Immune-driven serotype-specific evolution of dengue virus NS1: Insights from structural and epitope analyses of...

Despite the diagnostic and immunological importance of NS1, how evolutionary forces shapes NS1 evolution across different serotypes of dengue remain poorly understood.

NS1 sequences representing all four dengue virus serotypes were analyzed for haplotype diversity and selection pressure using codon-based models. Positively selected sites were structurally mapped onto dimeric, tetrameric and hexameric NS1 conformations. Diversifying sites were also overlapped with predicted B-cell epitopes. Structural mapping of selected sites was done on NS1-antbody structure. Minimum heavy atom distance was calculated of those sites from antibody-binding site and predicted affinity change (ΔΔG) was also calculated.

Haplotype frequency analysis showed high haplotype diversity and multiple co-circulating variants in DENV-2, moderate haplotype diversity in DENV-1 and 3, whereas DENV-4 showed lowest diversity. Selection pressure analyses identified several positively selected residues, predominantly localized within the wing and β-ladder domains. These sites also overlapped with some conserved predicted B-cell epitopes. Notably, none of the positively selected residues directly overlapped with the antigen-antibody binding interface and were associated with minimal predicted changes in binding affinity.

Our results indicate that NS1 evolution reflects strong functional constraint with localized, serotype-specific variation, underscoring the relevance of continued NS1 sequence monitoring for diagnostic robustness and the potential utility of conserved regions in vaccine design.