Temporal Phylodynamics of Coxsackievirus A6 VP1 in Shenzhen（2022-2024）

Temporal Phylodynamics of Coxsackievirus A6 VP1 in Shenzhen（2022-2024） | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Temporal Phylodynamics of Coxsackievirus A6 VP1 in Shenzhen（2022-2024） Yizhou Deng, Guiqing Yang, Qiumei Li, Feifei Wu, Min Ye, Fei Zhuo, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6951771/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 12 Nov, 2025 Read the published version in BMC Infectious Diseases → Version 1 posted 12 You are reading this latest preprint version Abstract Background Hand, foot, and mouth disease (HFMD) remains a significant childhood infection in the Asia-Pacific region; however, the emergence of Coxsackievirus A6 (CV-A6) since 2008 has reshaped its epidemiology. Despite extensive national surveillance, the evolutionary tempo-spatial dynamics of CV-A6 in southern China remain poorly characterized. This study aimed to delineate the temporal phylodynamics of the CV-A6 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. CV-A6 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 CV-A6 is now well established in Shenzhen, contributing to recurrent seasonal HFMD peaks in early summer. Its high evolutionary rate and rapid lineage turnover, coupled with limited geographic structuring, underscore the need for coordinated, cross-regional molecular surveillance. The conserved and variable VP1 residues identified in this study offer valuable candidate markers for molecular monitoring and vaccine antigen development. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Hand, foot, and mouth disease (HFMD) is a common acute infectious disease predominantly affecting children, with particularly high incidence rates in the Asia-Pacific region. In China, annual reported cases have often exceeded one million over the past two decades, with frequent occurrences of severe cases and fatalities, posing a significant burden on public health and the socio-economic system [1–3]. Historically, enterovirus A serotypes Enterovirus A71 (EV-A71) and Coxsackievirus A16 (CV-A16) were identified as the primary causative agents of HFMD [4]. However, since the first large-scale outbreak of Coxsackievirus A6 (CV-A6) in Finland in 2008 [5], the virus has rapidly spread across Europe, Asia, and the Americas, supplanting traditional serotypes to become the predominant pathogen in many regions [6,7]. Following the introduction of the inactivated EV-A71 vaccine into China's immunization program in 2016, the prevalence of EV-A71 and CV-A16 has significantly declined, whereas CV-A6 has exhibited rapid emergence [8,9]. Clinically, CV-A6 infection differs markedly from classical HFMD. Patients more frequently present with high fever and widespread vesicular or hemorrhagic rashes extending beyond the typical sites of the hands, feet, and mouth, often involving the trunk, buttocks, and distal limbs [10]. Onychomadesis (nail shedding) commonly occurs 2–3 weeks after recovery, with incidence rates ranging from 30% to 50% [11]. Moreover, there has been a noticeable increase in cases among adults and older children [12], and severe complications—such as aseptic meningitis, brainstem encephalitis, and myocardial involvement—have occasionally been reported [1]. These evolving epidemiological and clinical features underscore the need for enhanced surveillance and targeted clinical strategies to mitigate the growing impact of CV-A6 in HFMD. The VP1 capsid protein of CV-A6 plays a central role in viral pathogenicity, harboring most of the serotype-specific neutralizing epitopes. Due to its strong serotype correlation, VP1 is widely used as a molecular marker for virus typing, phylogenetic analysis, and mutation surveillance [13,14]. Phylogenetic analyses of VP1 sequences over the past decade consistently place nearly all global CV-A6 isolates within genotype D, predominantly subtype D3 [6,7,15]. The mean nucleotide divergence in D3 VP1 (6–8%) exceeds that observed in EV-A71 and CV-A16, suggesting a higher molecular evolutionary rate [7]. While comprehensive genomic studies have documented frequent recombination events in the non-structural regions of the CV-A6 genome, the VP1 region maintains a relatively conserved lineage structure, supporting its continued use in genotyping and viral source tracking [13]. Regional surveillance data from Beijing, Hong Kong, and Thailand demonstrate the persistent, localized circulation of subtype D3, with a gradual accumulation of characteristic mutations that form multiple sublineages and exhibit phylogenetic connections with neighboring countries [15,16,18]. Investigating VP1 sequence diversity, substitution rates, and lineage turnover dynamics is thus crucial for understanding the transmission patterns and adaptive evolution of CV-A6 [7,17]. However, spatiotemporal dynamics based on VP1 remain insufficiently studied in China [16]. To address this gap, we collected VP1 sequences from CV-A6 isolates obtained in Shenzhen's Luohu District between 2022 and 2024 and integrated them with global datasets. Using Bayesian molecular clock and discrete-time spatiotemporal models, we aim to reconstruct the virus's evolutionary history, infer transmission trajectories, and model population dynamics [17,18]. Additionally, we will evaluate the potential biological significance of key amino acid substitutions. These analyses will contribute to robust HFMD forecasting, early-warning systems, and precision control strategies. Methods Epidemiological Data and Sample Sources This study utilized pathogen surveillance and epidemiological data on hand, foot, and mouth disease (HFMD) obtained from the Infectious Disease Monitoring Information Reporting System of the Chinese Center for Disease Control and Prevention. In accordance with the Law of the People's Republic of China on the Prevention and Control of Infectious Diseases, HFMD is designated as a Class C notifiable disease. Medical institutions diagnosing HFMD cases are required to report them promptly to the national system [19]. Clinical specimens were primarily collected from outpatients at Luohu District People's Hospital in Shenzhen, all of whom were clinically diagnosed with HFMD and reported to the national infectious disease information system. To enhance sample diversity and representativeness, additional specimens were obtained from patients involved in HFMD outbreak clusters reported by schools in Luohu District. These samples were collected during the early phase of the outbreaks to ensure that the data accurately reflect current epidemic trends. Sample Selection and VP1 Gene Sequencing Throat swabs were processed using an automated nucleic acid extraction system (Zhongyuan Huiji Biotech, China), followed by commercial real-time quantitative PCR (qRT-PCR) assays (Jiangsu Shuoshi Biotech, China) for CV-A6 detection. Samples were selected for sequencing if qRT-PCR confirmed CV-A6 positivity with a cycle threshold (CT) value < 30, indicating sufficient viral load. To ensure comprehensive temporal coverage, specimens were collected throughout all seasons. Representative samples from outbreak clusters during peak incidence periods were prioritized, with a maximum of five samples sequenced per high-incidence month to reduce redundancy. A total of 50 samples were submitted to Shanghai Better Medical Technology Co., Ltd. for amplification and Sanger sequencing of the full-length VP1 gene (915 bp). High-quality VP1 sequences were successfully obtained from 47 samples. Rigorous quality control was conducted at each stage to ensure sequence integrity and accuracy. Nucleotide and Amino Acid Variation Analysis The CV-A6 prototype strain Gdula (GenBank: AY421764) was used as the reference for comparative analysis. The 47 VP1 gene sequences generated in this study were aligned at the nucleotide level using the MegAlign module of DNASTAR (Lasergene v7.1). Protein sequences were derived from the open reading frame, and both nucleotide and amino acid alignments were examined to identify variable sites. Emphasis was placed on nonsynonymous substitutions with a frequency > 5% or those located in known antigenic or functional domains, as these mutations may have biological relevance. Key mutations were mapped to structural or epitope data to assess their potential impact on viral phenotype, host receptor binding, or immune evasion. All alignments were performed using default parameters, and variant calling results were manually validated to ensure analytical accuracy. Dataset Construction and Temporal Signal Assessment To investigate the global phylogenetic structure and evolutionary dynamics of CV-A6, we retrieved all publicly available full-length VP1 gene sequences (915 bp) from the GenBank database using the keyword "Coxsackievirus A6." Sequence retrieval was completed on December 31, 2024. Sequences containing ambiguous nucleotides (e.g., "N") or misannotated as non–CV-A6 were excluded during the initial quality control process. Redundant sequences with ≥99.99% identity were removed using BioAider (v1.727) [20], followed by additional filtering to eliminate highly similar sequences from the same geographic region and collection year, thereby ensuring broad temporal and spatial coverage. The remaining sequences were aligned using MAFFT (v7.526) [21]. A maximum-likelihood phylogenetic tree was then reconstructed using IQ-TREE (v2.4.0) [22], with model selection guided by the Bayesian Information Criterion. Temporal signal was assessed using TreeTime [23] by regressing root-to-tip genetic distances against the sampling dates. The slope of the regression line represented the estimated nucleotide substitution rate, while the coefficient of determination (R²) quantified the strength of the temporal signal. To ensure suitability for molecular clock modeling, statistically significant outliers were removed, and only datasets with R² > 0.80 were retained for downstream phylodynamic analyses. The final dataset comprised 465 high-quality VP1 sequences originating from 12 countries and 23 provinces in China, providing robust spatial and temporal representation for molecular epidemiological investigation. Phylodynamic Analysis To estimate the evolutionary rate and epidemic dynamics of CV-A6, we performed a Bayesian phylodynamic analysis using the curated VP1 sequence dataset. The optimal nucleotide substitution model was identified using the ModelFinder module within PhyloSuite (v1.2.3) [24]. Among 24 candidate models, SYM+G4 was selected as the best-fitting model (Table 1). A time-scaled phylogenetic tree was inferred using BEAST v1.10.4, employing a Bayesian Markov Chain Monte Carlo (MCMC) approach under a relaxed molecular clock model, in conjunction with the Bayesian SkyGrid coalescent model [25,26]. The MCMC analysis was run for 200 million generations, with sampling performed every 20,000 generations. Convergence diagnostics were conducted in Tracer v1.7.2 [27], confirming that the effective sample size (ESS) for all key parameters exceeded 200. A Maximum Clade Credibility (MCC) tree was generated using TreeAnnotator (v1.10.4), and the resulting phylogeny was visualized with FigTree v1.4.4 [28]. This visualization provided detailed insights into the branching structure, estimated time to the most recent common ancestor (tMRCA), and the evolutionary trajectory of CV-A6. Table 1. Best-fit nucleotide substitution model identified by ModelFinder. ModelFinder will test up to 24 DNA models (sample size: 915 epsilon: 0.100) ... No. Model LnL df AIC AICc BIC 1 JC 39824.811 1011 81671.623 2127935.623 86543.555 2 JC+G4 35951.225 1012 73926.451 2124238.451 78803.202 3 TN+F 35650.495 1016 73332.991 2139876.991 78229.018 4 TN+F+G4 31803.118 1017 65640.236 2136252.236 70541.082 5 TNe 35645.958 1013 73317.916 2127681.916 78199.486 6 TNe+G4 31781.318 1014 65590.637 2124010.637 70477.026 7 K2P 35719.615 1012 73463.231 2123775.231 78339.982 8 K2P+G4 31787.078 1013 65600.155 2119964.155 70481.726 9 K2P 35719.615 1012 73463.231 2123775.231 78339.982 10 K2P+G4 31787.078 1013 65600.155 2119964.155 70481.726 11 F81+F 39880.447 1014 81788.894 2140208.894 86675.283 12 F81+F+G4 36026.881 1015 74083.762 2136563.762 78974.97 