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.

Introduction:Coxsackievirus A6 (CVA6) has emerged as a significant pathogen responsible for severe cases of hand, foot, and mouth disease (HFMD). This study aims to delineate the demographic characteristics and analyze the viral evolution of severe HFMD associated with CVA6, thereby assisting in its surveillance and management.Methods:In this investigation, 74 strains of CVA6 were isolated from samples collected from severe HFMD cases between 2012 and 2023. The VP1 gene sequences of CVA6 were amplified and analyzed to assess population historical dynamics and evolutionary characteristics using BEAST, DnaSP6, and PopART.Results:A significant portion (94.4%) of severe CVA6-associated HFMD cases (51 out of 54, with 20 lacking age information) were children under 5 years old. Among the 74 CVA6 strains analyzed, 72 belonged to the D3a sub-genotype, while only two strains were D2 sub-genotype. The average genetic distance between VP1 sequences prior to 2015 was 0.027, which increased to 0.051 when compared to sequences post-2015. Historical population dynamics analysis indicated three significant population expansions of severe CVA6-associated HFMD during 2012-2013, 2013-2014, and 2019-2020, resulting in the formation of 65 distinct haplotypes. Consistent with the MCC tree findings, transitioning between regional haplotypes required multiple base substitutions, showcasing an increase in population diversity during the evolutionary process (from 14 haplotypes in 2013 to 55 haplotypes over the subsequent decade).Conclusions:CVA6, associated with severe HFMD, is evolving and presents a risk of outbreak occurrence. Thus, enhanced surveillance of severe HFMD is imperative.

Abstract Echovirus 25 (E25), a member of the Enterovirus B (EV-B) family, 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 more 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.

分析江西一例免疫缺陷患者体内连续采集的15份粪便标本中分离到的Ⅲ型脊髓灰质炎病毒的全长VP1区基因特征.将从15份标本中分离到的14株Ⅲ型iVDPVs进行噬斑纯化,每个病毒随机挑取10个噬斑,接着进行逆转录-聚合酶链反应扩增,测定获取到的154株iVDPVs全长VP1区序列.通过系统发育分析和BEAST程序探究iVDPV病毒的进化特征,估算其进化速率和口服脊灰减毒活疫苗(attenuated oral polio vaccine,OPV)初始感染时间.14株iVDPVs与Ⅲ型脊髓灰质炎减毒疫苗株(Sabin Ⅲ)核苷酸和氨基酸同源性分别为97.8％～98.7％和97.6％～98.3％.相较于Sabin Ⅲ,14株iVDPVs VP1区第54位氨基酸发生了 A→V的突变,这可能导致温度敏感表型和衰减表型的改变.根据拓扑结构系统发育树被划分为3个Lineages,其中17049-1-8,17049-2-2和17049-2-9分别属于Lineage 1和Lineage 2,其余属于Lineage 3.具有Lineage 3序列特征的克隆是优势克隆,呈现出随时间持续分化的特征.BEAST程序估算的14株iVDPVs全长VP1区核苷酸平均进化速率为9.29×10-3/位点/年(95％置信区间:2.81×10-3-1.59×10-2),OPV初始感染时间为2012年11月 18 日.MCC 树(maximum clade credibility tree)显示前4个遗传分支的分化仅用了 40 d.14株Ⅲ型iVDPVs具有高度相关性,它们感染患儿后进化非常迅速,在慢性感染的早期便开始了谱系的分化.突变的不断积累和关键位点的改变可以引起iVDPVs神经毒力的变化,导致患儿出现AFP症状.iVDPV病例持续的排毒给脊灰根除目标的实现带来了巨大挑战,在脊髓灰质炎消灭的最后阶段及时调整免疫策略,继续维持较高的免疫覆盖率,保持iVDPV监测的灵敏性显得十分重要.

