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Hand, foot, and mouth disease (HFMD) is a common viral infection primarily affecting children. It causes vesicles on the skin and inside the mouth. Although most cases get better on their own, severe cases can lead to complications such as brain stem encephalitis, meningoencephalitis, acute flaccid paralysis, and pulmonary edema. Hand, foot, and mouth disease is caused by various enteroviruses, with enterovirus A71 (EV-A71) and coxsackievirus A16 being the most common. However, recent studies have shown a shift in the molecular epidemiology of HFMD-causing pathogens, with coxsackievirus A6 and coxsackievirus A10 causing more infections. In addition, extensive recombination events have been identified among enterovirus strains, which may have a role in faster evolution and extinction of dominant enterovirus serotypes. Other strains of enterovirus can also cause severe complications, and there has been an increase in mortality associated with brain stem encephalitis in children under 3 years of age and teenagers. Currently, there are no effective antiviral therapies available to treat enterovirus infections. Vaccines against EV-A71 have been approved and are now used in mainland China. Studying the changing epidemiology of HFMD pathogens and the evolution patterns of its causative agents is crucial in developing effective prevention and control strategies. Increased interest in the molecular epidemiology of HFMD causative agents has led to a better understanding of the critical drivers of HFMD outbreaks, which can inform efforts to prevent and control the disease.

Background: Hand, foot, and mouth disease (HFMD) is a global public health concern, notably within the Asia-Pacific region. Recently, the primary pathogen causing HFMD outbreaks across numerous countries, including China, is coxsackievirus (CV) A6, one of the most prevalent enteroviruses in the world. It is a new variant that has undergone genetic recombination and evolution, which might not only induce modifications in the clinical manifestations of HFMD but also heighten its pathogenicity because of nucleotide mutation accumulation., Objective: The study assessed the epidemiological characteristics of HFMD in China and characterized the molecular epidemiology of the major pathogen (CV-A6) causing HFMD. We attempted to establish the association between disease progression and viral genetic evolution through a molecular epidemiological study., Methods: Surveillance data from the Chinese Center for Disease Control and Prevention from 2021 to 2023 were used to analyze the epidemiological seasons and peaks of HFMD in Henan, China, and capture the results of HFMD pathogen typing. We analyzed the evolutionary characteristics of all full-length CV-A6 sequences in the NCBI database and the isolated sequences in Henan. To characterize the molecular evolution of CV-A6, time-scaled tree and historical population dynamics regarding CV-A6 sequences were estimated. Additionally, we analyzed the isolated strains for mutated or missing amino acid sites compared to the prototype CV-A6 strain., Results: The 2021-2023 epidemic seasons for HFMD in Henan usually lasted from June to August, with peaks around June and July. The monthly case reporting rate during the peak period ranged from 20.7% (4854/23,440) to 35% (12,135/34,706) of the total annual number of cases. Analysis of the pathogen composition of 2850 laboratory-confirmed cases identified 8 enterovirus serotypes, among which CV-A6 accounted for the highest proportion (652/2850, 22.88%). CV-A6 emerged as the major pathogen for HFMD in 2022 (203/732, 27.73%) and 2023 (262/708, 37.01%). We analyzed all CV-A6 full-length sequences in the NCBI database and the evolutionary features of viruses isolated in Henan. In China, the D3 subtype gradually appeared from 2011, and by 2019, all CV-A6 virus strains belonged to the D3 subtype. The VP1 sequences analyzed in Henan showed that its subtypes were consistent with the national subtypes. Furthermore, we analyzed the molecular evolutionary features of CV-A6 using Bayesian phylogeny and found that the most recent common ancestor of CV-A6 D3 dates back to 2006 in China, earlier than the 2011 HFMD outbreak. Moreover, the strains isolated in 2023 had mutations at several amino acid sites compared to the original strain., Conclusions: The CV-A6 virus may have been introduced and circulating covertly within China prior to the large-scale HFMD outbreak. Our laboratory testing data confirmed the fluctuation and periodic patterns of CV-A6 prevalence. Our study provides valuable insights into understanding the evolutionary dynamics of CV-A6., (© Yu Chen, Shouhang Chen, Yuanfang Shen, Zhi Li, Xiaolong Li, Yaodong Zhang, Xiaolong Zhang, Fang Wang, Yuefei Jin. Originally published in JMIR Public Health and Surveillance (https://publichealth.jmir.org).)

