First full-genome alignment representative for the genus <i>Pestivirus</i>

Members of the genus Pestivirus (family <i>Flaviviridae</i>) comprise economically important livestock pathogens like classical swine fever virus (CSFV) and bovine viral diarrhea virus (BVDV). Research over recent years revealed 11 recognized and eight proposed species. The single-stranded, positive-sense RNA genome encodes one large polyprotein that is processed by viral and cellular proteases into 12 mature proteins. In addition to its protein-coding function, the RNA genome contains secondary structures critical for various stages of the viral life cycle. Some of these structures, including the internal ribosome entry site (IRES) and a 3' stem-loop, essential for genome replication, have been studied in individual pestiviruses. Here, we present the first genome-wide multiple sequence alignment comprising all known pestivirus species (accepted and tentative) and a comprehensive analysis of phylogenetically conserved RNA secondary structures across the genus. Well-characterized elements, such as a 5' stem-loop, the IRES, and the 3' stem-loop SL I, were conserved in all pestiviruses, whereas additional 3' untranslated region structures were conserved only in subsets of species. We identified 29 novel conserved RNA secondary structures within the protein-coding region, with thus far unresolved functional importance. A miR-17 binding site, previously described in species A, B, and C, was detected in ten additional species but absent in species K, S, Q, and R. We identified a putative long-distance RNA interaction between the IRES and the 3' end of the genome. Together, these findings and the comprehensive MSA of all 19 pestivirus species provide a valuable resource for future research and diagnostic applications.