Introduction:Coxsackievirus A4 (CVA4) causes several diseases, including hand, foot, and mouth disease (HFMD) and herpangina. This study analyzed CVA4 isolates collected between 1996 and 2024 from the Xizang Autonomous Region to elucidate the phylogenetic characteristics and epidemiological patterns of this virus in high-altitude regions. Methods:VP1 coding region sequences of CVA4 were obtained through virus isolation and Sanger sequencing. MEGA software was used to construct a maximum likelihood phylogenetic tree based on the VP1 region. The BEAST toolkit was used to generate a maximum clade credibility tree and perform phylogeographic analysis. Results:From 2018 onwards, the prevalence of CVA4 among healthy carriers increased significantly, accounting for 62.12% of all detections. Genotyping revealed that most isolates belonged to genotype C, while the remainder were classified as D2. As the dominant genotype, genotype C has spread outward from Xigaze and Lhasa since 2011, leading to multiple asymptomatic infections in Shannan (2020), Xigaze and Lhasa (2023), and Ngari Prefecture (2024). Conclusions:Transmission of CVA4 genotype C among healthy children in high-altitude areas suggests strong environmental adaptability, highlighting the need to strengthen enterovirus surveillance and control, including targeted monitoring of CVA4, in these regions.

Coxsackievirus B1 (CV-B1) is primarily associated with meningitis but can also cause localized outbreaks of hand, foot, and mouth disease (HFMD). This study analyzed the genetic diversity of the VP1 gene in 39 strains of the CVB1 virus isolated from HFMD children across 15 provinces in China between 2010 and 2024, as well as 179 strains from 17 countries. Based on the average nucleotide difference of VP1 gene, we classified CVB1 virus into six genotypes A to F, Notably, genotype F is newly classified. Since 2010, genotype F guadually replaced genotype E as the dominant genotype in China and has further subdivided into three subtypes: F1, F2, and F3, with F3 being the most prevalent subtype in China currently. We specifically study the mild and severe cases within the F3 subtype. Temperature-sensitivity experiments revealed no differences between mild and severe cases of the F3 subtype, and they all belong to temperature-sensitive strains. Interestingly, we found that mild cases of the F3 subtype did not involve recombination, whereas all severe cases of the F3 subtype showed recombination with Coxsackievirus B4 (CVB4). CVB4 has consistently been the primary pathogen responsible for severe neonatal illnesses, suggesting that recombination between the F3 subtype and CVB4 may be associated with the development of severe HFMD. These findings provide fundamental scientific data for further investigation into the epidemiology and genetic characteristics of variants of Coxsackievirus B1 in China.

Hand, foot, and mouth disease (HFMD) is a common infectious disease in children, primarily caused by human enteroviruses (EVs). This study describes the epidemiological characteristics of HFMD in Taiyuan, China (2017-2024), also the genetic features of Coxsackievirus A6 (CVA6). The incidence of HFMD showed significant variations during this period. Male cases outnumbered females, with children aged 0-6 years accounting for 81.88% of all cases and exhibiting a distinct seasonal distribution pattern. Real-time reverse transcription polymerase chain reaction was performed on 4389 clinical specimens, identifying 1,920 human EVs-positive specimens. Positive cases included 690 CVA6, 530 CVA16, 65 CVA10, 152 Enterovirus A71, and 483 other human EVs. Pathogen spectrum analysis revealed that CVA6 maintained high prevalence from 2017 to 2023 but declined in 2024. Phylogenetic analysis of 194 complete VP1 sequences revealed all strains belonged to sub-genotype D3 within genotype D. Nucleic acid homology ranged from 91.4% to 100%, and amino acid similarity from 97.0% to 100%. A persistent, continuously circulating lineage existed within the D3a branch. The GH loop within VP1 revealed high sequence conservation and structural stability, confirming its suitability as a vaccine target. These findings enhance genetic data on CVA6, thereby providing crucial scientific basis for prevention and vaccine development.