13 HKY + F 35783.528 1015 73597.056 2136077.056 78488.264 14 HKY+F+G4 31829.335 1016 65690.67 2132234.67 70586.697 15 SYM 35597.181 1016 73226.362 2139770.362 78122.388 16 SYM+G4 31726.782 1017 65487.564 2136099.564 70388.41 17 TIM+F 35644.772 1017 73323.544 2143935.544 78224.39 18 TIM+F+G4 31798.421 1018 65632.843 2140316.843 70538.507 19 TVM + F 35734.022 1018 73504.044 2148188.044 78409.709 20 TVM+F+G4 31776.97 1019 65591.941 2144351.941 70502.424 21 TVME 35670.801 1015 73371.603 2135851.603 78262.811 22 TVMe+G4 31731.854 1016 65495.707 2132039.707 70391.734 23 GT+F 35601.09 1019 73240.18 2152000.18 78150.664 24 GTR+F+G4 31751.22 1020 65542.441 2148382.441 70457.743 Akaike Information Criterion: SYM+G4 Corrected Akaike Information Criterion: K2P+G4 Bayesian Information Criterion: SYM+G4 Best-fit model: SYM+G4 chosen according to BIC Results Epidemiological and Virological Analysis Between 2022 and 2024, a total of 195,951 notifiable infectious disease cases were reported in Luohu District, Shenzhen, of which 11,427 were cases of hand, foot, and mouth disease (HFMD). HFMD accounted for 3.11% (408 cases) of all reported notifiable diseases in 2022, 6.35% (6,332 cases) in 2023, and 5.64% (4,687 cases) in 2024. It consistently ranked among the top three most frequently reported infections and exhibited a generally increasing trend over the study period, with a notable peak in 2023. Monthly incidence data revealed a clear seasonal distribution (Figure 1). June emerged as the peak transmission month, contributing a total of 4,620 cases over the three-year period—representing 40.43% of all reported HFMD cases (4,620/11,427). June 2023 recorded the highest monthly incidence, with 3,185 cases. In contrast, the 2024 outbreak exhibited a flatter epidemic curve, with a prolonged transmission period from May to August, suggesting a broader seasonal window. To characterize the viral composition of HFMD cases, 719 clinical specimens collected between 2022 and 2024 were tested using real-time RT-PCR targeting five major enterovirus serotypes: EV-A71, CV-A16, CV-A4, CV-A6, and CV-A10. The overall enterovirus positivity rate was 76.91% (553/719), with CV-A6 identified as the predominant serotype, accounting for 30.87% (222/719) of all tested samples. A year-by-year analysis revealed a striking predominance of CV-A6 in 2023, with a positivity rate of 64.62% (190/294), indicating its dominance during that year's epidemic. In contrast, 2022 and 2024 showed greater serotype diversity: CV-A16 and CV-A10 were more prevalent in 2022, while CV-A16 reemerged prominently in 2024, accounting for nearly half of all positive detections (Figure 2). Monthly case counts illustrate seasonal trends, with consistent peaks observed in June across all three years. Serotype composition of laboratory-confirmed HFMD cases, showing year-specific dominance patterns of CV-A6, CV-A16, and other enteroviruses. Molecular Evolutionary Analysis To investigate the evolutionary dynamics of CV-A6 strains isolated in this study and their genetic relationships with strains from other regions and historical periods, we constructed a comprehensive VP1 sequence dataset comprising 512 sequences. This dataset included 47 newly sequenced isolates and representative global reference strains retrieved from GenBank. The sequences spanned from 1949 to 2024 and encompassed 23 provinces in China and 12 countries worldwide, ensuring broad temporal and geographical representation. Temporal signal strength was evaluated using TreeTime. Root-to-tip regression analysis demonstrated a strong linear relationship between genetic divergence and sampling time, with a coefficient of determination (R²) of 0.86 and no significant outliers identified (Figure 3B). These results indicate a robust temporal signal, supporting the application of molecular clock models for Bayesian phylogenetic reconstruction. The estimated nucleotide substitution rate was 4.49 × 10⁻³ substitutions per site per year, and the inferred time to the root was approximately 1942.5. Subsequently, a time-scaled Bayesian phylogenetic analysis was conducted using BEAST under the SYM+G4 nucleotide substitution model, applying a relaxed molecular clock and the Bayesian SkyGrid coalescent model. This analysis reconstructed the Maximum Clade Credibility (MCC) tree. The mean substitution rate for the VP1 region of CV-A6 was estimated at 4.778 × 10⁻³ substitutions per site per year (95% highest posterior density [HPD]: 4.3886 × 10⁻³–5.1626 × 10⁻³), and the time to the most recent common ancestor (tMRCA) was inferred to be approximately 1945 (95% HPD: 1913–1949) (Figures 3A, 3D). Bayesian SkyGrid demographic reconstruction revealed a rapid increase in the effective population size of CV-A6 beginning around 2008, followed by a plateau after 2010, indicating a convergence in genetic diversity over time (Figure 3C). Phylogenetic lineage analysis classified CV-A6 into six major genotypes: A, B, C, D1, D2, and D3. Of these, genotype D3 has emerged as the globally dominant lineage in recent years, characterized by a dense and compact clade structure. The tMRCA of genotype D3 was estimated at 1998 (95% HPD: 1995–2000), with a mean substitution rate of 4.192 × 10⁻³ substitutions per site per year (95% HPD: 1.653 × 10⁻³–7.651 × 10⁻³). All 47 CV-A6 isolates obtained in this study clustered within the D3 lineage and formed three distinct clades within the MCC tree. These clades were closely interspersed with strains from various regions of China, with no apparent geographic structuring. Instead, the phylogenetic pattern appeared more strongly associated with sampling time than with geographic origin, suggesting frequent interprovincial transmission and ongoing genetic exchange, with limited spatial differentiation among circulating strains. To contextualize the CV-A6 isolates from this study within a global evolutionary framework, a time-scaled circular phylogenetic tree was constructed based on VP1 sequences (Figure 4), incorporating 465 sequences with diverse temporal and geographic origins. The outer ring of the tree denotes the region of origin, while the inner ring represents sampling years, both color-coded for clarity. All isolates from Luohu District clustered within the D3 lineage and were dispersed among strains from multiple Chinese provinces, reflecting a temporally structured rather than geographically clustered pattern. This supports the hypothesis of widespread domestic circulation and a lack of strong geographic segregation. Notably, several cross-regional clustering patterns were observed. For example, a 2021 sequence from Hungary and a 2023 sequence from the United Kingdom were grouped with earlier Indian strains from 2013 and 2018, rather than with contemporaneous European isolates. These findings suggest the possibility of long-term cryptic transmission or regional reintroductions driven by cross-border human movement, highlighting the complex and dynamic nature of global CV-A6 dissemination. The inner ring of the phylogeny features a gradient from purple (earlier years) to red-orange (recent years), vividly illustrating the rapid post-2010 expansion of the D3 genotype. The increasing density of terminal branches is consistent with the Bayesian SkyGrid results, further supporting the recent dominance and adaptive success of the D3 lineage in human populations. Circular tree based on 465 VP1 sequences collected from 1949 to 2024. Outer ring colors indicate geographic origin; inner gradient indicates sampling year. Isolates from this study are highlighted and cluster within genotype D3. The tree shows widespread interregional mixing and recent global expansion of D3 lineage. Amino Acid Mutation Profile and Nucleotide Homology Analysis The complete VP1 gene of CV-A6 spans 915 nucleotides and encodes a 305-amino acid capsid protein. Amino acid sequences from the 47 isolates obtained in this study were inferred using MegAlign software. After eliminating redundant sequences with identical amino acid compositions, 16 representative strains containing unique mutations were selected. These sequences were aligned against the prototype CV-A6 strain Gdula (GenBank accession: AY421764.1), resulting in the identification of 31 amino acid substitution sites (Figure 5). Twelve amino acid sites—positions 5, 8, 10, 14, 32, 98, 160, 174, 194, 261, 279, and 305—exhibited consistent substitutions in all isolates compared with the Gdula reference strain. Additional variable sites included mutations such as S97N, S137N, N241D, V242I, H243R, and A283T. Several of these substitutions were located in surface-exposed regions or within the BC loop of the VP1 protein, which are commonly associated with antigenic properties; however, their functional relevance was not evaluated in this study. To assess genetic divergence, pairwise nucleotide distances were calculated among VP1 sequences. The genetic distance between the study isolates and the Gdula prototype ranged from 19.5% to 22.0%, indicating substantial divergence at the nucleotide level. In contrast, genetic distances among the 47 local isolates were markedly lower, all ≤6.7% (Figure 6), highlighting a high degree of sequence similarity among contemporary circulating strains and their clear evolutionary divergence from the historical prototype. Discussion Hand, foot, and mouth disease (HFMD) continues to impose a considerable public health burden among children in China [ 29 , 30 ]. Recent surveillance has revealed that Coxsackievirus A6 (CV-A6) has overtaken previously dominant serotypes in prevalence [ 31 , 32 ], highlighting the urgent need to elucidate its evolutionary dynamics and phenotypic plasticity. In this study, we analyzed 47 complete VP1 gene sequences collected in Luohu District, Shenzhen (2022–2024), alongside 465 international reference sequences. We reconstructed a dated phylogenetic tree of the CV-A6 D3 sublineage, estimated its evolutionary rate and effective population history, and examined the structural and functional significance of amino acid substitutions defining contemporary epidemic lineages. Our findings indicate that HFMD incidence in Luohu District consistently peaked in June across all three years. Among 719 laboratory-confirmed cases, CV-A6 accounted for 64.62% of positive detections in 2023, emerging as the predominant serotype. In contrast, both 2022 and 2024 exhibited co-circulation of multiple genotypes, with CV-A6 and CV-A16 being most prevalent. Multicenter studies across China have documented a gradual replacement of EV-A71 and CV-A16 by CV-A6 since 2012, with widespread outbreaks attributed to CV-A6 occurring in eastern and southern China by 2023 [ 32 – 34 ]. The majority of circulating strains cluster within the D3 sublineage, which is characterized by a higher evolutionary rate and enhanced immune escape potential [ 16 , 35 ]. The re-emergence of CV-A16 in 2024 may reflect dynamic genotype replacement: as herd immunity intensifies against a dominant serotype (e.g., CV-A6), alternative genotypes may spread more efficiently among susceptible populations [ 34 ]. Notably, no EV-A71-positive cases were identified in Luohu District during the study period, aligning with the high coverage of inactivated EV-A71 vaccination in Guangdong Province and its proven efficacy in preventing severe disease and mortality in children under five years of age [ 33 , 36 ]. However, the monovalent nature of the current EV-A71 vaccine, while effective in curbing EV-A71 transmission, may inadvertently facilitate the spread of non-EV-A71 genotypes—particularly CV-A6 and CV-A16—by reducing natural cross-protective immunity [ 36 , 38 ]. This phenomenon of serotype replacement has been reported across multiple Chinese provinces [ 32 ], underscoring the need for multivalent HFMD vaccines incorporating antigens from several key genotypes [ 39 ]. During the COVID-19 pandemic (2020–2021), the implementation of non-pharmaceutical interventions (NPIs) led to a marked reduction in HFMD incidence [ 40 , 41 ]. Following the relaxation of NPIs and the resumption of routine social and educational activities, a large cohort of immunologically naive children—previously unexposed to enteroviruses—became susceptible