Distinct evolutionary patterns and structural insights into A33 and A34 glycoprotein in parapoxvirus.

The Poxviridae family consists of large, double-stranded DNA viruses, with significant implications for human and animal health. Among these, the genus Parapoxvirus, which includes ORF virus (ORFV), bovine papular stomatitis virus (BPSV), and pseudocowpox virus (PCPV), demonstrates unique evolutionary characteristics. This study investigated the variability, evolutionary drivers, and structural characteristics of A33 and A34 proteins, key glycoproteins involved in viral dissemination and immune evasion. Our findings revealed that A33 and A34 exhibit higher variability in Parapoxvirus compared to other Poxviridae members, particularly in their extracellular domains. Codon usage analysis indicated selective pressures mainly shaping the codon usage bias (CUB), and selection pressure mapped identified positive selected sites concentrated in surface-exposed regions. Mutation analysis highlighted G > A and A > G transitions as predominant in ORFV, PCPV and BPSV. Phylogenetic analyses confirmed Parapoxvirus A33 and A34 form distinct clades, reflecting independent evolution within the genus. Despite sequence heterogeneity, structural modeling revealed conserved macrostructures with variable loop regions that likely enhance viral adaptability and host specificity. The conserved structural framework and identified diversity hotspots highlight A33 and A34 as promising subunit vaccine targets. This study advances our understanding of Parapoxvirus evolution and provides a foundation for developing effective control strategies.