AbstractBy identifying variations in viral RNA genomes, cutting-edge metagenome technology has potential to reshape current concepts about the evolution of RNA viruses. This technology, however, cannot process low-homology genomic regions properly, leaving the true diversity of RNA viruses unappreciated. To overcome this technological limitation we applied an advanced method, Fragmented and Primer-Ligated Double-stranded (ds) RNA Sequencing (FLDS), to screen RNA viruses from 155 fungal isolates, which allowed us to obtain complete viral genomes in a homology-independent manner. We created a high-quality catalog of 19 RNA viruses (12 viral species) that infect Aspergillus isolates. Among them, nine viruses were not detectable by the conventional methodology involving agarose gel electrophoresis of dsRNA, a hallmark of RNA virus infections. Segmented genome structures were determined in 42% of the viruses. Some RNA viruses had novel genome architectures; one contained a dual methyltransferase domain and another had a separated RNA-dependent RNA polymerase (RdRp) gene. A virus from a different fungal taxon (Pyricularia) had an RdRp sequence that was separated on different segments, suggesting that a divided RdRp is widely present among fungal viruses, despite the belief that all RNA viruses encode RdRp as a single gene. These findings illustrate the previously hidden diversity and evolution of RNA viruses, and prompt reconsideration of the structural plasticity of RdRp. By highlighting the limitations of conventional surveillance methods for RNA viruses, we showcase the potential of FLDS technology to broaden current knowledge about these viruses.Author SummaryThe development of RNA-seq technology has facilitated the discovery of RNA viruses in all types of biological samples. However, it is technically difficult to detect highly novel viruses using RNA-seq. We successfully reconstructed the genomes of multiple novel fungal RNA viruses by screening host fungi using a new technology, FLDS. Surprisingly, we identified two viral species whose RNA-dependent RNA polymerase (RdRp) proteins were separately encoded on different genome segments, overturning the commonly accepted view of the positional unity of RdRp proteins in viral genomes. This new perspective on divided RdRp proteins should hasten the discovery of viruses with unique RdRp structures that have been overlooked, and further advance current knowledge and understanding of the diversity and evolution of RNA viruses.
Sequence diversity and evolution of an iflavirus family associated with ticks
