This is the repository for all supplementary files of the publication: Triebel, S., Euelnfeld, T., Ontiveros, N. et al. First full-genome alignment representative for the genus Pestivirus. RNA - published in advance (2025). https://doi.org/10.1261/rna.080732.125 Abstract The members of the genus Pestivirus in the family Flaviviridae comprise economically important pathogens of livestock like classical swine fever (CSFV) and bovine viral diarrhea virus (BVDV). Intense research over the last years has revealed that at least 11 recognized and eight proposed pestivirus species exist. The single-stranded, positive-sense RNA genome encodes for one large polyprotein, which is processed by viral and cell-derived proteases into 12 mature proteins. Besides its protein-coding function, the RNA genome also contains RNA secondary structures with critical importance for various stages of the viral life cycle. Some of those RNA secondary structures, like the internal ribosome entry site (IRES) and a 3' stem-loop essential for genome replication, have already been studied for a few individual pestiviruses. In this study, we provide the first genome-wide multiple sequence alignment (MSA) including all known pestivirus species (accepted and tentative). Moreover, we performed a comprehensive analysis of RNA secondary structures phylogenetically conserved across the complete genus. While showing that well-described structures, like a 5' stem-loop structure, the IRES element, and the 3' stem loop SLI are conserved between all pestiviruses, other RNA secondary structures in the 3' untranslated region (UTR) were only conserved in subsets of the species. We identified 29 novel phylogenetically conserved RNA secondary structures in the protein-coding region, with thus far unresolved functional importance. The microRNA binding site for miR-17 was previously known in species A, B, and C; in this study, we identified it in ten additional species, but not in species K, S, Q, and R. Another interesting finding is the identification of a putative long-distance RNA interaction between the IRES and the 3' end of the genome. These results, together with the now available comprehensive multiple sequence alignment including all 19 pestivirus species, represent a valuable resource for future research and diagnostic purposes. The supplementary files include: F1 - original data set (fasta) F2 - pre-filtered data set (fasta) F3 - phylogenetic tree (nwk, nex) F4 - representative genomes (fasta) F5 - AnchoRNA output (nucleotide and protein sequences) (gff) F6 - nucleotide alignment (aln, fasta, stk; latter with additional annotations such as RNA secondary structures and genes) F7 - nucleotide alignment of the CDS (aln, fasta, stk; latter with additional annotations such as RNA secondary structures and genes) F8 - protein alignment (aln, fasta, stk) F9 - nucleotide alignment of the 5’ UTR (aln, fasta, stk) F10 - nucleotide alignment of the 3’ UTR (aln, fasta, stk) F11 - nucleotide alignment of the 3’ UTR of species A (stk) F12 - nucleotide alignment of the 3’ UTR of species C (stk) F13 - nucleotide alignment of the 3’ UTR of species K, S, and the Zikole strain (stk)