Validation of a molecular multiplex assay for the simultaneous detection and differentiation of bluetongue virus and epizootic haemorrhagic disease virus in biological samples.

Bluetongue virus (BTV) and epizootic haemorrhagic disease virus (EHDV) are Culicoides-transmitted viruses, circulating in multiple serotypes, that cause two relevant WOAH-listed diseases of ruminants. Following its first identification in Tunisia in 2021, a novel EHDV strain belonging to serotype 8 has been detected in cattle showing BTV-like symptoms in Italy and Spain in 2022, and soon after in Portugal and France. These are European regions with recurrent circulations of different BTV serotypes. Hence, in this study we describe the validation of a TaqMan RT-qPCR panBTV/panEHDV assay, based on well-established primers and probes sets, able to simultaneously detect and distinguish between BTV and EHDV. The implemented assay, characterized by high sensitivity, specificity and good reproducibility, can be successfully applied for the rapid and affordable diagnosis needed in the current epidemiological situation and represents a powerful tool to be employed in surveillance and control strategies with a significant reduction of costs.