Development of a multiplex real-time PCR assay for bovine immunodeficiency virus and bovine leukemia virus and its application to a large-Scale survey in Kyushu, Japan.

Bovine immunodeficiency virus (BIV) and bovine leukemia virus (BLV) are retroviruses infecting cattle, with BLV being a known cause of enzootic bovine leukosis. In contrast, BIV is considered non-pathogenic and has been less studied, despite its relevance to animal lentiviral evolution. This study aimed to develop and validate a multiplex real-time PCR assay for simultaneous detection of BIV proviral DNA and quantification of BLV proviral load. Novel primers and probes targeting the BIV pol gene were designed. The assay demonstrated a detection limit of 10 copies per reaction with a probability of 83%, comparable to conventional nested PCR. Importantly, the addition of BIV primers and probes did not interfere with the accurate quantification of BLV. Furthermore, BIV detection was unaffected even in samples with high BLV proviral loads, confirming the assay's robustness for simultaneous detection. The validated assay was applied to a molecular epidemiological survey of 6,051 cattle in the Kyushu region of Japan, conducted between 2021 and 2025. No BIV proviral DNA was detected in any sample, indicating a low prevalence of BIV in this population. This multiplex PCR assay provides a sensitive, efficient, and labor-saving tool for integrated surveillance of retroviral infections in cattle. Furthermore, it offers a valuable platform for future studies on the epidemiology and ecological role of BIV in bovine populations.