Poliovirus (PV), a historically significant enterovirus responsible for severe paralytic diseases, has seen its incidence dramatically reduced through widespread vaccination efforts, propelling global eradication initiatives. Despite the success of traditional oral poliovirus vaccines (OPVs) and inactivated poliovirus vaccines (IPVs), challenges such as vaccine-derived virus reversion and biosafety concerns during vaccine production persist. Virus-like particle (VLP) vaccines, which mimic native viral structures without containing viral genomes, offer enhanced safety profiles and robust immunogenicity, positioning them as promising candidates for next-generation poliovirus vaccines, especially in the post-certification era. This review systematically summarizes current progress in poliovirus VLP vaccine research, including the diverse expression systems employed for VLP production, strategies for peptide assembly and stabilization, and evaluations of antigenicity and immunogenicity. Additionally, it highlights structural analyses utilizing cutting-edge cryo-electron microscopy. By integrating recent developments in genetic engineering, structural biology, and immunology, this article discusses the advantages and challenges associated with poliovirus VLP vaccines and explores future directions aimed at supporting the global goal of a poliovirus-free world. This comprehensive overview aims to provide a theoretical foundation and technical guidance to facilitate the development and deployment of safer and more effective poliovirus vaccines.

Coxsackievirus A4 (CVA4) is an enterovirus associated with diverse clinical syndromes, yet its global evolution and transmission dynamics remain poorly defined. This study aimed to characterize the molecular epidemiology, evolutionary history, and phylodynamic patterns of CVA4 at both local and global scales. In 2024, 34 CVA4 infections were identified during a localized herpangina outbreak in Beijing, primarily affecting children under 5 years of age, with a pronounced peak in July. High detection and isolation rates, together with temporal clustering in mid-2024, indicate a genuine short-term expansion rather than sporadic detection. Phylogenetic analysis of global VP1 sequences resolved six genotypes (A-F), with Beijing strains clustering within genotype E and subgenotype F2, reflecting ongoing worldwide circulation. Bayesian phylodynamic analysis estimated a VP1 evolutionary rate of 1.626 × 10-3 substitutions per site per year (95% highest probability density [HPD], 1.428-1.827 × 10-3) and dated the CVA4 time to the most recent common ancestor back to March 1917 (95% HPD, June 1908-April 1926). Demographic reconstruction revealed fluctuating genetic diversity, with notable expansions in 2013 and 2016, as well as a modest increase in 2023 preceding the outbreak. Phylogeographic analyses identified France and China as key seeding regions. Recombination in the P2 and P3 regions, including evidence of CVA2-related donors, highlights recombination as a major contributor to CVA4 diversity. These findings improve our understanding of CVA4 evolution and transmission and underscore the need for enhanced genomic surveillance.

Nucleotide (nt) deletions in the VP1 region of poliovirus are extremely rare. However, as early as 2012, we detected a natural type 2 poliovirus strain in sewage, originating from the Sabin 2, which exhibited such deletions. Whole-genome analysis revealed that the virus genome is 7,436 nt in length, with three nucleotide deletions in positions 16-18 of the VP1 region (2,497-2,499 nt), resulting in a deletion of the amino acid at position six of the VP1 capsid protein. Notaly, the missing amino acids are not located at known attenuation or neutralization sites. In addition, the important attenuated sites at positions 481 and 2,909 remained unchanged. Only one substitution was observed at the known neutralizing antigen site: VP3-61 (Arg to Lys) on NAg3a. Recombinant analysis showed that the virus is a type 2/3 recombinant virus, with the crossover site located between nucleotides 6,981 and 7,439, spanning the 3Dpol region and the 3' untranslated region (UTR). The protein structure simulation showed that VP1 capsid protein of the PV-2 deletion variant was very similar to Sabin 2. In the VP1 region, the PV-2 deletion variant has only a triple nucleotide deletion, with no other mutations, indicates that the virus was in an early stage of evolution before being isolated from sewage. Furthermore, this variant is only a type 2/3 recombinant, not recombined with other non-polio enteroviruses, suggesting a short transmission and circulation time of the virus in the population. Therefore, we speculate that the nucleotide deletion in VP1 region of poliovirus may occur in the early evolutionary stage. Although the virus has not yet spread widely among humans, these findings highlight the importance of continuous environmental monitoring of poliovirus.

