Genotype-specific codon usage patterns differentiate swine-associated hepatitis E virus A3 and A4: Insights into evolutionary constraints

Hepatitis E virus (HEV) genotypes A3 and A4 are predominantly associated with swine and have been reported in zoonotic infections. Differences in codon usage between these genotypes within a common swine reservoir remain poorly characterized. This study analyzed 114 complete coding sequences of swine-associated HEV-A3 (n = 62) and HEV-A4 (n = 52). Nucleotide composition, relative synonymous codon usage (RSCU), effective number of codons (ENC), neutrality, parity rule 2 (PR2), dinucleotide abundance, codon adaptation index (CAI), principal component analysis (PCA), PERMANOVA, and site-specific selection analyses were performed for the complete coding region and separately for ORF1, ORF2, and ORF3. Both genotypes were GC-rich, with GC3 values of 57.66% in HEV-A3 and 59.89% in HEV-A4, while ORF3 showed higher GC3 than ORF1 and ORF2. RSCU analysis identified shared and genotype-specific synonymous codon preferences. Mean ENC values indicated moderate codon usage bias, with slightly lower values in HEV-A4 at the complete coding-region level. Neutrality and PR2 analyses indicated weak relationships and nucleotide asymmetry at synonymous third codon positions, respectively. CpG showed relative depletion in both genotypes. CAI differed between HEV-A3 and HEV-A4 at the complete coding-region level, and ORF-level analysis showed significant differences in ORF1 and ORF3 but not ORF2. Selection analyses indicated predominant purifying selection with localized pervasive and episodic diversifying signals that varied among ORFs. PCA and PERMANOVA supported significant differences in codon usage composition between HEV-A3 and HEV-A4 (R² = 0.24, p = 0.001). Overall, codon usage variation was genotype-specific and ORF-dependent, consistent with contributions from nucleotide composition, host-associated codon compatibility, and heterogeneous evolutionary constraints.

The online version contains supplementary material available at https://doi.org/10.1007/s13337-026-00986-6.