An orphan viral genome with unclear evolutionary status sheds light on a distinct lineage of flavi-like viruses infecting plants.

Advancements in high-throughput sequencing and associated bioinformatics methods have significantly expanded the RNA virus repertoire, including novel viruses with highly divergent genomes encoding "orphan" proteins that apparently lack homologous sequences. This absence of homologs in routine sequence similarity search complicates their taxonomic classification and raises a fundamental question: Do these orphan viral genomes represent viruses? In 2022, an orphan viral genome encoding a large polyprotein was identified in alfalfa () and thrips (), and named Snake River alfalfa virus (SRAV). SRAV was initially proposed as an uncommon flavi-like virus identified in a plant host distantly related to family . Subsequently, another research group showed its common occurrence in alfalfa but challenged its taxonomic position, suggesting it belongs to the family . In this study, a large-scale analysis of 77 publicly available small RNA datasets indicates that SRAV could be detected across various tissues and cultivars of alfalfa, and has a broad geographical distribution. Moreover, profiles of the SRAV-derived small interfering RNAs (vsiRNAs) exhibited typical characteristics of viruses in plant hosts. The evolutionary analysis suggests that SRAV represents a unique class of plant-hosted flavi-like viruses with an unusual genome organization and evolutionary status, distinct from previously identified flavi-like viruses documented to infect plants. The latter shows a close evolutionary relationship to flavi-like viruses primarily found in plant-feeding invertebrates and lacks evidence of triggering host RNA interference (RNAi) responses so far. Moreover, mining the transcriptome shotgun assembly (TSA) database identified two novel viral sequences with a similar genome organization and evolutionary status to SRAV. In summary, our study resolves the disagreement regarding the taxonomic status of SRAV and suggests the potential existence of two distinct clades of plant-hosted flavi-like viruses with independent evolutionary origins. Furthermore, our research provides the first evidence of plant-hosted flavi-like viruses triggering the host's RNAi antiviral response. The widespread occurrence of SRAV underscores its potential ecological significance in alfalfa, a crop of substantial economic importance.