Evolutionary Variation of Poxvirus Genome Architecture

Abstract Poxviruses (PXV) are large double-stranded DNA viruses (dsDNA) that infect and cause disease in a wide variety of hosts, including humans.. They are studied as models to understand host-specific disease and they have a wide variety of applications in biotechnology, including vaccines, oncolytic virotherapy, and immunotherapy. Despite a nearly fourfold variation in genome sizes across the Poxviridae family, the basic genome architectural features driving their evolution remain poorly understood. In this study of the summated poxvirus genome sequence database, we report that while only a weak negative correlation exist between viral coding densities and genome sizes, we detected a strong positive correlation between the number of assigned open reading frames (ORF) and genome sizes. We observe an inflection point at a genome size of 229 kb, above or below with the percentage of genes encoding secreted proteins increasing with genome size, and the percentage of predicted cytoplasmic proteins decreasing with genome size. Additionally, we observed a weak positive correlation between the potential of transcript overlap that would be expected to generate dsRNA and genome size. Unexpectedly, we observed a significant positive correlation between coding density and GC-content in mpox virus isolates ( R 2 = 0.799). Further analysis illustrated a rise in both GC-content and coding density of mpox isolates between 2022 and 2024. Codon usage bias was observed to be clustered by virus families, with tryptophan being the least utilized amino acid across all poxviruses. Protein distribution analysis revealed a right-skewed distribution of poxviral predicted ORFs, with the 150 - 250 amino acid cohort containing the highest number of viral proteins. Finally, we observe a significant positive correlation between ITR sizes and duplicate gene clusters ( R 2 = 0.679). Together, these architectural findings outline the constraints and adaptive pathways governing poxviral evolution and biology. Importance This study establishes a family genome design rulebook for poxviruses and illustrates how these rules might be linked with their biology and evolution. We demonstrate that genome growth is accompanied by a modest increase in noncoding DNA and an increase in extracellular proteome investment. We further demonstrate that ITR expansions correlate with duplicate gene clusters, rather than genome size, indicating that ITRs serve as hubs of innovation. Our results provide a standard for generating hypotheses and experiments with poxviruses. This work also introduces matrices for within-family viral comparisons.