Enterovirus C96 (EV-C96) is a recently discovered serotype belonging to enterovirus C species. It had been isolated from patients with acute flaccid paralysis, hand, foot, and mouth disease, diarrhea, healthy people, or environmental specimens. Despite increasing reports of the virus, the small number of full-length genomes available for EV-C96 has limited molecular epidemiological studies. In this study, newly collected rare EV-C96 strains in China from 1997 to 2020 were combined with sequences available in GenBank for comprehensive analyses. Sequence analysis revealed that the nucleotide sequence similarity of EV-C96 and the prototype strain (BAN00-10488) was 75%-81.8% and the amino acid sequence similarity was 85%-94.9%. EV-C96 had a high degree of genetic variation and could be divided into 15 genogroups. The mean evolutionary rate was 5.16 x 10-3 substitution/site/year, and the most recent common ancestor was dated to 1925. A recombination analysis revealed that EV-C96 may be a recombinant derived from other serotypes in the EV-C group in the nonstructural protein coding region. This comprehensive and integrated analysis of the whole genome sequence of EV-C96 provides valuable data for further studies on the molecular epidemiology of EV-C96 worldwide.

Objective:To analyze the pathogen spectrum and genetic characteristics of the main pathogens of hand, foot, and mouth disease (HFMD) in Chongqing from 2017 to 2018.Methods:Chongqing Children's Hospital was selected as the study object. Nucleic acid detection information of 1 071 patients diagnosed with HFMD from 2017 to 2018 was collected. The enterovirus serotypes of 810 samples were identified. There were 175 samples remaining unidentified for serotypes. Eighty-six samples were enterovirus-negative. Through virus isolation, nucleic acid concentration and gene sequencing, the above 261 samples were tested to obtain a complete pathogen spectrum of HFMD in this area. MEGA 7.0 software was used to construct phylogenetic trees of the four major pathogens for the study of genetic characteristics.Results:Except for 3 samples that were identified as Human parechovirus (HPeV), 258 samples were positive for enterovirus by the improved identification method. Among them, coxsackievirus A6 (CVA6) was the dominant serotype, followed by enterovirus A71 (EV-A71). There were also 13 serotypes of CVA16, CVA10 and other enteroviruses. The phylogenetic analysis results showed that the serotypes of CVA6 in Chongqing were all D3a sub-genotype, while the dominant gene subtypes of EV-A71, CVA10 and CVA16 were C4a, C2b and B1b, respectively. These four dominant genotypes co-evolved with the genotypes in other areas of China.Conclusions:This study improved the detection rate of enterovirus by optimizing the identification method, and further defined the pathogen spectrum of HFMD in Chongqing. The dominant pathogen of HFMD was CVA6 in Chongqing from 2017 to 2018, followed by EV-A71, and both serotypes were also the main pathogen causing severe HFMD in Chongqing area.

In 2013, a case of immunodeficiency vaccine-derived poliovirus (iVDPV) was identified in Jiangxi Province, China. In this study, we purified 14 type 3 original viral isolates from this case and characterized the molecular evolution of these iVDPVs for 298 days. Genetic variants were found in most of the original viral isolates, with complex genetic and evolutionary relationships among the variants. A phylogenetic tree constructed based on the P1 region showed that these iVDPVs were classified into lineage A and B. The dominant lineage B represents a major trend in virus evolution. The nucleotide substitution rate at the third codon position (3CP) estimated by the BEAST program was 1.76 x 10-2 substitutions/site/year (95% HPD: 1.23-2.39 x 10-2). The initial OPV dose was given dating back to March 2013, which was close to the time of the last OPV vaccination, suggesting that OPV infection may have originated with the last dose of vaccine. Recombinant analysis showed that these iVDPVs were inter-vaccine recombinants with two recombination patterns, S3/S2/S1 and S3/S2/S3/S2/S1. Whole genome sequence analysis revealed that key nucleotide sites (C472U, C2034U, U2493C) associated with the attenuated phenotype of Sabin 3 have been replaced. Temperature sensitivity test showed that all tested strains were temperature-sensitive, except for the variant Day11-5. Interestingly, we observed that the variant Day11-5 temperature resistance properties may be associated with the Lys to Met substitution at the VP2-162 site. Serological test and whole genome sequence analysis showed that the seropositivity rate remained high, and mutations in the antigenic sites did not significantly alter neutralization ability.