to infection, likely contributing to the HFMD surge observed in 2023 [ 37 , 42 ]. In Luohu District, a densely populated urban center with high levels of cross-border mobility, the effects of this "immunity debt" were particularly pronounced. Bayesian phylogenetic analysis using BEAST estimated the mean substitution rate of the CV-A6 VP1 region at 4.78 × 10⁻³ substitutions per site per year (95% highest posterior density [HPD]: 4.39–5.16 × 10⁻³). This rate is consistent with previously reported estimates ranging from 4.0 to 7.4 × 10⁻³ substitutions/site/year for CV-A6 in both global and Chinese datasets and places CV-A6 within the moderate-to-high evolutionary rate range among Enterovirus A species. These findings suggest that CV-A6 has a relatively high capacity for genetic drift [ 13 , 18 ]. The inferred time to the most recent common ancestor (tMRCA) for CV-A6 was dated to approximately 1945 (95% HPD: 1913–1949), in line with historical evidence of the virus being isolated as early as the mid-20th century [ 43 ]. Importantly, the tMRCA of the currently predominant D3 sublineage was estimated to be around 1998 (95% HPD: 1995–2000), supporting earlier studies indicating that D3 emerged in the late 1990s and subsequently replaced the D2 sublineage after 2010 [ 13 , 44 ]. These data suggest that D3 has established a stable phylogenetic lineage and undergone sustained adaptive expansion over the past two decades [ 44 , 45 ]. Bayesian SkyGrid analysis revealed an increase in CV-A6 population genetic diversity beginning around 2000, followed by a reduction starting in 2010—suggestive of a "post-bottleneck concentration." The progressive narrowing of the 95% HPD interval over time reflects a convergence in viral genetic diversity. This pattern, when interpreted alongside the maximum clade credibility (MCC) tree, appears to be driven by the rapid expansion and dominance of the D3 sublineage. These results imply a possible "selective sweep," potentially triggered by antigenic drift or recombination, that displaced other competing variants [ 13 , 18 ]. All 47 isolates analyzed in this study belonged to the D3 sublineage and were grouped into three closely related clusters on the MCC tree. Despite the predominance of Chinese sequences in each cluster, no clear geographic structuring was observed, indicating frequent interprovincial transmission of CV-A6 across China [ 45 , 46 ]. This observation aligns with the extensive domestic transportation network and high population mobility in the country [ 45 ]. Further supporting global transmission dynamics, CV-A6 strains isolated in Hungary and the United Kingdom between 2021 and 2023 clustered with Indian strains from 2013 and 2018, forming a shared lineage [ 44 , 47 ]. These patterns suggest multiple international introductions and possible undetected community transmission events. The observed "year-dominated, geography-attenuated" dissemination highlights the limitations of relying solely on local surveillance systems to detect interregional and cross-border transmission risks in a timely manner [ 47 ]. The amino acid substitutions identified in the VP1 region exhibited distinct signatures characteristic of the D3 sublineage. Twelve fixed substitutions were consistently observed across all isolates, potentially serving as molecular markers of the D3 lineage's evolutionary trajectory. Several mutations were located in key functional regions of the VP1 protein and are hypothesized to influence viral antigenicity, receptor binding, and pathogenicity [ 14 , 48 , 49 ]. For example, the S97N mutation—commonly observed in recent D3 epidemic strains—is situated in a surface-exposed domain. Evidence suggests this substitution may alter local charge distribution, potentially reducing the binding affinity of neutralizing antibodies and enhancing immune evasion [ 14 , 51 ]. The S137N substitution, also widespread among HFMD outbreak strains globally, may represent an important site of antigenic drift, challenging existing population immunity [ 7 , 52 ]. Other mutations—N241D, V242I, and H243R—located in the VP1 BC loop, are implicated in receptor-binding functions and may influence viral adhesion, transmissibility, and tissue tropism [ 50 , 51 ]. Additionally, the A283T substitution has frequently been associated with more severe HFMD cases and may contribute to prolonged disease duration or heightened virulence [ 53 , 54 ]. Beyond these, several other substitutions—such as E90D, T102A, and A255T—were identified, though their biological significance remains unclear [ 7 , 50 ]. The possibility of synergistic interactions among these mutations, particularly when co-occurring with functionally relevant sites such as S97N, S137N, and A283T, underscores the need for ongoing molecular and epidemiological surveillance. Functional characterization through in vitro and in vivo assays will be essential to monitor the emergence of phenotypic variants with potential clinical or epidemiological significance [ 7 , 14 ]. Nucleotide homology analysis of the VP1 region further confirmed that the isolates obtained in this study exhibited substantial genetic divergence from the Gdula prototype strain, while demonstrating high genetic homogeneity among themselves [ 50 ]. This pattern supports the hypothesis that circulating strains likely originated from closely related transmission chains, and indicates that the locally prevalent CV-A6 D3 sublineage is characterized by phylogenetic stability and sustained evolutionary continuity—features that merit continued surveillance in the context of prevention strategies and vaccine development [ 54 , 55 ]. At a practical level, this study provides molecular evidence to inform the development of multivalent HFMD vaccines and to support the monitoring of vaccine-induced selection pressure. The genetic diversity of CV-A6 appears largely confined to a single, rapidly evolving lineage, emphasizing the need to include CV-A6 antigens in future vaccine formulations. Additionally, diagnostic platforms must be updated to detect key immune escape mutations in order to maintain sensitivity and diagnostic accuracy. From a public health policy perspective, the consistent seasonal peak in HFMD cases during June and the high infection rates among children underscore the necessity of reinforcing seasonal health education, strengthening hygiene protocols in school and childcare settings, and establishing real-time interprovincial viral genome data sharing mechanisms to facilitate timely risk assessment and coordinated responses. Despite the strengths of this comprehensive molecular epidemiological analysis of CV-A6 in southern China, several limitations should be acknowledged. First, all samples were collected exclusively from Luohu District, Shenzhen, which may constrain the generalizability of the findings to the broader Guangdong Province or national context. Second, the analysis was restricted to the VP1 gene, omitting mutational and recombination data from non-structural regions (e.g., 3Dpol, 5′UTR), which may harbor determinants of replication efficiency or virulence. Third, while the dataset spanned multiple months and epidemic phases, the relatively modest sample size (n = 47) may have limited the detection of low-frequency mutations or subtle genotype-phenotype correlations. Lastly, several functional interpretations of amino acid substitutions were based on previously published studies and remain unverified through experimental validation. Thus, conclusions regarding the pathogenic or immunological impact of these mutations should be interpreted cautiously. To address these limitations, future studies should expand the geographic scope of sampling to include additional regions within Guangdong Province and other domestic and international locations with high CV-A6 prevalence. Whole-genome sequencing is recommended to uncover recombination events and assess evolutionary dynamics across the full viral genome. Moreover, structural and functional analyses—including animal model experiments—are warranted to evaluate the biological effects of key mutations on viral replication and pathogenicity. Sustained interprovincial and international collaboration in surveillance and data sharing is essential. Establishing a real-time, nationwide and global molecular surveillance and early warning system for CV-A6 would facilitate the timely detection of emerging high-risk variants and guide appropriate public health responses. Additionally, the development of spatiotemporal models incorporating meteorological variables and human mobility data is recommended to enhance the accuracy of HFMD epidemic forecasts. These models can support informed allocation of public health resources and optimize the timing of vaccination campaigns. Conclusions This integrated epidemiological and phylodynamic investigation shows that Coxsackievirus A6 is now the principal driver of HFMD in Shenzhen, exhibiting marked June seasonality and accounting for nearly two-thirds of virologically confirmed cases in 2023. Bayesian analyses dating the VP1 tMRCA to the mid-1940s, a mean evolutionary rate of ≈ 4.8 × 10⁻³ substitutions/site/year, and a post-2008 expansion of the effective population size collectively point to sustained, rapid viral diversification. All contemporary isolates clustered within genotype D3 and formed temporally, rather than geographically, structured clades interspersed with strains from multiple Chinese provinces, indicating frequent interprovincial dissemination and limited local sequestration. Thirty-one amino-acid substitutions—including twelve fixed relative to the prototype strain and several variable, surface-exposed sites—underscore continuing antigenic drift with potential implications for immune escape. Together, these findings highlight the importance of coupling high-resolution genomic surveillance with routine HFMD reporting to detect emergent lineages promptly, refine risk-based early-warning systems, and inform regionally tailored control measures and future vaccine design. Abbreviations CV Coxsackievirus EV enterovirus HFMD hand, foot, and mouth disease qRT-PCR real-time quantitative PCR HPD highest posterior density MCC maximum clade credibility MCMC Markov chain Monte Carlo tMRCA time of the most recent common ancestor Declarations Clinical trial registration Not applicable Ethics approval and consent to participate This study received ethical approval from the Committee for Ethical Review of Center for Disease Prevention and Control of Luohu District (Approval No.: LHCDCIRB2025-003A). The requirement for informed consent was formally waived as the research utilized fully anonymized residual diagnostic samples collected during routine public health surveillance, in compliance with Article 9 of China's Regulations on Human Genetic Resources Management (State Council Decree No. 717). All experimental protocols strictly adhered to the principles of the Declaration of Helsinki (2013 revision). Consent for publication Not applicable Availability of data and materials All VP1 sequences isolated in this study are deposited in GenBank under accessions PV804734-PV804780 Competing interests The authors declare that they have no competing interests in this section. Funding This study was conducted with the support of the Luohu District Soft Science Research Program(LX202302009). Authors' contributions YZ.D. designed the study. Sample collection and laboratory testing were conducted by YZ.D., QM.L., and FF.W. Data curation was performed by YZ.D., QM.L., and YM.W. The project was supervised by GQ.Y., M.Y., and F.Z. The initial draft of the manuscript was written by YZ.D. and critically reviewed and revised by GQ.Y., M.Y., and F.Z. Acknowledgements The authors gratefully acknowledge the staff of Luohu District People's Hospital for their contributions to sample collection and case reporting. References Zhu P, Ji W, Li D, Li Z, Chen Y, Dai B, Han S, Chen S, Jin Y, Duan G. Current status of hand-foot-and-mouth disease. J Biomed Sci. 2023 Feb 24;30(1):15. doi: 10.1186/s12929-023-00908-4. PMID: 36829162; PMCID: PMC9951172. Huang, C. Y., Su, S. B.,& Chen, K. T. (2024). A review of enterovirus-associated hand-foot and mouth disease: preventive strategies and the need for a global enterovirus surveillance network. Pathogens and Global Health, 118 (7–8), 538–548. https://doi.org/10.1080/20477724.2024.2400424 Zhang, W., Rui, J., Cheng, X. et al. Transmissibility of hand, foot, and mouth disease in 97 counties of China. 