Abstract
BACKGROUND: Hand, foot, and mouth disease (HFMD) remains a significant childhood infection in the Asia-Pacific region; however, the emergence of Coxsackievirus A6 (CVA6) since 2008 has reshaped its epidemiology. Despite extensive national surveillance, the evolutionary tempo-spatial dynamics of CVA6 in southern China remain poorly characterized. This study aimed to delineate the temporal phylodynamics of the CVA6 VP1 region in Shenzhen and identify lineage-defining mutations that may inform early-warning systems and control strategies. METHODS: Epidemiological surveillance data (2022-2024) were integrated with VP1 sequences from 47 Shenzhen isolates and 465 global reference strains (1949-2024). Real-time RT-PCR was used to determine serotype composition. Bayesian relaxed-clock, SkyGrid, and discrete phylogeographic models were applied to reconstruct substitution rates, lineage turnover, and transmission routes. Amino acid variability was mapped onto the VP1 protein structure. RESULTS: Among 195,951 notifiable disease cases, 11,427 (5.8%) were identified as HFMD, with seasonal peaks consistently observed in June and a maximum of 3,185 cases recorded in June 2023. CVA6 was detected in 30.9% of tested specimens and predominated in 2023 (64.6%). Root-to-tip regression showed a strong temporal signal (R² = 0.86), and the mean VP1 substitution rate was 4.78 × 10⁻³ substitutions/site/year (95% highest posterior density [HPD]: 4.39-5.16 × 10⁻³), with the estimated time to the most recent common ancestor (tMRCA) around 1945. All Shenzhen isolates belonged to genotype D3 and formed three temporally structured clades interspersed with isolates from various Chinese provinces, suggesting frequent interprovincial transmission and limited geographic clustering. SkyGrid analysis indicated a rapid expansion in the effective population size beginning in 2008, which stabilized after 2010. A total of 31 VP1 amino acid substitutions were identified; twelve (e.g., positions 5, 8, 10, 14, 32, 98, 160, 174, 194, 261, 279, 305) were fixed compared to the prototype strain Gdula, while six variable sites (e.g., S97N, N241D) were located in surface-exposed or BC-loop regions. Pairwise nucleotide distances among local strains were ≤ 6.7%, compared to 19.5-22.0% divergence from Gdula. CONCLUSIONS: Genotype D3 of Coxsackievirus A6 appears to be a significant contributor to seasonal HFMD peaks in Shenzhen, particularly in early summer. Its high evolutionary rate, rapid lineage turnover, and limited geographic structuring emphasize the importance of coordinated, cross-regional molecular surveillance. The study identifies 31 amino acid substitutions, including surface-exposed sites, which may indicate ongoing antigenic drift and potential immune escape. These findings underscore the need for high-resolution genomic surveillance, early-warning systems, and region-specific control measures for effective management of HFMD. CLINICAL TRIAL REGISTRATION: Not applicable.