The neurotropic Borna disease virus (BDV) causes typically a persistent virus infection of the central nervous system. In order to investigate whether an adapted virus replication contributes to BDV persistence in vivo, a fast and reliable real-time RT-PCR assay was constructed to quantify the amounts of leader-containing (leBDV) as a marker for virus replication, genomic (vBDV) and nucleoprotein-(BDV-N +ssRNA)-specific RNA. Therefore, leBDV, vBDV and BDV-N +ssRNA values were determined in experimentally infected Lewis rats between 14 and 90 days post infection (dpi). Surprisingly low leBDV values were found compared to vBDV and the abundantly expressed BDV-N transcripts. vBDV multiplied only in the acute phase of infection followed by constant expression until 90 dpi. Ratios of vBDV to leBDV were 401:1 at 14 dpi and diminished to 209:1 at 90 dpi, indicating a regulated co-expression of replicative intermediates as a potential prerequisite for viral persistence.