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Pathogens 2024, 13, 1020. https://doi.org/10.3390/pathogens13111020 Additional Declarations No competing interests reported. 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01:23:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6951771/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6951771/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12879-025-12003-0","type":"published","date":"2025-11-12T15:57:36+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":88491149,"identity":"34507e31-9103-4df7-a5b3-830352f9b40f","added_by":"auto","created_at":"2025-08-07 04:18:11","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":47744,"visible":true,"origin":"","legend":"\u003cp\u003eMonthly distribution of HFMD cases reported in Luohu District, 2022–2024.\u003c/p\u003e\n\u003cp\u003eMonthly case counts illustrate seasonal trends, with consistent peaks observed in June across all three years.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6951771/v1/0d296a242cc95c2e7f4c2110.png"},{"id":88490137,"identity":"dfcf2266-4833-480f-b941-0750c078cb56","added_by":"auto","created_at":"2025-08-07 04:10:11","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":22780,"visible":true,"origin":"","legend":"\u003cp\u003eAnnual distribution of enterovirus types detected in HFMD cases (2022–2024).\u003cbr\u003e\nSerotype composition of laboratory-confirmed HFMD cases, showing year-specific dominance patterns of CV-A6, CV-A16, and other enteroviruses.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-6951771/v1/62e353b9e7fd5b0a050c85f6.png"},{"id":88490144,"identity":"91bfb6df-3080-4adc-a8cd-a6c0ab5a16c1","added_by":"auto","created_at":"2025-08-07 04:10:11","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":199764,"visible":true,"origin":"","legend":"\u003cp\u003eMolecular evolutionary analysis of CV-A6 based on VP1 gene sequences.\u003c/p\u003e\n\u003cp\u003e(A) Maximum clade credibility (MCC) tree annotated with genotypes A–D3. The D3 genotype (red) forms the dominant, rapidly expanding lineage.\u003c/p\u003e\n\u003cp\u003e(B) Temporal signal regression of root-to-tip genetic distance versus sampling date (R² = 0.86), confirming suitability for molecular clock modeling.\u003c/p\u003e\n\u003cp\u003e(C) Bayesian SkyGrid plot showing changes in effective population size over time, with rapid expansion starting in 2008 and plateauing after 2010.\u003c/p\u003e\n\u003cp\u003e(D) Time-scaled MCC tree of D3 genotype, showing fine-scale branching, temporal structure, and 95% HPD intervals at key nodes.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6951771/v1/dc2e395f6e4a34496d6603e3.png"},{"id":88490145,"identity":"583af0d4-7080-4123-80f9-35c6b522f45f","added_by":"auto","created_at":"2025-08-07 04:10:11","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":738506,"visible":true,"origin":"","legend":"\u003cp\u003eTime-scaled phylogenetic tree of global CV-A6 VP1 sequences.\u003c/p\u003e\n\u003cp\u003eCircular tree based on 465 VP1 sequences collected from 1949 to 2024. Outer ring colors indicate geographic origin; inner gradient indicates sampling year. Isolates from this study are highlighted and cluster within genotype D3. The tree shows widespread interregional mixing and recent global expansion of D3 lineage.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6951771/v1/77d127be3fe9cb7f4dc756e3.png"},{"id":88491150,"identity":"ab9df502-16e4-4c9f-982a-249e8506d223","added_by":"auto","created_at":"2025-08-07 04:18:11","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":183806,"visible":true,"origin":"","legend":"\u003cp\u003eAmino acid mutation sites and genetic differences analysis of the VP1 gene. Analysis of amino acid mutations in the VP1 gene of the isolated strains of CV-A6\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6951771/v1/0a14c713ff86500e48ee5f59.png"},{"id":88490143,"identity":"0b13285c-60ef-4ffb-b0e4-5320da919844","added_by":"auto","created_at":"2025-08-07 04:10:11","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":140633,"visible":true,"origin":"","legend":"\u003cp\u003eEstimates of evolutionary divergence among VP1 sequences of CV-A6 strains. The matrix displays pairwise nucleotide divergence values among 48 CV-A6 isolates, including the prototype strain (AY421764.1). Values are represented both numerically and by color intensity. The analysis was performed using the Maximum Composite Likelihood model.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6951771/v1/14ae7fdcfd37f2dcb4928578.png"},{"id":96105150,"identity":"902079b7-fb97-44da-b07a-c2abf99caa98","added_by":"auto","created_at":"2025-11-17 16:09:21","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1831370,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6951771/v1/526b6e34-8ca7-44fc-8e89-e6e4e3efc991.pdf"},{"id":88490135,"identity":"8637623e-502f-4a28-80a1-5d21a914a806","added_by":"auto","created_at":"2025-08-07 04:10:11","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":16794,"visible":true,"origin":"","legend":"","description":"","filename":"CoxsackievirusA6sequencenumbersforphylogeneticevolutionaryanalysis..xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6951771/v1/4e333f19d15aa3551b830c37.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eTemporal Phylodynamics of Coxsackievirus A6 VP1 in Shenzhen（2022-2024）\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHand, foot, and mouth disease (HFMD) is a common acute infectious disease predominantly affecting children, with particularly high incidence rates in the Asia-Pacific region. In China, annual reported cases have often exceeded one million over the past two decades, with frequent occurrences of severe cases and fatalities, posing a significant burden on public health and the socio-economic system [1\u0026ndash;3].\u003c/p\u003e\n\u003cp\u003eHistorically, enterovirus A serotypes Enterovirus A71 (EV-A71) and Coxsackievirus A16 (CV-A16) were identified as the primary causative agents of HFMD [4]. However, since the first large-scale outbreak of\u0026nbsp;Coxsackievirus A6 (CV-A6) in Finland in 2008 [5], the virus has rapidly spread across Europe, Asia, and the Americas, supplanting traditional serotypes to become the predominant pathogen in many regions [6,7]. Following the introduction of the inactivated EV-A71 vaccine into China\u0026apos;s immunization program in 2016, the prevalence of EV-A71 and CV-A16 has significantly declined, whereas CV-A6 has exhibited rapid emergence [8,9].\u003c/p\u003e\n\u003cp\u003eClinically, CV-A6 infection differs markedly from classical HFMD. Patients more frequently present with high fever and widespread vesicular or hemorrhagic rashes extending beyond the typical sites of the hands, feet, and mouth, often involving the trunk, buttocks, and distal limbs [10]. Onychomadesis (nail shedding) commonly occurs 2\u0026ndash;3 weeks after recovery, with incidence rates ranging from 30% to 50% [11]. Moreover, there has been a noticeable increase in cases among adults and older children [12], and severe complications\u0026mdash;such as aseptic meningitis, brainstem encephalitis, and myocardial involvement\u0026mdash;have occasionally been reported [1]. These evolving epidemiological and clinical features underscore the need for enhanced surveillance and targeted clinical strategies to mitigate the growing impact of CV-A6 in HFMD.\u003c/p\u003e\n\u003cp\u003eThe VP1 capsid protein of CV-A6 plays a central role in viral pathogenicity, harboring most of the serotype-specific neutralizing epitopes. Due to its strong serotype correlation, VP1 is widely used as a molecular marker for virus typing, phylogenetic analysis, and mutation surveillance [13,14]. Phylogenetic analyses of VP1 sequences over the past decade consistently place nearly all global CV-A6 isolates within genotype D, predominantly subtype D3 [6,7,15]. The mean nucleotide divergence in D3 VP1 (6\u0026ndash;8%) exceeds that observed in EV-A71 and CV-A16, suggesting a higher molecular evolutionary rate [7].\u003c/p\u003e\n\u003cp\u003eWhile comprehensive genomic studies have documented frequent recombination events in the non-structural regions of the CV-A6 genome, the VP1 region maintains a relatively conserved lineage structure, supporting its continued use in genotyping and viral source tracking [13]. Regional surveillance data from Beijing, Hong Kong, and Thailand demonstrate the persistent, localized circulation of subtype D3, with a gradual accumulation of characteristic mutations that form multiple sublineages and exhibit phylogenetic connections with neighboring countries [15,16,18].\u003c/p\u003e\n\u003cp\u003eInvestigating VP1 sequence diversity, substitution rates, and lineage turnover dynamics is thus crucial for understanding the transmission patterns and adaptive evolution of CV-A6 [7,17]. However, spatiotemporal dynamics based on VP1 remain insufficiently studied in China [16]. To address this gap, we collected VP1 sequences from CV-A6 isolates obtained in Shenzhen\u0026apos;s Luohu District between 2022 and 2024 and integrated them with global datasets. Using Bayesian molecular clock and discrete-time spatiotemporal models, we aim to reconstruct the virus\u0026apos;s evolutionary history, infer transmission trajectories, and model population dynamics [17,18]. Additionally, we will evaluate the potential biological significance of key amino acid substitutions. These analyses will contribute to robust HFMD forecasting, early-warning systems, and precision control strategies.\u003c/p\u003e"},{"header":"Methods","content":"\u003ch3\u003eEpidemiological Data and Sample Sources\u003c/h3\u003e\n\u003cp\u003eThis study utilized pathogen surveillance and epidemiological data on hand, foot, and mouth disease (HFMD) obtained from the Infectious Disease Monitoring Information Reporting System of the Chinese Center for Disease Control and Prevention. In accordance with the Law of the People\u0026apos;s Republic of China on the Prevention and Control of Infectious Diseases, HFMD is designated as a Class C notifiable disease. Medical institutions diagnosing HFMD cases are required to report them promptly to the national system [19].\u003c/p\u003e\n\u003cp\u003eClinical specimens were primarily collected from outpatients at Luohu District People\u0026apos;s Hospital in Shenzhen, all of whom were clinically diagnosed with HFMD and reported to the national infectious disease information system. To enhance sample diversity and representativeness, additional specimens were obtained from patients involved in HFMD outbreak clusters reported by schools in Luohu District. These samples were collected during the early phase of the outbreaks to ensure that the data accurately reflect current epidemic trends.\u003c/p\u003e\n\u003ch3\u003eSample Selection and VP1 Gene Sequencing\u003c/h3\u003e\n\u003cp\u003eThroat swabs were processed using an automated nucleic acid extraction system (Zhongyuan Huiji Biotech, China), followed by commercial real-time quantitative PCR (qRT-PCR) assays (Jiangsu Shuoshi Biotech, China) for CV-A6 detection.\u003c/p\u003e\n\u003cp\u003eSamples were selected for sequencing if qRT-PCR confirmed CV-A6 positivity with a cycle threshold (CT) value \u0026lt; 30, indicating sufficient viral load. To ensure comprehensive temporal coverage, specimens were collected throughout all seasons. Representative samples from outbreak clusters during peak incidence periods were prioritized, with a maximum of five samples sequenced per high-incidence month to reduce redundancy.\u003c/p\u003e\n\u003cp\u003eA total of 50 samples were submitted to Shanghai Better Medical Technology Co., Ltd. for amplification and Sanger sequencing of the full-length VP1 gene (915 bp). High-quality VP1 sequences were successfully obtained from 47 samples. Rigorous quality control was conducted at each stage to ensure sequence integrity and accuracy.