidalpha1</i>), also known as equine abortion virus. We compared the sensitivity and specificity of the 3 qPCR primer-probe sets to determine the most reliable set. Sets gB1H and gB1P, which target the glycoprotein B (<i>gB</i>) gene of EqAHV1, detected all 10 copies and even lower copy numbers. In contrast, set gC1 (ISO 17025-accredited method used at the WOAH reference laboratory), which targets the glycoprotein C (<i>gC</i>) gene, failed to detect ≤10 copies of EqAHV1. Our results showed the lower sensitivity of gC1, which was not improved by modification of primer and probe concentrations. gB1P detected not only EqAHV1 but also equid alphaherpesvirus 4 (EqAHV4; <i>Orthoherpesviridae</i>, <i>Varicellovirus equidalpha4</i>), likely owing to an erroneous amplification of the homologous EqAHV4 <i>gB</i> gene, indicating that gB1P is not suitable for the detection of EqAHV1 with high specificity. We then compared gB1H with gB1D, a set recommended in the previous version of the Manual, using 120 nasal swabs collected from febrile horses. gB1H had slightly higher sensitivity than gB1D. gB1H proved to be the most reliable primer-probe set for detecting EqAHV1, with high sensitivity and specificity. Nevertheless, individual laboratories are encouraged to validate these methods under their own conditions before implementation.