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

BoDV-1 (Borna disease virus 1) is a negative-sense RNA virus that establishes persistent infection in the mammalian central nervous system and has recently been associated with fatal encephalitis in humans. During replication, BoDV-1 produces three types of RNA: genomic RNA, antigenomic RNA, and mRNA. However, conventional RT-qPCR cannot distinguish these RNA species. This study aimed to develop a strand-specific RT-qPCR assay capable of detecting and quantifying each viral RNA individually. Standard RNAs were prepared using the 5′- and 3′-terminal 1,200-nucleotide regions of the viral genome, the full-length N mRNA, and the 3′ region of L mRNA. Strand specificity was achieved by using tagged RT primers and increasing the reverse transcription temperature to 57°C. Vero cells infected with BoDV-1 were monitored for one month. The assay specifically detected genomic and antigenomic RNAs, whereas N mRNA could not be completely distinguished from antigenomic RNA because of their highly similar sequences. Therefore, N mRNA levels were estimated by subtracting the antigenomic RNA signal. Viral RNA levels increased during infection, while the antigenome-to-genome ratio remained constant, suggesting stable viral replication. In contrast, the mRNA-to-genome ratio gradually decreased, indicating transcriptional suppression during persistent infection. Limitations of the study include the use of synthetic standard RNAs, experiments performed only in Vero cells, and the analysis of only N mRNA among the viral transcripts.
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