\u003c/p\u003e\n\u003ch3\u003eNucleotide and Amino Acid Variation Analysis\u003c/h3\u003e\n\u003cp\u003eThe CV-A6 prototype strain Gdula (GenBank: AY421764) was used as the reference for comparative analysis. The 47 VP1 gene sequences generated in this study were aligned at the nucleotide level using the MegAlign module of DNASTAR (Lasergene v7.1). Protein sequences were derived from the open reading frame, and both nucleotide and amino acid alignments were examined to identify variable sites. Emphasis was placed on nonsynonymous substitutions with a frequency \u0026gt; 5% or those located in known antigenic or functional domains, as these mutations may have biological relevance.\u003c/p\u003e\n\u003cp\u003eKey mutations were mapped to structural or epitope data to assess their potential impact on viral phenotype, host receptor binding, or immune evasion. All alignments were performed using default parameters, and variant calling results were manually validated to ensure analytical accuracy.\u003c/p\u003e\n\u003ch3\u003eDataset Construction and Temporal Signal Assessment\u003c/h3\u003e\n\u003cp\u003eTo investigate the global phylogenetic structure and evolutionary dynamics of CV-A6, we retrieved all publicly available full-length VP1 gene sequences (915 bp) from the GenBank database using the keyword \u0026quot;Coxsackievirus A6.\u0026quot; Sequence retrieval was completed on December 31, 2024. Sequences containing ambiguous nucleotides (e.g., \u0026quot;N\u0026quot;) or misannotated as non\u0026ndash;CV-A6 were excluded during the initial quality control process. Redundant sequences with \u0026ge;99.99% identity were removed using BioAider (v1.727) [20], followed by additional filtering to eliminate highly similar sequences from the same geographic region and collection year, thereby ensuring broad temporal and spatial coverage.\u003c/p\u003e\n\u003cp\u003eThe remaining sequences were aligned using MAFFT (v7.526) [21]. A maximum-likelihood phylogenetic tree was then reconstructed using IQ-TREE (v2.4.0) [22], with model selection guided by the Bayesian Information Criterion. Temporal signal was assessed using TreeTime [23] by regressing root-to-tip genetic distances against the sampling dates. The slope of the regression line represented the estimated nucleotide substitution rate, while the coefficient of determination (R\u0026sup2;) quantified the strength of the temporal signal. To ensure suitability for molecular clock modeling, statistically significant outliers were removed, and only datasets with R\u0026sup2; \u0026gt; 0.80 were retained for downstream phylodynamic analyses.\u003c/p\u003e\n\u003cp\u003eThe final dataset comprised 465 high-quality VP1 sequences originating from 12 countries and 23 provinces in China, providing robust spatial and temporal representation for molecular epidemiological investigation.\u003c/p\u003e\n\u003ch3\u003ePhylodynamic Analysis\u003c/h3\u003e\n\u003cp\u003eTo estimate the evolutionary rate and epidemic dynamics of CV-A6, we performed a Bayesian phylodynamic analysis using the curated VP1 sequence dataset. The optimal nucleotide substitution model was identified using the ModelFinder module within PhyloSuite (v1.2.3) [24]. Among 24 candidate models, SYM+G4 was selected as the best-fitting model (Table 1).\u003c/p\u003e\n\u003cp\u003eA time-scaled phylogenetic tree was inferred using BEAST v1.10.4, employing a Bayesian Markov Chain Monte Carlo (MCMC) approach under a relaxed molecular clock model, in conjunction with the Bayesian SkyGrid coalescent model [25,26]. The MCMC analysis was run for 200 million generations, with sampling performed every 20,000 generations. Convergence diagnostics were conducted in Tracer v1.7.2 [27], confirming that the effective sample size (ESS) for all key parameters exceeded 200.\u003c/p\u003e\n\u003cp\u003eA Maximum Clade Credibility (MCC) tree was generated using TreeAnnotator (v1.10.4), and the resulting phylogeny was visualized with FigTree v1.4.4 [28]. This visualization provided detailed insights into the branching structure, estimated time to the most recent common ancestor (tMRCA), and the evolutionary trajectory of CV-A6.\u003c/p\u003e\n\u003cp\u003eTable 1. Best-fit nucleotide substitution model identified by ModelFinder.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eModelFinder will test up to 24 DNA models (sample size: 915 epsilon: 0.100) ...\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"612\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 48px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNo.\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 128px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eModel\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 122px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eLnL\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u003cstrong\u003edf\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 78px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAIC\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 92px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAICc\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 78px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBIC\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eJC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e39824.811\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e1011\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e81671.623\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e2127935.623\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e86543.555\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eJC+G4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e35951.225\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e1012\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e73926.451\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e2124238.451\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e78803.202\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eTN+F\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e35650.495\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e1016\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e73332.991\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e2139876.991\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e78229.018\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eTN+F+G4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e31803.118\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e1017\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e65640.236\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e2136252.236\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e70541.082\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eTNe\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e35645.958\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e1013\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e73317.916\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e2127681.916\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e78199.486\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eTNe+G4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e31781.318\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e1014\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e65590.637\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e2124010.637\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e70477.026\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eK2P\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e35719.615\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e1012\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e73463.231\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e2123775.231\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e78339.982\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eK2P+G4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e31787.078\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e1013\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e65600.155\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e2119964.155\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e70481.726\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eK2P\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e35719.615\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e1012\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e73463.231\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e2123775.231\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e78339.982\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eK2P+G4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e31787.078\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e1013\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e65600.155\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e2119964.155\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e70481.726\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eF81+F\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e39880.447\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e1014\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e81788.894\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e2140208.894\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e86675.283\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eF81+F+G4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e36026.881\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e1015\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e74083.762\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e2136563.762\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e78974.97\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eHKY + F\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e35783.528\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e1015\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e73597.056\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e2136077.056\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e78488.264\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eHKY+F+G4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e31829.335\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e1016\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e65690.67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e2132234.67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e70586.697\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eSYM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e35597.181\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e1016\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e73226.362\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e2139770.362\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e78122.388\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eSYM+G4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e31726.782\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e1017\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e65487.564\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e2136099.564\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e70388.41\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eTIM+F\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e35644.772\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e1017\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e73323.544\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e2143935.544\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e78224.39\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eTIM+F+G4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e31798.421\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e1018\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e65632.843\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e2140316.843\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e70538.507\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eTVM + F\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e35734.022\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e1018\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e73504.044\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e2148188.044\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e78409.709\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eTVM+F+G4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e31776.97\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e1019\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e65591.941\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e2144351.941\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e70502.424\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eTVME\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e35670.801\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e1015\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e73371.603\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e2135851.603\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e78262.811\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eTVMe+G4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e31731.854\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e1016\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e65495.707\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e2132039.707\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e70391.734\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eGT+F\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e35601.09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e1019\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e73240.18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e2152000.18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e78150.664\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eGTR+F+G4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e31751.22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e1020\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e65542.441\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e2148382.441\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e70457.743\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eAkaike Information Criterion: SYM+G4\u003c/p\u003e\n\u003cp\u003eCorrected Akaike Information Criterion: K2P+G4\u003c/p\u003e\n\u003cp\u003eBayesian Information Criterion: SYM+G4\u003c/p\u003e\n\u003cp\u003eBest-fit model: SYM+G4 chosen according to BIC\u0026nbsp;\u003c/p\u003e"},{"header":"Results","content":"\u003ch4\u003eEpidemiological and Virological Analysis\u003c/h4\u003e\n\u003cp\u003eBetween 2022 and 2024, a total of 195,951 notifiable infectious disease cases were reported in Luohu District, Shenzhen, of which 11,427 were cases of hand, foot, and mouth disease (HFMD). HFMD accounted for 3.11% (408 cases) of all reported notifiable diseases in 2022, 6.35% (6,332 cases) in 2023, and 5.64% (4,687 cases) in 2024. It consistently ranked among the top three most frequently reported infections and exhibited a generally increasing trend over the study period, with a notable peak in 2023.\u003c/p\u003e\n\u003cp\u003eMonthly incidence data revealed a clear seasonal distribution (Figure 1). June emerged as the peak transmission month, contributing a total of 4,620 cases over the three-year period\u0026mdash;representing 40.43% of all reported HFMD cases (4,620/11,427). June 2023 recorded the highest monthly incidence, with 3,185 cases. In contrast, the 2024 outbreak exhibited a flatter epidemic curve, with a prolonged transmission period from May to August, suggesting a broader seasonal window.\u003c/p\u003e\n\u003cp\u003eTo characterize the viral composition of HFMD cases, 719 clinical specimens collected between 2022 and 2024 were tested using real-time RT-PCR targeting five major enterovirus serotypes: EV-A71, CV-A16, CV-A4, CV-A6, and CV-A10. The overall enterovirus positivity rate was 76.91% (553/719), with CV-A6 identified as the predominant serotype, accounting for 30.87% (222/719) of all tested samples.\u003c/p\u003e\n\u003cp\u003eA year-by-year analysis revealed a striking predominance of CV-A6 in 2023, with a positivity rate of 64.62% (190/294), indicating its dominance during that year\u0026apos;s epidemic. In contrast, 2022 and 2024 showed greater serotype diversity: CV-A16 and CV-A10 were more prevalent in 2022, while CV-A16 reemerged prominently in 2024, accounting for nearly half of all positive detections (Figure 2).\u003c/p\u003e\n\u003cp\u003eMonthly case counts illustrate seasonal trends, with consistent peaks observed in June across all three years.\u003c/p\u003e\n\u003cp\u003eSerotype composition of laboratory-confirmed HFMD cases, showing year-specific dominance patterns of CV-A6, CV-A16, and other enteroviruses.\u0026nbsp;\u003c/p\u003e\n\u003ch3\u003eMolecular Evolutionary Analysis\u003c/h3\u003e\n\u003cp\u003eTo investigate the evolutionary dynamics of CV-A6 strains isolated in this study and their genetic relationships with strains from other regions and historical periods, we constructed a comprehensive VP1 sequence dataset comprising 512 sequences. This dataset included 47 newly sequenced isolates and representative global reference strains retrieved from GenBank. The sequences spanned from 1949 to 2024 and encompassed 23 provinces in China and 12 countries worldwide, ensuring broad temporal and geographical representation.\u003c/p\u003e\n\u003cp\u003eTemporal signal strength was evaluated using TreeTime. Root-to-tip regression analysis demonstrated a strong linear relationship between genetic divergence and sampling time, with a coefficient of determination (R\u0026sup2;) of 0.86 and no significant outliers identified (Figure 3B). These results indicate a robust temporal signal, supporting the application of molecular clock models for Bayesian phylogenetic reconstruction. The estimated nucleotide substitution rate was 4.49 \u0026times; 10⁻\u0026sup3; substitutions per site per year, and the inferred time to the root was approximately 1942.5.\u003c/p\u003e\n\u003cp\u003eSubsequently, a time-scaled Bayesian phylogenetic analysis was conducted using BEAST under the SYM+G4 nucleotide substitution model, applying a relaxed molecular clock and the Bayesian SkyGrid coalescent model. This analysis reconstructed the Maximum Clade Credibility (MCC) tree. The mean substitution rate for the VP1 region of CV-A6 was estimated at 4.778 \u0026times; 10⁻\u0026sup3; substitutions per site per year (95% highest posterior density [HPD]: 4.3886 \u0026times; 10⁻\u0026sup3;\u0026ndash;5.1626 \u0026times; 10⁻\u0026sup3;), and the time to the most recent common ancestor (tMRCA) was inferred to be approximately 1945 (95% HPD: 1913\u0026ndash;1949) (Figures 3A, 3D).\u003c/p\u003e\n\u003cp\u003eBayesian SkyGrid demographic reconstruction revealed a rapid increase in the effective population size of CV-A6 beginning around 2008, followed by a plateau after 2010, indicating a convergence in genetic diversity over time (Figure 3C).\u003c/p\u003e\n\u003cp\u003ePhylogenetic lineage analysis classified CV-A6 into six major genotypes: A, B, C, D1, D2, and D3. Of these, genotype D3 has emerged as the globally dominant lineage in recent years, characterized by a dense and compact clade structure. The tMRCA of genotype D3 was estimated at 1998 (95% HPD: 1995\u0026ndash;2000), with a mean substitution rate of 4.192 \u0026times; 10⁻\u0026sup3; substitutions per site per year (95% HPD: 1.653 \u0026times; 10⁻\u0026sup3;\u0026ndash;7.651 \u0026times; 10⁻\u0026sup3;).\u003c/p\u003e\n\u003cp\u003eAll 47 CV-A6 isolates obtained in this study clustered within the D3 lineage and formed three distinct clades within the MCC tree. These clades were closely interspersed with strains from various regions of China, with no apparent geographic structuring. Instead, the phylogenetic pattern appeared more strongly associated with sampling time than with geographic origin, suggesting frequent interprovincial transmission and ongoing genetic exchange, with limited spatial differentiation among circulating strains.\u003c/p\u003e\n\u003cp\u003eTo contextualize the CV-A6 isolates from this study within a global evolutionary framework, a time-scaled circular phylogenetic tree was constructed based on VP1 sequences (Figure 4), incorporating 465 sequences with diverse temporal and geographic origins. The outer ring of the tree denotes the region of origin, while the inner ring represents sampling years, both color-coded for clarity.\u003c/p\u003e\n\u003cp\u003eAll isolates from Luohu District clustered within the D3 lineage and were dispersed among strains from multiple Chinese provinces, reflecting a temporally structured rather than geographically clustered pattern. This supports the hypothesis of widespread domestic circulation and a lack of strong geographic segregation.\u003c/p\u003e\n\u003cp\u003eNotably, several cross-regional clustering patterns were observed. For example, a 2021 sequence from Hungary and a 2023 sequence from the United Kingdom were grouped with earlier Indian strains from 2013 and 2018, rather than with contemporaneous European isolates. These findings suggest the possibility of long-term cryptic transmission or regional reintroductions driven by cross-border human movement, highlighting the complex and dynamic nature of global CV-A6 dissemination.\u003c/p\u003e\n\u003cp\u003eThe inner ring of the phylogeny features a gradient from purple (earlier years) to red-orange (recent years), vividly illustrating the rapid post-2010 expansion of the D3 genotype. The increasing density of terminal branches is consistent with the Bayesian SkyGrid results, further supporting the recent dominance and adaptive success of the D3 lineage in human populations.\u003c/p\u003e\n\u003cp\u003eCircular tree based on 465 VP1 sequences collected from 1949 to 2024. Outer ring colors indicate geographic origin; inner gradient indicates sampling year. Isolates from this study are highlighted and cluster within genotype D3. The tree shows widespread interregional mixing and recent global expansion of D3 lineage.\u0026nbsp;\u003c/p\u003e\n\u003ch3\u003eAmino Acid Mutation Profile and Nucleotide Homology Analysis\u003c/h3\u003e\n\u003cp\u003eThe complete VP1 gene of CV-A6 spans 915 nucleotides and encodes a 305-amino acid capsid protein. Amino acid sequences from the 47 isolates obtained in this study were inferred using MegAlign software. After eliminating redundant sequences with identical amino acid compositions, 16 representative strains containing unique mutations were selected. These sequences were aligned against the prototype CV-A6 strain Gdula (GenBank accession: AY421764.1), resulting in the identification of 31 amino acid substitution sites (Figure 5).\u003c/p\u003e\n\u003cp\u003eTwelve amino acid sites\u0026mdash;positions 5, 8, 10, 14, 32, 98, 160, 174, 194, 261, 279, and 305\u0026mdash;exhibited consistent substitutions in all isolates compared with the Gdula reference strain. Additional variable sites included mutations such as S97N, S137N, N241D, V242I, H243R, and A283T. Several of these substitutions were located in surface-exposed regions or within the BC loop of the VP1 protein, which are commonly associated with antigenic properties; however, their functional relevance was not evaluated in this study.\u003c/p\u003e\n\u003cp\u003eTo assess genetic divergence, pairwise nucleotide distances were calculated among VP1 sequences. The genetic distance between the study isolates and the Gdula prototype ranged from 19.5% to 22.0%, indicating substantial divergence at the nucleotide level. In contrast, genetic distances among the 47 local isolates were markedly lower, all \u0026le;6.7% (Figure 6), highlighting a high degree of sequence similarity among contemporary circulating strains and their clear evolutionary divergence from the historical prototype.\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eHand, foot, and mouth disease (HFMD) continues to impose a considerable public health burden among children in China [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Recent surveillance has revealed that Coxsackievirus A6 (CV-A6) has overtaken previously dominant serotypes in prevalence [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], highlighting the urgent need to elucidate its evolutionary dynamics and phenotypic plasticity. In this study, we analyzed 47 complete VP1 gene sequences collected in Luohu District, Shenzhen (2022\u0026ndash;2024), alongside 465 international reference sequences. We reconstructed a dated phylogenetic tree of the CV-A6 D3 sublineage, estimated its evolutionary rate and effective population history, and examined the structural and functional significance of amino acid substitutions defining contemporary epidemic lineages.