Viromics of extant insect orders unveil the evolution of the flavi-like superfamily.

Insects are the most diversified and species-rich group of animals and harbor an immense diversity of viruses. Several taxa in the flavi-like superfamily, such as the genus , are associated with insects; however, systematic studies on insect virus genetic diversity are lacking, limiting our understanding of the evolution of the flavi-like superfamily. Here, we examined the diversity of flavi-like viruses within the most complete and up-to-date insect transcriptome collection comprising 1,243 insect species by employing a RdRp profile hidden Markov model search. We identified seventy-six viral sequences in sixty-one species belonging to seventeen insect, one entognathan, and one arachnidan orders. Phylogenetic analyses revealed that twenty-seven sequences fell within the phylogeny but did not group with established genera. Despite the large diversity of insect hosts studied, we only detected one virus in a blood-feeding insect, which branched within the genus , indicating that this genus likely diversified only in hematophagous arthropods. Nine new jingmenviruses with novel host associations were identified. One of the jingmenviruses established a deep rooting lineage additional to the insect- and tick-associated clades. Segment co-segregation phylogenies support the separation of tick- and insect-associated groups within jingmenviruses, with evidence for segment reassortment. In addition, fourteen viruses grouped with unclassified flaviviruses encompassing genome length of up to 20 kb. Species-specific clades for Hymenopteran- and Orthopteran-associated viruses were identified. Forty-nine viruses populated three highly diversified clades in distant relationship to , a plant-infecting virus family, suggesting the detection of three previously unknown insect-associated families that contributed to tombusvirus evolution.