Hand, Foot and Mouth Disease (HFMD) is a highly contagious viral illness primarily affecting children globally. A significant epidemiological transition has been noted in mainland China, characterized by a substantial increase in HFMD cases caused by non-Enterovirus A71 (EV-A71) and non-Coxsackievirus A16 (CVA16) enteroviruses (EVs). Our study conducts a retrospective examination of 36,461 EV-positive specimens collected from Guangdong, China, from 2013 to 2021. Epidemiological trends suggest that, following 2013, Coxsackievirus A6 (CVA6) and Coxsackievirus A10 (CVA10) have emerged as the primary etiological agents for HFMD. In stark contrast, the incidence of EV-A71 has sharply declined, nearing extinction after 2018. Notably, cases of CVA10 infection were considerably younger, with a median age of 1.8 years, compared to 2.3 years for those with EV-A71 infections, possibly indicating accumulated EV-A71-specific herd immunity among young children. Through extensive genomic sequencing and analysis, we identified the N136D mutation in the 2 A protein, contributing to a predominant subcluster within genogroup C of CVA10 circulating in Guangdong since 2017. Additionally, a high frequency of recombination events was observed in genogroup F of CVA10, suggesting that the prevalence of this lineage might be underrecognized. The dynamic landscape of EV genotypes, along with their potential to cause outbreaks, underscores the need to broaden surveillance efforts to include a more diverse spectrum of EV genotypes. Moreover, given the shifting dominance of EV genotypes, it may be prudent to re-evaluate and optimize existing vaccination strategies, which are currently focused primarily target EV-A71., (© 2024. The Author(s).)

Hand, foot, and mouth disease (HFMD) caused by various enteroviruses is a major public health concern globally. Human enterovirus 71(EVA71), coxsackievirus A16 (CVA16), coxsackievirus A6 (CVA6), and coxsackievirus A10 (CVA10) are four major enteroviruses responsible for HFMD. Rapid, accurate, and specific point-of-care (POC) detection of the four enteroviruses is crucial for the prevention and control of HFMD. Here, we developed two multiplex high-fidelity DNA polymerase loop-mediated isothermal amplification (mHiFi-LAMP) assays for simultaneous detection of EVA71, CVA16, CVA6, and CVA10. The assays have good specificity and exhibit high sensitivity, with limits of detection (LOD) of 11.2, 49.6, 11.4, and 20.5 copies per 25 μL reaction for EVA71, CVA16, CVA6, and CVA10, respectively. The mHiFi-LAMP assays showed an excellent clinical performance (sensitivity 100.0%, specificity 83.3%, n = 47) when compared with four singleplex RT-qPCR assays (sensitivity 93.1%, specificity 100%). In particular, the HiFi-LAMP assays exhibited better performance (sensitivity 100.0%, specificity 100%) for CVA16 and CVA6 than the RT-qPCR assays (sensitivity 75.0-92.3%, specificity 100%). Furthermore, the mHiFi-LAMP assays detected all clinical samples positive for the four enteroviruses within 30 min, obviously shorter than about 1-1.5 h by the RT-qPCR assays. The new mHiFi-LAMP assays can be used as a robust point-of-care testing (POCT) tool to facilitate surveillance of HFMD at rural and remote communities and resource-limited settings., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH, DE part of Springer Nature.)

