Pervasive short hairpin folding in non-encapsidated RNA viruses

Genome-wide RNA secondary structure can influence viral replication, stability, translation, and interactions with host defenses, yet its distribution and significance across RNA viruses remain poorly understood. We developed the pervasive hairpin (peHaPi) metric to quantify densely distributed stem-loop structures and applied it to 817,234 GenBank virus sequences. This revealed a distinctive RNA architecture characterized by numerous short, highly uniform hairpins spanning most of the genome. The structure was strongly enriched in capsidless RNA viruses, encompassing nearly all ormycoviruses and a substantial fraction of narnaviruses, although it also occurred in several other RNA and DNA virus groups. Comparative analyses showed that increasing peHaPi scores appeared associated with more stable and constrained predicted RNA structures and altered codon base-pairing patterns, indicating selective constraints imposed by pervasive folding. Relationships between peHaPi and RdRp sequence similarity differed among virus groups, consistent with independent evolutionary origins of this structural phenotype. Analysis of virus-derived small RNAs provided no evidence that peHaPi(+) viruses elicit a reduced siRNA response, arguing against siRNA evasion as its primary function. Finally, combining peHaPi classification with DNA-read subtraction identified candidate RNA virus contigs lacking detectable protein homology or recognizable RdRp signatures across multiple independent datasets. These findings reveal pervasive short-hairpin organization as a novel feature of predominantly capsidless viruses and establish genome-wide RNA structure as a complementary signature for discovering highly divergent ormyco- and narnaviruses.