\u003c/p\u003e\u003cp\u003eOur findings indicate that HFMD incidence in Luohu District consistently peaked in June across all three years. Among 719 laboratory-confirmed cases, CV-A6 accounted for 64.62% of positive detections in 2023, emerging as the predominant serotype. In contrast, both 2022 and 2024 exhibited co-circulation of multiple genotypes, with CV-A6 and CV-A16 being most prevalent. Multicenter studies across China have documented a gradual replacement of EV-A71 and CV-A16 by CV-A6 since 2012, with widespread outbreaks attributed to CV-A6 occurring in eastern and southern China by 2023 [\u003cspan additionalcitationids=\"CR33\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The majority of circulating strains cluster within the D3 sublineage, which is characterized by a higher evolutionary rate and enhanced immune escape potential [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe re-emergence of CV-A16 in 2024 may reflect dynamic genotype replacement: as herd immunity intensifies against a dominant serotype (e.g., CV-A6), alternative genotypes may spread more efficiently among susceptible populations [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Notably, no EV-A71-positive cases were identified in Luohu District during the study period, aligning with the high coverage of inactivated EV-A71 vaccination in Guangdong Province and its proven efficacy in preventing severe disease and mortality in children under five years of age [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. However, the monovalent nature of the current EV-A71 vaccine, while effective in curbing EV-A71 transmission, may inadvertently facilitate the spread of non-EV-A71 genotypes\u0026mdash;particularly CV-A6 and CV-A16\u0026mdash;by reducing natural cross-protective immunity [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. This phenomenon of serotype replacement has been reported across multiple Chinese provinces [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], underscoring the need for multivalent HFMD vaccines incorporating antigens from several key genotypes [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eDuring the COVID-19 pandemic (2020\u0026ndash;2021), the implementation of non-pharmaceutical interventions (NPIs) led to a marked reduction in HFMD incidence [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Following the relaxation of NPIs and the resumption of routine social and educational activities, a large cohort of immunologically naive children\u0026mdash;previously unexposed to enteroviruses\u0026mdash;became susceptible to infection, likely contributing to the HFMD surge observed in 2023 [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. In Luohu District, a densely populated urban center with high levels of cross-border mobility, the effects of this \"immunity debt\" were particularly pronounced.\u003c/p\u003e\u003cp\u003eBayesian phylogenetic analysis using BEAST estimated the mean substitution rate of the CV-A6 VP1 region at 4.78 \u0026times; 10⁻\u0026sup3; substitutions per site per year (95% highest posterior density [HPD]: 4.39\u0026ndash;5.16 \u0026times; 10⁻\u0026sup3;). This rate is consistent with previously reported estimates ranging from 4.0 to 7.4 \u0026times; 10⁻\u0026sup3; substitutions/site/year for CV-A6 in both global and Chinese datasets and places CV-A6 within the moderate-to-high evolutionary rate range among Enterovirus A species. These findings suggest that CV-A6 has a relatively high capacity for genetic drift [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe inferred time to the most recent common ancestor (tMRCA) for CV-A6 was dated to approximately 1945 (95% HPD: 1913\u0026ndash;1949), in line with historical evidence of the virus being isolated as early as the mid-20th century [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Importantly, the tMRCA of the currently predominant D3 sublineage was estimated to be around 1998 (95% HPD: 1995\u0026ndash;2000), supporting earlier studies indicating that D3 emerged in the late 1990s and subsequently replaced the D2 sublineage after 2010 [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. These data suggest that D3 has established a stable phylogenetic lineage and undergone sustained adaptive expansion over the past two decades [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eBayesian SkyGrid analysis revealed an increase in CV-A6 population genetic diversity beginning around 2000, followed by a reduction starting in 2010\u0026mdash;suggestive of a \"post-bottleneck concentration.\" The progressive narrowing of the 95% HPD interval over time reflects a convergence in viral genetic diversity. This pattern, when interpreted alongside the maximum clade credibility (MCC) tree, appears to be driven by the rapid expansion and dominance of the D3 sublineage. These results imply a possible \"selective sweep,\" potentially triggered by antigenic drift or recombination, that displaced other competing variants [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAll 47 isolates analyzed in this study belonged to the D3 sublineage and were grouped into three closely related clusters on the MCC tree. Despite the predominance of Chinese sequences in each cluster, no clear geographic structuring was observed, indicating frequent interprovincial transmission of CV-A6 across China [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. This observation aligns with the extensive domestic transportation network and high population mobility in the country [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eFurther supporting global transmission dynamics, CV-A6 strains isolated in Hungary and the United Kingdom between 2021 and 2023 clustered with Indian strains from 2013 and 2018, forming a shared lineage [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. These patterns suggest multiple international introductions and possible undetected community transmission events. The observed \"year-dominated, geography-attenuated\" dissemination highlights the limitations of relying solely on local surveillance systems to detect interregional and cross-border transmission risks in a timely manner [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe amino acid substitutions identified in the VP1 region exhibited distinct signatures characteristic of the D3 sublineage. Twelve fixed substitutions were consistently observed across all isolates, potentially serving as molecular markers of the D3 lineage's evolutionary trajectory. Several mutations were located in key functional regions of the VP1 protein and are hypothesized to influence viral antigenicity, receptor binding, and pathogenicity [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eFor example, the S97N mutation\u0026mdash;commonly observed in recent D3 epidemic strains\u0026mdash;is situated in a surface-exposed domain. Evidence suggests this substitution may alter local charge distribution, potentially reducing the binding affinity of neutralizing antibodies and enhancing immune evasion [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. The S137N substitution, also widespread among HFMD outbreak strains globally, may represent an important site of antigenic drift, challenging existing population immunity [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eOther mutations\u0026mdash;N241D, V242I, and H243R\u0026mdash;located in the VP1 BC loop, are implicated in receptor-binding functions and may influence viral adhesion, transmissibility, and tissue tropism [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Additionally, the A283T substitution has frequently been associated with more severe HFMD cases and may contribute to prolonged disease duration or heightened virulence [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eBeyond these, several other substitutions\u0026mdash;such as E90D, T102A, and A255T\u0026mdash;were identified, though their biological significance remains unclear [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. The possibility of synergistic interactions among these mutations, particularly when co-occurring with functionally relevant sites such as S97N, S137N, and A283T, underscores the need for ongoing molecular and epidemiological surveillance. Functional characterization through in vitro and in vivo assays will be essential to monitor the emergence of phenotypic variants with potential clinical or epidemiological significance [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eNucleotide homology analysis of the VP1 region further confirmed that the isolates obtained in this study exhibited substantial genetic divergence from the Gdula prototype strain, while demonstrating high genetic homogeneity among themselves [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. This pattern supports the hypothesis that circulating strains likely originated from closely related transmission chains, and indicates that the locally prevalent CV-A6 D3 sublineage is characterized by phylogenetic stability and sustained evolutionary continuity\u0026mdash;features that merit continued surveillance in the context of prevention strategies and vaccine development [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAt a practical level, this study provides molecular evidence to inform the development of multivalent HFMD vaccines and to support the monitoring of vaccine-induced selection pressure. The genetic diversity of CV-A6 appears largely confined to a single, rapidly evolving lineage, emphasizing the need to include CV-A6 antigens in future vaccine formulations. Additionally, diagnostic platforms must be updated to detect key immune escape mutations in order to maintain sensitivity and diagnostic accuracy.\u003c/p\u003e\u003cp\u003eFrom a public health policy perspective, the consistent seasonal peak in HFMD cases during June and the high infection rates among children underscore the necessity of reinforcing seasonal health education, strengthening hygiene protocols in school and childcare settings, and establishing real-time interprovincial viral genome data sharing mechanisms to facilitate timely risk assessment and coordinated responses.\u003c/p\u003e\u003cp\u003eDespite the strengths of this comprehensive molecular epidemiological analysis of CV-A6 in southern China, several limitations should be acknowledged. First, all samples were collected exclusively from Luohu District, Shenzhen, which may constrain the generalizability of the findings to the broader Guangdong Province or national context. Second, the analysis was restricted to the VP1 gene, omitting mutational and recombination data from non-structural regions (e.g., 3Dpol, 5\u0026prime;UTR), which may harbor determinants of replication efficiency or virulence. Third, while the dataset spanned multiple months and epidemic phases, the relatively modest sample size (n\u0026thinsp;=\u0026thinsp;47) may have limited the detection of low-frequency mutations or subtle genotype-phenotype correlations. Lastly, several functional interpretations of amino acid substitutions were based on previously published studies and remain unverified through experimental validation. Thus, conclusions regarding the pathogenic or immunological impact of these mutations should be interpreted cautiously.\u003c/p\u003e\u003cp\u003eTo address these limitations, future studies should expand the geographic scope of sampling to include additional regions within Guangdong Province and other domestic and international locations with high CV-A6 prevalence. Whole-genome sequencing is recommended to uncover recombination events and assess evolutionary dynamics across the full viral genome. Moreover, structural and functional analyses\u0026mdash;including animal model experiments\u0026mdash;are warranted to evaluate the biological effects of key mutations on viral replication and pathogenicity.