Phylogenetic and genome-wide analysis of codon usage reveals geographic and host-specific adaptation in Vaccinia virus.

Vaccinia virus (VACV) is a member of the Poxviridae family and served as the vaccine strain used to eradicate smallpox. As a large double-stranded DNA virus, VACV exhibits a broad host range and remains a model organism for studying viral evolution and gene expression. Codon usage bias (CUB) is a fundamental feature of genome evolution that reflects the interplay between mutational pressure and natural selection. In this study, we systematically analyzed codon usage patterns in 107 strains of Vaccinia virus (VACV) to uncover the driving forces behind its genomic evolution and host adaptation. Our findings revealed a strong A/T bias across all codon positions, particularly at the third codon position (AT3 = 66.59%), suggesting a mutational or selective preference for A/T-ending codons. RSCU analysis confirmed the predominance of A/T-ending codons, while dinucleotide abundance showed balanced usage without significant suppression, unlike host genomes such as Homo sapiens and Bos taurus. CAI and RCDI analyses demonstrated that VACV codon usage is better adapted to Homo sapiens, highlighting potential host-specific evolutionary pressures. The PR2 and neutrality plots further emphasized the dominant role of natural selection, with minor contributions from mutational bias. Correspondence analysis indicated that geographic origin had limited influence on codon usage variation, while correlation and ENC-GC3s analyses reinforced the interplay between mutational pressure and selection. Phylogenetic analysis revealed regional clustering of strains, particularly from Brazil and the USA, indicating localized viral evolution. Overall, our results suggest that natural selection is the primary force shaping VACV codon usage, facilitating its efficient replication and adaptation across host environments.