Development of a Conventional Reverse Transcription-PCR System for Broad Detection of Bovine Rhinitis A Virus

Bovine rhinitis A virus (BRAV) is a virus of the genus Aphthovirus, family Picornaviridae. BRAV has recently attracted interest as a causative agent in bovine respiratory disease complex (BRDC). However, only a few PCR systems, designed based on the limited genome information, are available to detect BRAV. Therefore, we aimed to construct a new reverse transcription-PCR (RT-PCR) system for the broad detection of BRAV and compared its sensitivity and specificity with two existing real-time RT-PCR systems. Initially, we designed a new primer pair targeting the highly conserved 3D^pol region. The detection limit of RT-PCR using the primers against strain H-1 was 10^-2 TCID₅₀/mL, and the system was able to detect gene amounts equivalent to those detectable by existing systems. Moreover, the new system could detect synthetic DNA referring to the emerging BRAV3 strain NSWL8. The new RT-PCR system did not show cross-reactivity against non-BRAV BRDC-related viruses or taxonomically related viruses. To assess the validity of the new RT-PCR system for analysing clinical samples, nasal swabs, conjunctival swabs, and lung homogenates were collected from dairy and beef cattle in the field from 2019 to 2023 in Japan. Among the 125 clinical samples, the new RT-PCR detected BRAV from 15 samples, a higher number than two existing real-time RT-PCR systems, which identified only two and six positives. In conclusion, this RT-PCR system was demonstrated to detect currently circulating BRAVs in a highly sensitive and broad manner.