Akanksha Alampally

Domain-Specific Evolution of Influenza A H3N2 Hemagglutinin Revealed by c/µ and Molecular Clock Analyses

Akanksha Alampally, Rowan University Dr.Chun Wu Department of Chemistry and Biochemistry & Department of Biological & Biomedical Sciences, Rowan University, Glassboro, NJ 08028, USA

Influenza A H3N2 is a major driver of seasonal epidemics, causing millions of severe cases and up to 650,000 global deaths annually. Despite yearly vaccinations, rapid antigenic drift often reduces vaccine effectiveness, underscoring the need to better understand viral evolution. To address this, we analyzed 8,768 hemagglutinin (HA) sequences spanning 1975-2011 using the substitution-to-mutation rate ratio (c/µ), a framework that directly links substitution mutations to the genotype-phenotype relationship, providing critical insight into how influenza adapts under host immune pressure, offering a predictive tool for vaccine design, and validated against other RNA viruses including SARS-CoV-2 and HIV-1. Our results revealed striking domain-specific heterogeneity in HA. The receptor-binding domain (RBD) exhibited the highest average c/µ (~9.65), reflecting intense positive selection and immune-driven antigenic drift, with key substitutions (N137Y, P143S, G158E, S159N) exceeding c/µ > 50. Elevated ratios in the vestigial esterase and F′ subdomains suggested adaptive fine-tuning, while HA2 regions-including the fusion peptide, transmembrane, and cytoplasmic domains-showed lower ratios (c/µ ~2-3), consistent with purifying selection on structurally constrained sites. A high ratio (T4C, c/µ ~50) in the 3′UTR suggested potential regulatory effects on HA expression. Molecular clock analysis estimated a mean substitution rate of ~2.9 × 10⁻³ substitutions/site/year, with the RBD accumulating ~2-3 changes per year-nearly three times faster than HA2. Divergence dating placed the MRCA of H3N2 HA around 1968-1970, consistent with its pandemic origin. These findings align with the Near-Neutral Balanced Selectionist Theory (NNBST), which posits that evolution is shaped by a balance of nearly neutral mutations. This perspective contrasts with Selectionist Theory (ST), which emphasizes adaptation; Kimura's Neutral Theory (KNT), which attributes change to random drift; and Ohta's Nearly-Neutral Theory (ONNT), which highlights slightly deleterious mutations. By integrating c/µ analysis with structural mapping, our study underscores the balance of constraint and adaptability in HA, identifying evolutionary hotspots that extend beyond current vaccine targets. Together, our c/µ framework can aid in anticipating antigenic drift and improving influenza vaccine strategies to reduce global morbidity and mortality. Keywords: Influenza A H3N2, hemagglutinin, antigenic drift, c/µ ratio, molecular clock, evolutionary theory, vaccine design.