A Self-Regulatory Viral Small RNA Embedded in the Promoter Region of a Novel Transcript Facilitates Bombyx mori Bidensovirus Replication

Bombyx mori bidensovirus (BmBDV) is a bipartite single-stranded DNA virus that causes severe losses in sericulture, yet its transcriptional complexity and possible self-regulatory viral small RNAs remain unexplored. Here, transcriptome and small RNA sequencing of BmBDV-infected midguts was performed to identify novel viral transcripts and functional virus-derived small RNAs (vsRNAs). A previously unrecognized transcript (transcript IV, nt 290–2929) was discovered on the VD1 segment, sharing its 5′ promoter region with NS2 and its 3′ polyadenylation signal with VP. Notably, the most abundant viral small RNA, a 20-nt vsRNA designated vsRNA-18895 (TPM up to 11,750), mapped to the promoter-overlap region shared by NS1, NS2 and transcript IV (VD1 nt 596–615). In vivo agomir/antagomir experiments showed that injection of a double-stranded RNA corresponding to vsRNA-18895 (an agomir) significantly upregulated NS1, NS2 and transcript IV (up to 103.52-, 102.47- and 101.87-fold, respectively) and enhanced VD1 replication (103.26-fold), whereas injection of a single-strand RNA complementary to vsRNA-18895 (an antagomir) suppressed them; VD2 and other viral genes (VP, PolB, NS3 and P133) were unaffected. Because the abundance of vsRNA-18895 itself was not directly measured following antagomir injection, this treatment may either reduce, or merely interfere with, the activity of the small RNA. These findings are consistent with a positive feedback loop in which a virus-encoded small RNA embedded in the promoter region of a novel transcript facilitates BmBDV replication, providing functional evidence of a self-regulatory vsRNA in an invertebrate single-stranded DNA virus. Because a complementary antisense small RNA produced from the same locus was of comparable abundance, a possible contribution of that strand to the observed effects cannot be excluded.