In silico detection and characterization of novel Picornavirales mined from publicly available insect RNA-seq datasets

Authors: LAHEY, ZACHARY - Oak Ridge Institute For Science And Education (ORISE); JONES, TARA-KAY - Corteva Agriscience; Simmons, Alvin; Andreason, Sharon

Submitted to: Microbiology Spectrum
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/26/2025
Publication Date: 12/17/2025
Citation: Lahey, Z., Jones, T.L., Simmons, A.M., Andreason, S.A. 2025. In silico detection and characterization of novel Picornavirales mined from publicly available insect RNA-seq datasets. Microbiology Spectrum. https://doi.org/10.1128/spectrum.01701-25.
DOI: https://doi.org/10.1128/spectrum.01701-25

Interpretive Summary: A wide variety of viruses infect insects, and sometimes these viruses are inadvertently discovered during insect sequencing experiments. In this study, a bioinformatics pipeline was developed for the detection of viruses in insect sequencing datasets. Publicly available datasets were screened using the pipeline, and 40 new insect-infecting viruses were discovered. These discoveries and the new pipeline will be of interest and useful to the scientific community and provides a new means of discovery of viruses that can be explored for pest control.

Technical Abstract: Picornavirales is a hyperdiverse order of viruses that infect assorted hosts, including arthropods. Members contain a positive-sense, single-stranded RNA genome with a polyadenylated tail. As such, they are often sequenced as ‘bycatch’ in RNA-seq experiments targeting other organisms. We developed a bioinformatic pipeline to assemble and annotate genomes of the order Picornavirales using publicly available insect metatranscriptome datasets. Using our pipeline, 40 novel viruses were discovered, including putative new members of the families Dicistroviridae, Iflaviridae, and Polycipiviridae. The hosts of the new picornaviruses include beetles, crickets, parasitic wasps, sap-sucking hemipterans, and thrips. We validated each virus as new in silico by performing a sequence similarity search of each virus against the NCBI non-redundant database and by phylogenetic reconstruction of the RNA-dependent RNA polymerase amino acid sequence from each novel virus and other members of the order Picornavirales. Three taxa classified as iflaviruses that infect Lepidoptera (butterflies and moths) render Iflaviridae paraphyletic, and their taxonomic status should be reevaluated. Two of the newly identified iflaviruses are dicistronic, the first genomes with this phenotype in the family Iflaviridae. A newly characterized psyllid iflavirus displays an inverted genome architecture and forms a clade with other psyllid-infecting iflaviruses that share this feature. We also detected and sequenced two of the novel iflaviruses, GnV2 and DcV1, in wild-captured Graminella nigrifrons and commercial Delphastus catalinae, demonstrating their presence in populations unrelated to those previously sequenced. Our pipeline provides an additional method by which novel viruses can be discovered, assembled, annotated, and submitted to nucleotide sequence archives.