\u003c/p\u003e\u003cp\u003eSustained interprovincial and international collaboration in surveillance and data sharing is essential. Establishing a real-time, nationwide and global molecular surveillance and early warning system for CV-A6 would facilitate the timely detection of emerging high-risk variants and guide appropriate public health responses. Additionally, the development of spatiotemporal models incorporating meteorological variables and human mobility data is recommended to enhance the accuracy of HFMD epidemic forecasts. These models can support informed allocation of public health resources and optimize the timing of vaccination campaigns.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis integrated epidemiological and phylodynamic investigation shows that Coxsackievirus A6 is now the principal driver of HFMD in Shenzhen, exhibiting marked June seasonality and accounting for nearly two-thirds of virologically confirmed cases in 2023. Bayesian analyses dating the VP1 tMRCA to the mid-1940s, a mean evolutionary rate of \u0026asymp;\u0026thinsp;4.8 \u0026times; 10⁻\u0026sup3; substitutions/site/year, and a post-2008 expansion of the effective population size collectively point to sustained, rapid viral diversification. All contemporary isolates clustered within genotype D3 and formed temporally, rather than geographically, structured clades interspersed with strains from multiple Chinese provinces, indicating frequent interprovincial dissemination and limited local sequestration. Thirty-one amino-acid substitutions\u0026mdash;including twelve fixed relative to the prototype strain and several variable, surface-exposed sites\u0026mdash;underscore continuing antigenic drift with potential implications for immune escape. Together, these findings highlight the importance of coupling high-resolution genomic surveillance with routine HFMD reporting to detect emergent lineages promptly, refine risk-based early-warning systems, and inform regionally tailored control measures and future vaccine design.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eCV\u003c/p\u003e\n\u003cp\u003eCoxsackievirus\u003c/p\u003e\n\u003cp\u003eEV\u003c/p\u003e\n\u003cp\u003eenterovirus\u003c/p\u003e\n\u003cp\u003eHFMD\u003c/p\u003e\n\u003cp\u003ehand, foot, and mouth disease\u003c/p\u003e\n\u003cp\u003eqRT-PCR\u003c/p\u003e\n\u003cp\u003ereal-time quantitative PCR\u003c/p\u003e\n\u003cp\u003eHPD\u003c/p\u003e\n\u003cp\u003ehighest posterior density\u003c/p\u003e\n\u003cp\u003eMCC\u003c/p\u003e\n\u003cp\u003emaximum clade credibility\u003c/p\u003e\n\u003cp\u003eMCMC\u003c/p\u003e\n\u003cp\u003eMarkov chain Monte Carlo\u003c/p\u003e\n\u003cp\u003etMRCA\u003c/p\u003e\n\u003cp\u003etime of the most recent common ancestor\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eClinical trial registration\u003c/p\u003e\n\u003cp\u003eNot applicable\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eEthics approval and consent to participate\u003c/p\u003e\n\u003cp\u003eThis study received ethical approval from the Committee for Ethical Review of Center for Disease Prevention and Control of Luohu District (Approval No.: LHCDCIRB2025-003A). The requirement for informed consent was formally waived as the research utilized fully anonymized residual diagnostic samples collected during routine public health surveillance, in compliance with Article 9 of China\u0026apos;s Regulations on Human Genetic Resources Management (State Council Decree No. 717). All experimental protocols strictly adhered to the principles of the Declaration of Helsinki (2013 revision).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eConsent for publication\u003c/p\u003e\n\u003cp\u003eNot applicable\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAvailability of data and materials\u003c/p\u003e\n\u003cp\u003eAll VP1 sequences isolated in this study are deposited in GenBank under accessions PV804734-PV804780 \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCompeting interests\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests in this section.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eThis study was conducted with the support of the Luohu District Soft Science Research Program(LX202302009).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAuthors\u0026apos; contributions\u003c/p\u003e\n\u003cp\u003eYZ.D. designed the study. Sample collection and laboratory testing were conducted by YZ.D., QM.L., and FF.W. Data curation was performed by YZ.D., QM.L., and YM.W. The project was supervised by GQ.Y., M.Y., and F.Z. The initial draft of the manuscript was written by YZ.D. and critically reviewed and revised by GQ.Y., M.Y., and F.Z.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAcknowledgements\u003c/p\u003e\n\u003cp\u003eThe authors gratefully acknowledge the staff of Luohu District People\u0026apos;s Hospital for their contributions to sample collection and case reporting.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eZhu P, Ji W, Li D, Li Z, Chen Y, Dai B, Han S, Chen S, Jin Y, Duan G. Current status of hand-foot-and-mouth disease. J Biomed Sci. 2023 Feb 24;30(1):15. doi: 10.1186/s12929-023-00908-4. PMID: 36829162; PMCID: PMC9951172.\u003c/li\u003e\n\u003cli\u003eHuang, C. Y., Su, S. B.,\u0026amp; Chen, K. T. (2024). A review of enterovirus-associated hand-foot and mouth disease: preventive strategies and the need for a global enterovirus surveillance network. 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Epidemiological and genetic characteristics of enteroviruses associated with hand, foot, and mouth disease in Jiaxing, China from 2019 to 2022. Sci Rep 15, 14546 (2025). https://doi.org/10.1038/s41598-025-99251-x\u003c/li\u003e\n\u003cli\u003eXu, L., Zheng, Q., Li, S. et al. Atomic structures of Coxsackievirus A6 and its complex with a neutralizing antibody. Nat Commun 8, 505 (2017). https://doi.org/10.1038/s41467-017-00477-9\u003c/li\u003e\n\u003cli\u003eLee, J.-E.; Kim, M.-J.; Lim, M.-H.; Han, S.-J.; Kim, J.-Y.; Kim, S.-H.; Ha, Y.-D.; Gang, G.-L.; Chung, Y.-S.; Seo, J.-M. Epidemiological and Genetic Characterization of Coxsackievirus A6-Associated Hand, Foot, and Mouth Disease in Gwangju, South Korea, in 2022. Viruses 2024, 16, 476. https://doi.org/10.3390/v16030476\u003c/li\u003e\n\u003cli\u003eYang X, Li Y, et al. Clinical features and phylogenetic analysis of severe hand-foot-and-mouth disease caused by Coxsackievirus A6. Infect Genet Evol. 2020;77:104054. https://doi.org/10.1016/j.meegid.2019.104054\u003c/li\u003e\n\u003cli\u003eHuanhuan Lu, Jinbo Xiao, Wenhui Wang, Dongmei Yan, Tianjiao Ji, Qian Yang, Haiyan Wei, Yanhua Du, Yunting Zeng, Jun Guo, Jianhua Chen, Hanri Zeng, Yingying Liu, Shuaifeng Zhou, Hong Ji, Jianxing Wang, Xiaofang Zhou, Yong Zhang. Evolutionary Diversity of Coxsackievirus A6 Causing Severe Hand, Foot, and Mouth Disease \u0026mdash; China, 2012\u0026ndash;2023[J]. China CDC Weekly, 2024, 6(20): 442-449. doi: 10.46234/ccdcw2024.086\u003c/li\u003e\n\u003cli\u003eJoyce, A.M.; Hill, J.D.; Tsoleridis, T.; Astbury, S.; Berry, L.; Howson-Wells, H.C.; Allen, N.; Canning, B.; Jones, C.B.; Clark, G.; et al. Coxsackievirus A6 U.K. Genetic and Clinical Epidemiology Pre- and Post-SARS-CoV-2 Emergence. Pathogens 2024, 13, 1020. https://doi.org/10.3390/pathogens13111020\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-infectious-diseases","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"infd","sideBox":"Learn more about [BMC Infectious Diseases](http://bmcinfectdis.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/infd","title":"BMC Infectious Diseases","twitterHandle":"#bmcinfectdis","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6951771/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6951771/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eHand, foot, and mouth disease (HFMD) remains a significant childhood infection in the Asia-Pacific region; however, the emergence of Coxsackievirus A6 (CV-A6) since 2008 has reshaped its epidemiology. Despite extensive national surveillance, the evolutionary tempo-spatial dynamics of CV-A6 in southern China remain poorly characterized. This study aimed to delineate the temporal phylodynamics of the CV-A6 VP1 region in Shenzhen and identify lineage-defining mutations that may inform early-warning systems and control strategies.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eEpidemiological surveillance data (2022\u0026ndash;2024) were integrated with VP1 sequences from 47 Shenzhen isolates and 465 global reference strains (1949\u0026ndash;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.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eAmong 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. CV-A6 was detected in 30.9% of tested specimens and predominated in 2023 (64.6%). Root-to-tip regression showed a strong temporal signal (R\u0026sup2; = 0.86), and the mean VP1 substitution rate was 4.78 \u0026times; 10⁻\u0026sup3; substitutions/site/year (95% highest posterior density [HPD]: 4.39\u0026ndash;5.16 \u0026times; 10⁻\u0026sup3;), 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 \u0026le;\u0026thinsp;6.7%, compared to 19.5\u0026ndash;22.0% divergence from Gdula.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eGenotype D3 of CV-A6 is now well established in Shenzhen, contributing to recurrent seasonal HFMD peaks in early summer. Its high evolutionary rate and rapid lineage turnover, coupled with limited geographic structuring, underscore the need for coordinated, cross-regional molecular surveillance. The conserved and variable VP1 residues identified in this study offer valuable candidate markers for molecular monitoring and vaccine antigen development.\u003c/p\u003e","manuscriptTitle":"Temporal Phylodynamics of Coxsackievirus A6 VP1 in Shenzhen（2022-2024）","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-07 04:10:06","doi":"10.21203/rs.3.rs-6951771/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-09-04T07:55:33+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-22T04:22:27+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-22T03:57:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"287219125410580939437807723682575487944","date":"2025-08-13T14:23:32+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-13T06:51:44+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"172613605876189270644917508429328643492","date":"2025-08-12T14:07:19+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"99520593269220568502216501900223301703","date":"2025-08-11T18:28:19+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-04T05:07:26+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-30T09:23:48+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-07-07T18:16:36+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-07T01:03:55+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Infectious Diseases","date":"2025-07-07T01:00:40+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-infectious-diseases","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"infd","sideBox":"Learn more about [BMC Infectious Diseases](http://bmcinfectdis.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/infd","title":"BMC Infectious Diseases","twitterHandle":"#bmcinfectdis","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4e36bb5c-7cf9-49ec-a645-12b8ca96cf55","owner":[],"postedDate":"August 7th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-11-17T16:04:12+00:00","versionOfRecord":{"articleIdentity":"rs-6951771","link":"https://doi.org/10.1186/s12879-025-12003-0","journal":{"identity":"bmc-infectious-diseases","isVorOnly":false,"title":"BMC Infectious Diseases"},"publishedOn":"2025-11-12 15:57:36","publishedOnDateReadable":"November 12th, 2025"},"versionCreatedAt":"2025-08-07 04:10:06","video":"","vorDoi":"10.1186/s12879-025-12003-0","vorDoiUrl":"https://doi.org/10.1186/s12879-025-12003-0","workflowStages":[]},"version":"v1","identity":"rs-6951771","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6951771","identity":"rs-6951771","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}