Natural selection shapes codon usage and host adaptation of NS1 in mosquito-borne pathogenic flaviviruses

The NS1 protein of nine mosquito-borne flaviviruses, including Dengue virus 1-4, Japanese encephalitis virus, West Nile virus, Yellow fever virus, Tembusu virus, and Zika virus, shows distinct codon usage and evolutionary traits. Codon usage analysis shows notable base composition bias and non-conservatism in NS1, with distinct evolutionary traits from its ORF. Analysis of relative synonymous codon usage (RSCU) indicates that the NS1 genes exhibit non-conservative RSCU patterns within different mosquito-borne pathogenic flaviviruses. Principal component analysis (PCA) based on the RSCU values, effective number of codons (ENC)-GC3, and parity rule 2 analysis (PR2) plot analyses demonstrate the similarity in codon usage patterns of NS1 genes among different mosquito-borne pathogenic flaviviruses. The ENC-GC3 and PR2 results, along with neutrality and selection pressure analyses, confirm that natural selection, especially purifying selection, plays a primary role in shaping NS1 codon usage. In addition, NS1 is subject to stronger positive selection than ORF, resulting in higher host adaptability in its codon bias, such as higher CAI index, hydrophilicity, aromaticity, and low CpG usage. These features indicate that the codon usage pattern of NS1 plays a crucial role in viral adaptation and immune evasion mechanisms, supporting the design and optimization of NS1-based vaccines.