Diagram of the hybrid BCoV DI RNA and the head-to-tail strategy used to detect the synthesis of (−)-strand DI RNA.

<p>(A) Upper panel: the composition of naturally occurring BCoV DI RNA relative to the full-length BCoV genome. Lower panel: the modified BCoV DI RNA construct BM 25A used in this study. The 65-nt leader sequence is illustrated with a filled rectangle. (B) Strategy for determining the synthesis of (–)-strand DI RNA. RNA extracted at 8 hpt of DI RNA transcript was treated with TAP and head-to-tail ligated. MHV-A59 3′ UTR-negative-strand-specific primer 1 MHV3UTR3(–) was used for RT with the ligated RNA as template. MHV-A59 3′ UTR-negative-strand-specific primer 2 MHV3UTR6(–) and BCoV 5′ UTR-positive-strand specific primer 3 BCoV23-40(+) were used for PCR.