Lineage Diversification and Evolutionary Dynamics of the Hemagglutinin–Neuraminidase Gene in Mumps Virus Genotype G

Mumps virus (MuV) genotype G is widely represented among circulating strains, but the evolutionary patterns of the hemagglutinin–neuraminidase (HN) gene remain incompletely understood. In this study, we analyzed publicly available full-length genotype G HN sequences using phylogenetic, phylodynamic, codon-based selection, and structure-guided epitope prediction approaches. The genotype G HN sequences were categorized into Clade 1, an operationally defined Diverse group, and Clade 2. Clade 1, which was composed mainly of Japanese strains, showed a relatively structured pattern over time. In contrast, Clade 2 showed more recent diversification and an overall increase in relative genetic diversity, although this phylodynamic pattern was sensitive to sampling structure. The Diverse group was phylogenetically heterogeneous, and its Bayesian skyline estimates were not used for biological interpretation because repeated analyses showed unstable posterior behavior. Root-to-tip regression supported temporal structure in the complete dataset, with the strongest signal in Clade 2. Bayesian molecular dating estimated the time to the most recent common ancestor of the sampled genotype G HN sequences at approximately 1932, and the mean evolutionary rate was 4.925 × 10−4 substitutions/site/year. Although the HN protein is a major surface antigen and a target of neutralizing antibodies, codon-based analyses showed no robust evidence of positive selection using multiple methods. Instead, many codon sites were inferred to be under purifying selection, suggesting that genotype G HN evolution is largely constrained by the need to maintain protein function. Predicted B-cell epitope regions were broadly similar among representative genotype G strains. Overall, these findings indicate that genotype G HN lineages have followed distinct evolutionary patterns, while the HN gene remains mainly shaped by purifying selection. These findings may help improve our understanding of MuV genotype G HN gene evolution and support future molecular surveillance.