Development and validation of a real time RT-qPCR assay for detection of the emerging Bluetongue virus serotype 5...

Bluetongue (BT) is a WOAH-notifiable economically important disease of ruminants caused by Bluetongue virus (BTV), transmitted by Culicoides spp. biting midges. Over the past two years, Italy has experienced a marked re-emergence of BT, with thousands of outbreaks reported due to the simultaneous circulation of several BTV strains belonging to serotypes 3, 4, and 8. Moreover, in September 2025, BTV-5 was detected in Sardinia, marking its first occurrence in Europe. Following the first identification by Whole Genome Sequencing, the development of a reliable real-time RT-qPCR-based assay capable of typing the novel BTV-5 ITA 2025 strain was essential, as currently available molecular typing methods targeting BTV segment 2, which encodes the outer capsid protein VP2, are unable to detect this newly emerging strain. Therefore, in this study we developed, optimised, and validated a real-time RT-PCR assay for the detection and typing of BTV-5 ITA 2025 in field samples. The assay is characterised by high sensitivity and specificity, as well as good reproducibility, and can be effectively applied for BTV-5 ITA 2025 diagnosis in the current epidemiological context, supporting surveillance and control strategies.