Coxsackievirus A10 (CVA10) has been reported frequently in many infectious diseases and cases associated with hand, foot, and mouth disease (HFMD) emerging increasingly in recent years. Based on the National HFMD System Surveillance, 180 CVA10 strains were isolated from the mainland of China between 2008 and 2023. These strains were analyzed alongside 103 representative full VP1 sequences obtained from GenBank, with a focus on global-scale phylogenetic analysis and spatiotemporal dynamics of CVA10. Eight genotypes (A-H) were defined, of which the genotype C was the dominant gene subtype in Chinese mainland. Bayesian analysis indicated that the most renascent common ancestor (tMRCA) of CVA10 originated in 1932 (95% HPD:1867-1958), with a high evolutionary rate of 3.32 × 10-3 substitutions/site/year (95% HPD: 2.62 × 10-3 to 3.40 × 10-3). By analyzing the spatial propagation paths, the global CVA10 exhibited distinct regional characteristics. Though the origin of CVA10 could be in the USA, regional dissemination was mainly located around the Asia-Europe region. The spatiotemporal dynamics of CVA10 exhibited frequent viral traffic among localities, and virus from East and South China have played a central role in spreading around the mainland of China. Our phylogenetic description and phylogeographic analyses indicate the importance of large spatial- and temporal-scale studies in understanding epidemiological dynamics of CVA10, particularly the diffusion routes will be of great importance to global control efforts.

Enterovirus D68 (EV-D68) is a significant global pathogen associated with severe respiratory infections and acute flaccid myelitis in children. Currently, there are no vaccines or antiviral drugs available for EV-D68, and a robust model to elucidate the pathogenesis of EV-D68 and evaluate treatment methods is lacking. We developed a mouse-adapted EV-D68 strain that caused progressive limb paralysis after intramuscular inoculation in 7-day-old mice. Viral load analysis showed that the skeletal muscle and spinal cord had the highest titers and most severe injuries. RNA sequencing of the infected muscle, brain, spinal cord, and lung tissues revealed differentially expressed genes (DEGs) associated with viral infection and pathogenesis. DEGs were significantly enriched in various pathways associated with antiviral immunity, interferon responses, and cytokine signaling. In the spinal cord, DEGs highlighted mitochondrial dysfunction and oxidative stress as crucial contributors to neural damage. Flow cytometry analysis of spinal cord cells showed that EV-D68 activates the immune system, leading to systemic inflammation and significant increases in CD8+ and CD4+ T cells, but limited neutrophil and monocyte infiltration. This mouse model provides a valuable tool for studying EV-D68 pathogenesis and evaluating antiviral and vaccine efficacy, thereby advancing the understanding of its neuropathological mechanisms. Importance: We developed a novel mouse model of EV-D68 that provides a valuable tool for studying its pathogenesis and evaluating antiviral and vaccine efficacy, deepening the understanding of its neuropathological basis.

Echovirus 30 (E30) is a positive-sense RNA virus in the Picornaviridae family, specifically within the Enterovirus genu. It is a known pathogen that causes severe infectious diseases in humans. However, mechanisms underlying viral infection remain poorly understood. Therefore, this study aims to elucidate the mechanisms underlying E30 infection. A whole-genome CRISPR/Cas9 gene knockout screen was employed, and it was observed that knocking out ADP-ribosylation factor GTPase activating protein 1 (ARFGAP1) significantly reduced E30 replication. Subsequent analysis revealed that ARFGAP1 influencing the early stages and internalization of viral infection. Transcriptomic analysis further demonstrated that ARFGAP1 plays a crucial role in vesicular transport. Animal infection studies demonstrated that QS11-mediated inhibition of ARFGAP1 significantly decreased viral replication in mice with homozygous gene knock-out of both the human neonatal Fc receptor and the interferon-alpha/beta receptor (hFcRn-IFNAR-/- mice), mitigated tissue damage, and enhanced survival rates. This study identifies ARFGAP1 as a crucial host factor that promotes E30 infection, offering in depth understanding of the viral infection mechanisms and potential antiviral therapies.

In recent years, coxsackievirus A6 (CVA6) has surpassed enterovirus A71 to become the main pathogen causing severe Hand, Foot, and Mouth disease (HFMD) in China with a substantial disease burden. However, there is currently no commercial CVA6 vaccine. The D3a genotype of CVA6 is the predominant genotype in China. In this study, virus-like particles (VLPs) and mRNA vaccines based on the CVA6 sub-genotype D3a were successfully developed. The immunogenicity and protective effects of the VLP of CVA6 combined with Al(OH)3 and CpG adjuvant indicated that VLP-induced neutralizing antibodies against three CVA6 sub-genotype (D2, D3a, and D3b) strains in Institute of Cancer Research (ICR) mice, and the combination of the two adjuvants enhanced cellular immunity. Passive immunization with serum from mice immunized with VLPs protected suckling mice against CVA6 lethal challenge in both antiserum transfer and maternal immunization experiments. The immunogenicity and protective effects of the mRNA vaccine of CVA6 indicate that it induces robust T-cell immunity. T-cell immunity was found to cross-protect against coxsackievirus A10 infection in mice. This is the first trial of a CVA6 mRNA vaccine worldwide and the first comparison of the immunogenicity and protective effects of VLP and mRNA vaccines based on D3a CVA6. The study provides a theoretical basis for the development of enteroviruses vaccines and the formulation of immunization strategies.

