Multilayered nucleotide organization reveals purifying selection and host-driven adaptation in CPV and FPV.

Since feline panleukopenia virus (FPV) is considered the most likely ancestor of canine parvovirus (CPV), comprehensive comparisons of nucleotide organization in corresponding viral genes between CPV and FPV may provide novel insights into the evolutionary dynamics underlying the divergence of these two viruses. Here, we characterize the evolutionary patterns of CPV and FPV genes across multiple levels of nucleotide organization. Both viruses exhibited highly conserved nucleotide usage at nonsynonymous sites, with Ka/Ks patterns consistent with strong purifying selection, whereas synonymous sites showed greater variability. CpG dinucleotides were markedly underrepresented across all four viral genes, suggesting host-associated selective pressure and/or intrinsic nucleotide compositional constraints. Extensive nonrandom biases in synonymous codon usage, codon neighboring nucleotide context, and codon pair usage further revealed fine-scale genomic optimization shaped by natural selection and nucleotide compositional constraints. Structural protein genes (VP1 and VP2) displayed stronger codon usage bias and higher tRNA adaptation than nonstructural genes. Moreover, CPV genes showed greater translational adaptation to feline hosts than to canine hosts. These findings highlight how closely related parvoviruses exploit flexible nucleotide organization to facilitate host adaptation while maintaining essential protein functions.