Molecular Evolution in the Second Hypervariable Region of the G Gene for Human Respiratory Syncytial Virus Lineage A.D - Worldwide, 2010-2024.

Introduction: Human respiratory syncytial virus subgroup A (HRSV-A) lineage A.D is characterized by a unique 72-nucleotide duplication in the second hypervariable region of the G-gene (G-HVR2) and has become the predominant global circulating strain since 2010. Molecular evolutionary features of this region should be monitored. Methods: A global HRSV-A G-HVR2 sequence dataset (2010-2024) was assembled that included 144 Chinese sequences and the molecular characteristics and evolutionary patterns in lineage A.D were analyzed. Results: Molecular clock analysis based on G-HVR2 estimated that HRSV-A lineage A.D originated in 2006, with subsequent diversification into four distinct lineages. A.D.4 and A.D.5 were grouped as A.D.4/A.D.5. Lineages circulate globally with temporal fluctuations and sequential dominance shifts. The evolutionary rate of A.D was high, with the highest rate observed in A.D.4/A.D.5. Amino acid analysis indicated five shared, lineage-specific mutations in A.D.1 and A.D.4/A.D.5 that coincided with high-frequency mutation sites. Nine positively-selected sites were predicted across all lineages, three of which resided within the 72-nucleotide duplication. A.D.1 and A.D.4/A.D.5 lost conserved N- and O-glycosylation sites. A 72-nucleotide duplication in lineage A.D introduced two extra β-strands and two unique α-helical motifs relative to the prototype strain. Conclusion: Adaptive evolution in the G-HVR2 region of HRSV-A lineage A.D was evident, likely facilitating the rapid transmission of this lineage. Sustained monitoring of lineage-specific evolution in this region is critical for targeted prevention and control of HRSV infections.