Data Mining Sheds Light on a Novel Family of Plant-Associated Negative-Sense RNA Viruses Linked to Lispiviruses

Large-scale mining of public transcriptomic datasets can reveal viral diversity that remains invisible to conventional virus-surveillance approaches, while increasingly powerful structure-prediction methods provide a complementary route to characterizing highly divergent viral proteins. Here, we combine sequence detection, phylogenetic analysis, structural prediction, and host-association analyses to investigate the cryptic diversity and biology of a new clade of plant-associated lispi-like viruses. Analyses of RNA-sequencing datasets identified and enabled assembly of 87 coding-complete lispi-like virus genome sequences associated with 75 plant hosts, expanding the known diversity of this new group by approximately 40-fold. The viruses share a conserved four-cistron genome organization, 3′-N(P1)-P2-P3-L(P4)-5′, in which the first cistron is referred to interchangeably as N or P1 and the fourth as L or P4. Structural analyses generate testable structural hypotheses for the four conserved proteins. N (P1) adopts a canonical negative-strand RNA virus nucleocapsid architecture with conserved RNA-interacting residues and a predicted RNA-packaging configuration. P2 is exceptionally divergent, although a subset of structures resembles the ITPase/HAM1 fold. P3 forms a conserved trimeric coiled-coil architecture reminiscent of a viral fusion-protein stalk, but lacks the family-wide sequence features expected of a canonical membrane glycoprotein. L (P4) contains a structurally resolved Mononegavirales-type RNA-dependent RNA polymerase (RdRp) core with invariant catalytic motifs, including the characteristic GDN signature, whereas its accessory regions are substantially more divergent. Phylogenetic insights form a distinct monophyletic lineage sister to the predominantly invertebrate-associated Lispiviridae, supporting their recognition as the new proposed family Masuviridae, comprising 15 tentative genera. Genus-level clustering is accompanied by marked differences in host association, ranging from strong specialization to broader host ranges. Retrospective screening of public sequencing libraries further identified lispi-like virus sequences in 1536 libraries representing 134 plant species, 11 plant families and 182 geographic locations, highlighting a substantial and geographically widespread cryptic virome. Together, these results establish Masuviridae as a deeply divergent lineage of plant-associated negative-sense RNA viruses and illustrate how the integration of sequence, structural, and large-scale transcriptomic analyses can move viral dark matter from detection towards evolutionary and functional characterization.