Phylogenetic classification of a group of self-replicating RNAs that are common in co-infections with poleroviruses

•Autonomously replicating RNAs are widespread in co-infections with poleroviruses.•These RNAs form a monophyletic group and are most closely related to tombusviruses.•Autonomous replication is confirmed and production of a subgenomic RNA is observed. Tombusvirus-like associated RNAs (tlaRNAs) are positive-sense single-stranded RNAs found in plants co-infected with viruses of the genus Polerovirus. TlaRNAs depend upon capsid proteins supplied in trans by the co-infecting polerovirus for vector transmission and intra-host systemic movement. Here, the full-length genomes of five tlaRNAs were determined using a combination of RT-PCR and next-generation sequencing, and evidence is provided for an additional tlaRNA associated with potato leafroll virus. Phylogenetic analyses based on conserved domains of the RdRp placed tlaRNAs as a monophyletic clade clustering with members of the family Tombusviridae and comprising three different subclades. Full-length clones of tlaRNAs from two of three subclades were confirmed to replicate autonomously, and each produces a subgenomic RNA during infection.