Development of a strand-specific RT-qPCR assay for detecting and quantifying Borna disease virus RNAs in infected cells

Borna disease virus 1 (BoDV-1) is a nonsegmented negative-strand RNA virus (NSV) that persists in the central nervous system of a broad range of mammalian species. Considering the recent reports linking BoDV-1 to fatal encephalitis in humans, a deeper understanding of its viral life cycle is required. Similar to other NSVs, BoDV-1 produces three types of viral RNAs in infected cells: genome, antigenome, and mRNA. Although a conventional reverse transcription quantitative real-time PCR (RT-qPCR) assay is used to detect viral RNAs, this assay cannot distinguish between RNA species because of primer-independent cDNA synthesis during the reverse transcription (RT) reaction. In this study, we developed a strand-specific RT-qPCR (ssRT-qPCR) assay, in which an RT-primer fused with a non-viral tag sequence at the 5'-end is used in the RT reaction. This assay successfully detected and quantified three types of BoDV-1 RNAs distinctively. Furthermore, using this assay, we measured the intracellular kinetics of the BoDV-1 RNA species, revealing that BoDV-1 may control transcription, but not replication, to establish and maintain persistent infection. Overall, this novel ssRT-qPCR assay is a powerful tool for understanding the life cycle of BoDV-1.