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

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