Knowles, N. J. | bioRxiv Science

bioRxiv2026

Comprehensive hallmark gene sequence, genomic and structural analysis of Picornavirales viruses clarifies new and existing taxa

The order Picornavirales is a group of highly diverse RNA viruses that includes many pathogens of significance to human and veterinary health, agriculture and the wider environment. However, the wide range of viruses assigned to the order, together with their genomic variability, and the recent description of numerous "picorna-like" viruses derived from metagenomic analyses of environmental samples, challenges the existing taxonomic classification of members of the order and the criteria for their classification. Here, we combine the existing gold standard, hallmark RNA-dependent RNA-polymerase (RdRp) gene sequence-based analysis with helicase sequence-based phylogeny, RdRp structural prediction through the use of ColabFold and Fold Tree, and analysis of coding complete genomes using GRAViTy-V2, to genetically classify 525 Picornavirales genomes and recently described "picorna-like" viruses. All analyses were conducted with a bespoke, fully automated pipeline for retrieval of genomes, domain classification and extraction, phylogenetic analysis, and output conditioning, which is available as open-source software. Our results reveal broad support for existing families as well as for six novel families, and 32 new genera. In instances where inconsistencies were found between classification methods, we demonstrate how examination of the pipelines output may be used to reconcile differences with respect to the genomic features quantified by the analysis. Automated multimodal taxonomic analysis may save significant resources over manual methods and better define demarcation criteria for families and genera.

microbiology↗

bioRxiv2021

Mutagenesis Mapping of RNA Structures within the Foot-and-Mouth Disease Virus Genome Reveals Functional Elements Localised in the Polymerase (3Dpol) Encoding Region.

AbstractRNA structure plays a crucial role in the replication of positive sense RNA viruses and can form functional elements within the untranslated regions (UTRs) and the protein coding sequences (or open reading frames (ORFs)). While RNA structures in the UTRs of several picornaviruses have been functionally characterised, the roles of putative RNA structures predicted for the ORF remain largely undefined. Here we have undertaken a bioinformatic analysis of the foot-and-mouth disease virus (FMDV) genome and predicted the existence of 53 evolutionarily conserved RNA structures within the ORF. Forty-five (45) of these structures were located in the regions encoding the non-structural proteins (nsps). To investigate if the structures in the regions encoding the nsps are required for FMDV replication we used a mutagenesis method, CDLR mapping, where sequential coding segments were shuffled to minimise RNA secondary structures while preserving protein coding, native dinucleotide frequencies and codon usage. To examine the impact of these changes on replicative fitness, mutated sequences were inserted into an FMDV sub-genomic replicon. We found that three of the RNA structures, all at the 3 termini of the FMDV ORF, were critical for replicon replication. Contrastingly, disruption of the other 42 conserved RNA structures that lie within the regions encoding the nsps had no effect on replicon replication, suggesting that these structures are not required for initiating translation or replication of viral RNA. Conserved RNA structures that are not essential for virus replication could provide ideal targets for the rational attenuation of a wide range of FMDV strains. ImportanceSome RNA structures formed by the genomes of RNA viruses are critical for viral replication. Our study shows that of 45 conserved RNA structures located within the regions of the foot-and-mouth disease virus (FMDV) genome that encode the non-structural proteins, only three are essential for replication of an FMDV sub-genomic replicon. Replicons replication is dependent on RNA translation and synthesis; thus, our results suggest that the three RNA structures are critical for either initiation of viral RNA translation and/or viral RNA synthesis. Although further studies are required to identify if the remaining 42 RNA structures have other roles in virus replication, they may provide targets for the rational large-scale attenuation of a wide range of FMDV strains. FMDV causes a highly contagious disease posing a constant threat to global livestock industries. Such weakened FMDV strains could be investigated as live-attenuated vaccines or could enhance biosecurity of conventional inactivated vaccine production.

evolutionary biology↗