As the proportion of non-enterovirus 71 and non-coxsackievirus A16 which proportion of composition in the hand, foot, and mouth pathogenic spectrum gradually increases worldwide, the attention paid to other enteroviruses has increased. As a member of the species enterovirus A, coxsackievirus A14 (CVA14) has been epidemic around the world until now since it has been isolated. However, studies on CVA14 are poor and the effective population size, evolutionary dynamics, and recombination patterns of CVA14 are not well understood. In this study, 15 CVA14 strains were isolated from HFMD patients in mainland China from 2009 to 2019, and the complete sequences of CVA14 in GenBank as research objects were analyzed. CVA14 was divided into seven genotypes A-G based on an average nucleotide difference of the full-length VP1 coding region of more than 15%. Compared with the CVA14 prototype strain, the 15 CVA14 strains showed 84.0–84.7% nucleotide identity in the complete genome and 96.9–97.6% amino acid identity in the encoding region. Phylodynamic analysis based on 15 CVA14 strains and 22 full-length VP1 sequences in GenBank showed a mean substitution rate of 5.35 × 10−3 substitutions/site/year (95% HPD: 4.03–6.89 × 10−3) and the most recent common ancestor (tMRCA) of CVA14 dates back to 1942 (95% HPD: 1930–1950). The Bayesian skyline showed that the effective population size had experienced a decrease–increase–decrease fluctuation since 2004. The phylogeographic analysis indicated two and three possible migration paths in the world and mainland China, respectively. Four recombination patterns with others of species enterovirus A were observed in 15 CVA14 strains, among which coxsackievirus A2 (CVA2), coxsackievirus A4 (CVA4), coxsackievirus A6 (CVA6), coxsackievirus A8 (CVA8), and coxsackievirus A12 (CVA12) may act as recombinant donors in multiple regions. This study has filled the gap in the molecular epidemiological characteristics of CVA14, enriched the global CVA14 sequence database, and laid the epidemiological foundation for the future study of CVA14 worldwide.

Echovirus, a member of the Enterovirus B (EV-B) family, has led to numerous outbreaks and pandemics, causing a broad spectrum of diseases. Based on the national hand, foot, and mouth disease (HFMD) surveillance system, seven strains of echovirus 33 (E33) were isolated from Mainland of China between 2010 and 2018. The whole genomes of these strains were isolated and sequenced, and phylogenetic trees were constructed based on the gene sequences in different regions of the EV-B prototype strains. It was found that E33 may be recombined in the P2 and P3 regions. Five genotypes (A–E) were defined based on the entire VP1 region of E33, of which the C gene subtype was the dominant gene subtype at present. Recombinant analysis showed that genotype C strains likely recombined with EV-B80, EV-B85, E13, and CVA9 in the P2 and P3 regions, while genotype E had the possibility of recombination with CVB3, E3, E6, and E4. Results of Bayesian analysis indicated that E33 may have appeared around 1955 (95% confidence interval: 1945–1959), with a high evolutionary rate of 1.11 × 10−2 substitution/site/year (95% highest posterior density (HPD): 8.17 × 10−3 to 1.4 × 10−2 substitution/site/year). According to spatial transmission route analysis, two significant transmission routes were identified: from Australia to India and from Oman to Thailand, which the E33 strain in Mainland of China likely introduced from Mexico and India. In conclusion, our study fills the gaps in the evolutionary analysis of E33 and can provide important data for enterovirus surveillance.

Coxsackievirus B5 (CVB5) is an important enterovirus B species (EV-Bs) type. We used the full-length genomic sequences of 53 viral sequences from the national hand, foot, and mouth disease surveillance network in the Chinese mainland (2001–2021). Among them, 69 entire VP1 coding region nucleotide sequences were used for CVB5 genotyping and genetic evolution analysis. Phylogenetic analysis based on a data set of 448 complete VP1 sequences showed that CVB5 could be divided into four genotypes (A-D) worldwide. Sequences from this study belonged to genotypes B and D, which dominated transmission in the Chinese mainland. Two transmission lineages of CVB5 have been discovered in the Chinese mainland, lineage 2 was predominant. Markov chain Monte Carlo analysis indicated that the tMRCA of CVB5 in the Chinese mainland could be traced to 1955, while the global trend could be traced to 1862, 93 years earlier than China. The evolution rate of CVB5 was higher in the Chinese mainland than worldwide. The spatiotemporal dynamics analysis of CVB5 assessed that virus transportation events were relatively active in Central, Northeast, North and Northwest China. Recombination analysis revealed frequent intertypic recombination in the non-structural region of CVB5 genotypes B and D with the other EV-Bs, revealing eight recombination lineages. Our study showed the molecular evolution and phylogeography of CVB5 that could provide valuable information for disease prevention.