Molecular Evolution of the HMPV-A Fusion Gene Reveals Lineage-Specific Diversification and Structural Conservation

Human metapneumovirus genotype A (HMPV-A) comprises genetically diverse lineages, but whether F-gene diversification is accompanied by changes in the conserved fusion (F) protein remains unclear. We analyzed 500 PARNAS-selected HMPV-A F gene sequences from 25 countries collected between 1982 and 2025 using time-scaled phylogenetics, Bayesian skyline analysis, evolutionary-rate and patristic-distance estimation, codon-based selection analysis, sequence-identity profiling, and structural epitope modeling. Three phylogenetically distinct clusters were identified. Cluster 1 diverged from the common ancestor of Clusters 2 and 3 around 1927. Cluster 3 comprised 435 representative sequences and showed the highest estimated evolutionary rate, greatest phylogenetic diversity, and the most pronounced recent demographic fluctuations. The overall evolutionary rate was 9.20 × 10−4 substitutions/site/year. Despite nucleotide-level diversification, whole-protein identity exceeded 97% in all clusters, 299 codons (approximately 55%) were consistently under negative selection, whereas positive-selection signals were limited and method-specific, with MEME identifying codons 188 and 271 and FUBAR identifying codon 528. Major neutralizing antibody-binding regions were largely conserved, and the six modeled representatives showed similar global F-protein backbone structures and predicted epitope-surface distributions, although A61S and K143T occurred within neutralizing antibody-binding sites and require experimental evaluation. These findings demonstrate lineage-specific F gene diversification under strong functional constraint and support the HMPV F protein as a broadly conserved intervention target.