Transcriptional mapping of the messenger and leader RNAs of orchid fleck virus, a bisegmented negative-strand RNA virus

•Six polyadenylated mRNAs were detected in orchid fleck virus (OFV)-infected plants.•The transcription start and termination sites in the OFV genome were mapped.•One to three non-viral adenine nucleotides were present at the 5' end of each OFV mRNA.•Putative plus- and minus-strand leader RNAs were found to be transcribed and polyadenylated.

The transcriptional strategy of orchid fleck virus (OFV), which has a two-segmented negative-strand RNA genome and resembles plant nucleorhabdoviruses, remains unexplored. In this study, the transcripts of six genes encoded by OFV RNA1 and RNA2 in the poly(A)-enriched RNA fraction from infected plants were molecularly characterized. All of the OFV mRNAs were initiated at a start sequence 3'-UU-5' with one to three non-viral adenine nucleotides which were added at the 5' end of each mRNA, whereas their 3' termini ended with a 5'-AUUUAAA(U/G)AAAA(A)n-3' sequence. We also identified the presence of polyadenylated short transcripts derived from the 3'-terminal leader regions of both genomic and antigenomic strands, providing the first example of plus- and minus-strand leader RNAs in a segmented minus-strand RNA virus. The similarity in the transcriptional strategy between this bipartite OFV and monopartite rhabdoviruses, especially nucleorhabdoviruses (family Rhabdoviridae) is additional support for their close relationship.