Enterovirus D68 (EV-D68), a serotype of the enterovirus species D, has garnered significant attention due to outbreaks reported in 2014, 2016, and 2018. In this study, 36 Chinese EV-D68 strains were isolated, sequenced, and combined with all EV-D68 VP1 sequences from GenBank to form a data set of 1679 sequences. This data set served as the basis for phylogenetic, evolutionary dynamics, phylogeographic, and key amino acid site mutation analyses of EV-D68. Based on the VP1 region, EV-D68 is classified into four genotypes (A-D), and seven subgenotypes (B1-B3, D1-D4), with B3 and D3 being the predominant subgenotypes. Bayesian skyline plots indicated that genotypes B and D experienced multiple population expansions, aligning with reported EV-D68 outbreaks. Phylogeographic analyses of the B3 subgenotypes revealed sequences from Europe and North America clustering into a single evolutionary branch, suggesting significant transmission between these regions. Additionally, mutation analysis identified VP1-98 as a high-frequency mutation site, differing significantly between the previously prevalent A and C genotypes and the currently prevalent B and D genotypes. However, the functional implications of this mutation require further investigation. This study provides a solid theoretical basis for epidemiological research, disease surveillance, and prevention efforts related to EV-D68.

Hand, foot, and mouth disease (HFMD) represents a globally prevalent infectious disease that is caused by enteroviruses. Enterovirus A71 (EV-A71), coxsackievirus A16 (CVA16), and coxsackievirus A6 (CVA6) are recognized as the predominant causative agents of HFMD. CVA16 is a member of the genus Enterovirus within the family Picornaviridae. B1a and B1b are the most prevalent subgenotypes, whereas the B1c subgenotype is relatively scarce. In this study, a comprehensive analysis was conducted on 15 of CVA16 B1c strains isolated from samples of patients diagnosed with HFMD in Jixi (Heilongjiang Province, China) in 2022. Subsequently, whole genome sequencing of these strains was carried out. Phylogenetic origin and potential recombination events were analyzed by aligning sequences of isolated of CVA16 B1c strains with related sequences deposited in GenBank. The CVA16 B1c isolates examined in this study exhibited a high degree of similarities. Specifically, the nucleotide similarity within the VP1 region ranged from 99.6 to 100%. The average nucleotide substitution rate of CVA16 B1c viruses worldwide was estimated to be 5.14 × 10−3 (4.13–6.27 × 10−3) substitution/site/year, and the most recent common ancestor could be traced back to 2003. The earliest CVA16 B1c strain isolated in China was traced back to 2011. Transmission pathway analysis suggested that Chinese strains may have originated in India. Recombination analysis showed that CVA16 B1c strains likely undergone recombination events with EV-A71 and CVA4. In conclusion, the analysis of a cluster of CVA16 B1c cases detected for the first time in Heilongjiang Province not only expanded the gene sequence library of CVA16 B1c strains in China but also offered an epidemiological basis for further investigations into the antigen–antibody interactions and pathogenicity of CVA16 B1c.

