<i>In silico</i> detection and characterization of novel <i>Picornavirales</i> mined from publicly available insect RNA-seq data sets

<i>Picornavirales</i> is a hyperdiverse order of viruses that infect assorted hosts, including arthropods. Members of this group 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 simple-to-follow bioinformatic pipeline to assemble and annotate genomes of the order <i>Picornavirales</i> using publicly available insect metatranscriptome data sets. Using our pipeline, 40 novel viruses were discovered, including putative new members of the families <i>Dicistroviridae</i>, <i>Iflaviridae</i>, and <i>Polycipiviridae</i>. The hosts of the new picornaviruses include beetles (two families), crickets, parasitic wasps, sap-sucking hemipterans (members of all suborders), and thrips. We validated each virus as new <i>in silico</i> by performing a sequence similarity (BLAST) 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 <i>Picornavirales</i>. Three taxa classified as iflaviruses by the International Committee on Taxonomy of Viruses that infect Lepidoptera (butterflies and moths) render <i>Iflaviridae</i> 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 <i>Iflaviridae</i>. 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 <i>Graminella nigrifrons</i> and purchased <i>Delphastus catalinae</i>, demonstrating their presence in populations beyond those previously sequenced. Our pipeline provides an additional method by which novel viruses can be discovered, assembled, annotated, and submitted to nucleotide sequence archives.IMPORTANCEInsects are the most abundant group of animals on Earth and serve as reservoirs for myriad microscopic organisms, including viruses. Just a fraction of this viral diversity has been characterized despite some having considerable consequences to human and animal health, agricultural production, and overall ecological stability. In this study, we illustrate a bioinformatic viral discovery pipeline using single-stranded, positive-sense RNA viruses of the order <i>Picornavirales</i> as a case study. Picornaviruses are some of the 'dark taxa' of the virosphere that are ubiquitous in arthropods, including the vectors of serious plant diseases. Discoveries of these viruses have been accelerated by the advent of next-generation sequencing technologies, opening potential research avenues that could be exploited for pest control.