Since the establishment of the Chinese acute flaccid paralysis (AFP) case surveillance system in 1999, around 7,200 strains of poliovirus (PV) type 1 have been identified and isolated. Among these, the VP1 region of 5,649 strains has been sequenced. Based on the existing VP1 region sequence library, four strains of type 1 PV with six-nucleotide deletion in the VP1 region, identified from AFP cases, healthy children, and environmental sewage samples, were identified, and their biological characteristics were investigated. Whole-genome sequence analysis showed that the similarity with the Sabin 1 strain was 99.5-99.8%, and the mutation rate in the VP1 region was only 0.11-0.55%, indicating that these strains are not vaccine-derived PVs. The missing nucleotide is located at positions 2,783-2,788 in the VP1 region, resulting in the deletion of amino acids 102 and 103 at neutralizing antigen site 1 (N-Ag I) in the BC loop. It is worth noting that the neutralization test results showed that the two strains detected from AFP and healthy children evaded immune recognition, whereas the other two from environmental sewage did not. Molecular docking and neutralization antigen site analyzes indicate that the deletion of nucleotides 2,783-2,788 in N-Ag I is not a critical factor leading to the development of neutralization escape variants.IMPORTANCEInterestingly, we observed that the VP3-60 mutation in N-Ag IIIa may be the main reason for the immune evasion of these two viruses. In addition, based on the temperature sensitivity experiments, the four viruses exhibited similar temperature sensitivity to the Sabin 1 strain and their replication ability at 39.5°C was comparable to vaccine-derived polioviruses. Although variants with six-nucleotide deletion (2,783-2,788 nt) in the VP1 region do not cause significant biological changes, they can still spread in the environment and among populations, posing a certain risk of transmission.

Multiple subtypes of avian influenza virus (AIV), including H5N1, H5N6, and H5N8 viruses, are currently co-circulating in wild birds and poultry and causing sporadic human infections. Vaccine development is essential for pandemic preparedness. In this study, we constructed a candidate vaccine virus (CVV) using reverse genetics (RG) based on the sequence of the first human-infected H5N8 subtype AIV, A/Astrakhan/3212/2020 (H5N8). We evaluated the immunogenicity of the rH5N8/PR8 vaccine strain in combination with Alum, ISA51, and MF59 adjuvants, and we optimized immunization strategies including dosage, administration route, and immunization interval in BALB/c mice. Our results demonstrated that a 10 μg dose of inactivated rH5N8/PR8 with MF59 adjuvant, administered intramuscularly twice at 7-day intervals, induced the strongest immune response and effectively protected mice against challenge with wild-type H5N8 AIVs. Since pandemic influenza vaccines typically require tailored vaccination doses and routes specific to their characteristics, this study provides valuable insights for the development of similar vaccine strains with pandemic potential.

Hand, foot, and mouth disease (HFMD) caused by a group of enteroviruses is a global public health problem. In recent years, coxsackievirus A6 (CVA6) has emerged as an important HFMD agent. Previous studies have shown that mutations of glycine 64 in RNA-dependent RNA polymerase (3D polymerase), which is central to viral replication, cause phenotypic changes such as ribavirin resistance, increased replication fidelity, and virulence attenuation in poliovirus and enterovirus A71. In this study, we constructed CVA6 mutants with G64R, G64S, and G64T substitutions by site-directed mutagenesis in full-length cDNA of an infectious CVA6 strain cloned in pcDNA3.1. Viral RNA was obtained by in vitro transcription, and the rescued virus strains were propagated in RD cells. Sequencing after six passages revealed that G64S and G64T mutations were stably inherited, whereas G64R was genetically unstable and reversed to the wild type. Comparison of the biological characteristics of the wild-type and mutant CVA6 strains in an in vivo model (one-day-old ICR mice) revealed that the pathogenicity of CVA6-G64S and CVA6-G64T was significantly reduced compared to wild-type CVA6. In vitro experiments indicated the mutant CVA6-G64S and CVA6-G64T strains had increased resistance to 0.8 mM ribavirin and a decreased replication rate in the presence of 0.8 mM guanidine hydrochloride. Our results show that mutation of residue 64 reduces CVA6 susceptibility to ribavirin and increases CVA6 susceptibility to guanidine hydrochloride, together with increased replication fidelity and attenuated viral pathogenicity, thus laying a foundation for the development of safe and effective live attenuated CVA6 vaccine.

Coxsackievirus A16 (CVA16) is a major pathogen that causes hand, foot, and mouth disease (HFMD). The recombination form (RF) shifts and global transmission dynamics of CVA16 remain unknown. In this retrospective study, global sequences of CVA16 were retrieved from the GenBank database and analyzed using comprehensive phylogenetic inference, RF surveys, and population structure. A total of 1,663 sequences were collected, forming a 442-sequences dataset for VP1 coding region analysis and a 345-sequences dataset for RF identification. Based on the VP1 coding region used for serotyping, three genotypes (A, B, and D), two subgenotypes of genotype B (B1 and B2), and three clusters of subgenotype B1 (B1a, B1b, and B1c) were identified. Cluster B1b has dominated the global epidemics, B2 disappeared in 2000, and D is an emerging genotype dating back to August 2002. Globally, four oscillation phases of CVA16 evolution, with a peak in 2013, and three migration pathways were identified. Europe, China, and Japan have served as the seeds for the global transmission of CVA16. Based on the 3D coding region of the RFs, five clusters of RFs (RF-A to -E) were identified. The shift in RFs from RF-B and RF-C to RF-D was accompanied by a change in genotype from B2 to B1a and B1c and then to B1b. In conclusion, the evolution and population dynamics of CVA16, especially the coevolution of 3D and VP1 genes, revealed that genotype evolution and RF replacement were synergistic rather than stochastic.

Hand, foot, and mouth disease (HFMD), a common childhood infection caused by enterovirus, poses a serious public health concern in China. We collected and analyzed epidemiological data on 62,133 HFMD cases in Shenyang City, Liaoning Province, from 2013 to 2023. The average annual incidence was 76.12 per 100,000 person-years; 99.45% of cases were mild, while 0.55% were severe. Only one patient died. HFMD infections peaked annually in July. Children in kindergartens and scattered children accounted for 44.6% and 42.2% of cases, respectively. Real-time RT-PCR detection of enteroviruses in 5534 patient samples revealed 3780 positives, of which 25.1% were CVA16-positive. Positives were randomly sampled, yielding 240 VP1 sequences of CVA16. Phylogenetic tree results showed that all VP1 sequences belonged to the B1 sub-genogroup. However, the sub-genogroup prevalence varied over time: from 2013 to 2014 and 2019 to 2021, the predominant sub-genogroup was B1a, while it was B1b from 2015 to 2018. Further phylogenetic analyses showed substantial divergence between B1a branches in CVA16, suggesting possible turnover of the B1a sub-genogroup in CVA16 due to evolution. This study provides epidemiological data on HFMD in Shenyang, and provides a phylogenetic analysis of CVA16, offering a theoretical basis for preventing and controlling HFMD in Shenyang City.

Background: The increasing incidence of hand, foot, and mouth disease (HFMD) associated with Coxsackievirus A6 (CVA6) has become a very significant public health problem. The aim of this study is to investigate the recombination, geographic transmission, and evolutionary characteristics of the global CVA6. Methods: From 2019 to 2022, 73 full-length CVA6 sequences were obtained from HFMD patients in China and analyzed in combination with 1032 published whole genome sequences. Based on this dataset, the phylogenetic features, recombinant diversity, Bayesian phylodynamic characteristics, and key amino acid variations in CVA6 were analyzed. Results: The four genotypes of CVA6, A, D, E, and F, are divided into 24 recombinant forms (RFs, RF -A - RF -X) based on differences in the P3 coding region. The eastern China region plays a key role in the dissemination of CVA6 in China. VP1 -137 and VP1 -138 are located in the DE loop on the surface of the CVA6 VP1 protein, with the former being a highly variable site and the latter having more non-synonymous substitutions. Conclusions: Based on whole genome sequences, this study contributes to the CVA6 monitoring, early warning, and the pathogenic mechanism by studying recombination diversity, geographical transmission characteristics, and the variation of important amino acid sites.

Echovirus 25 (E25), a member of the Enterovirus B (EV-B) species, can cause aseptic meningitis (AM), viral meningitis (VM), and acute flaccid paralysis (AFP). However, systematic studies on the molecular epidemiology of E25, especially those concerning its evolution and recombination, are lacking. In this study, 18 strains of E25, isolated from seven provinces of China between 2009 and 2018, were collected based on the Chinese hand, foot, and mouth disease (HFMD) surveillance network, and 95 sequences downloaded from GenBank were also screened. Based on the phylogenetic analysis of 113 full-length VP1 sequences worldwide, globally occurring E25 strains were classified into 9 genotypes (A–I), and genotype F was the dominant genotype in the Chinese mainland. The average nucleotide substitution rate of E25 was 6.08 × 10–3 substitutions/site/year, and six important transmission routes were identified worldwide. Seventeen recombination patterns were determined, of which genotype F can be divided into 9 recombination patterns. A positive selector site was found in the capsid protein region of genotype F. Recombination analysis and pressure selection analysis for genotype F showed multiple recombination patterns and evolution characteristics, which may be responsible for it being the dominant genotype in the Chinese mainland. This study provides a theoretical basis for the subsequent prevention and control of E25.