This study aimed to prepare two hairpin-structure DNA probes by conjugating carminic acid (CA) or hemin into two ends of specific genes of coxsackievirus A16 (CV-A16) and enterovirus A71 (EV-A71) (probe CV-A16-CA and probe EV-A71-hemin ). Then, probe CV-A16-CA and probe EV-A71-hemin as the signal molecules were adsorbed onto NH 2 -MIL-53 (Al) (MOF). Based on these biocomposites, an electrochemical biosensor with dual-signal outputs for simultaneous assay of CV-A16 and EV-A71 was constructed. The stem-loops of probes switched both CA and hemin monomer to dimer, reducing the electrical activity of both CA and hemin. Subsequently, the target-induced opening of the stem-loop switched both CA and hemin dimers to monomers, resulting in two nonoverlapping increasing electrical signals. This sensitively reflected the concentration of target CV-A16 and target EV-A17 ranging from 10 -10 to 10 -15  M with a detection limit of 0.19 and 0.24 fM. This strategy was mainly applied to the simultaneous determination of target CV-A16 and target EV-A17 in 100% serum with satisfactory results. The MOF combined with the high loading capacity broke through the intrinsic limitation on sensitivity using the traditional methods. An increase of three orders of magnitude was observed. This study involved simple one-step detection, and only a simple replacement of a gene could trigger its potential in clinical and diagnostic applications., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The objective of this work was to explore the application value of a new type of fluorescent nucleic acid isothermal amplification (SAT) to detect EV/EV71/CA16-SAT in children with hand-foot-mouth disease (HFMD). For this purpose, from March 2017 to September 2019, Chengdu Children's Specialized Hospital collected throat swabs from children with clinical manifestations of hand, foot and mouth disease, and used SAT technology to screen and detect universal enterovirus (EV) nucleic acid (There were 1860 children with EV-RNA) positive. Patients who are EV-RNA positive at any time: first use the same throat swab specimen to detect EV71/CA16-RNA; secondly, collect venous blood and use the colloidal gold method to detect IgM antibodies in EV71/CA16 serum. The patients with positive EV71/CA16-RNA or EV71/CA16-IgM (or both) were repeated the above two methods 2 weeks and 4 weeks after standard treatment for review and comprehensive analysis. Results showed that 763 cases were enrolled for the first time: 59.76% were male and 40.24% were female; the age ranged from 1 month to 13 years, of which 69.06% were from 1 to 4 years old; CA16-RNA positive 56.23%, EV71-RNA positive 21.89%, CA16/EV71 -RNA were all positive in 1.57%; CA16-IgM was positive in 64.48%, EV71-IgM was positive in 54.26%, and CA16/EV71-IgM were both positive in 18.74%. After 2 weeks, 722 cases were reexamined: 26.73% were positive for CA16-RNA, 7.89% were positive for EV71-RNA, 0.28% were both positive for CA16/EV71-RNA; 66.21% were positive for CA16-IgM, 51.52% were positive for EV71-IgM, and IgM were all positive in 17.73%. Four weeks later, 489 cases were reexamined: among them, CA16-RNA positive 5.73% of which were positive for EV71 color RNA (0.005%), and 12.68% of them were all positive for EV71lym. The strategy of combining SAT technology and colloidal gold method to detect EV/EV71/CA16 nucleic acid (RNA) and serum IgM antibody in children HFMD can improve the early detection rate and accuracy of HFMD; According to the comprehensive analysis of the detection results of children with HFMD at the early stage, 2 weeks and 4 weeks of the present study, it is suggested that EV/EV71/CA16-SAT nucleic acid detection can be used to judge the prognosis, follow-up treatment, set isolation time, return students to school, and community management in children with HFMD. and prevention and control have more clinical application value.

Background: Enterovirus 71(EV71)-associated hand, foot and mouth disease (HFMD) decreased dramatically in Beijing from 2009 to 2019. This study was to investigate the epidemiological characteristics, evolutionary dynamics, geographic diffusion pathway, and other features of EV71 in Beijing, China., Methods: We conducted a retrospective study of EV71-associated HFMD and its causative agent in Beijing, China, from 2009 to 2019. Phylogenetic and phylogeographic methods based on the EV71 genome were used to determine the evolution features, origin, and spatiotemporal dynamics. Positive selection sites in the VP1 gene were identified and exhibited in the tertiary structure. Bayesian birth-death skyline model was used to estimate the effective reproductive number (Re)., Results: EV71-associated HFMD decreased greatly in Beijing. From 2009 to 2019, EV71 strains prevalent in Beijing shared high homology in each gene segment and evolved with a rate of 4.99*10 - 3 substitutions per site per year. The genetic diversity of EV71 first increased and peaked in 2012 and then decreased with fluctuations. The time to the most recent common ancestor (TMRCA) of EV71 in Beijing was estimated around 2003 when the EV71 strains were transmitted to Beijing from east China. Beijing played a crucial role in seeding EV71 to central China as well. Two residues (E145Q/G, A293S) under positive selection were detected from both the VP1 dataset and the P1 dataset. They were embedded within the loop of the VP1 capsid and were exposed externally. Mean Re estimate of EV71 in Beijing was about 1.007., Conclusion: In recent years, EV71 was not the primary causative agent of HFMD in Beijing. The low Re estimate of EV71 in Beijing implied that strategies for preventing and controlling HFMD were performed effectively. Beijing and east China played a crucial role in disseminating EV71 to other regions in China., (© 2023. The Author(s).)

Hand, foot and mouth disease (HFMD) is a public health threat worldwide, particularly in the Asia-Pacific region. Enterovirus A71 (EV-A71), coxsackievirus A16 (CVA16), and CVA6 are the major pathogens causing HFMD outbreaks in several countries, including Thailand. We retrieved 385 VP1 nucleotide sequences, comprising 228 EV-A71, 33 CVA16, and 124 CVA6, deposited in the databases between 2000 and 2022 for molecular evolutionary characterization using Bayesian phylogeny. All EV-A71 identified belonged to genotype B, subgenotypes B4, and B5, and to genotype C, subgenotypes C1, C2, C4a, C4b, and C5. The analyzes demonstrated these viruses' co-circulation and subgenotypic changes throughout the past two decades. The CVA16 was grouped in genotype B1, predominantly subgenotype B1a, and the CVA6 was grouped in subgenotype D3, clades 1-4. The tMRCA of EV-A71 genotypes B and C, CVA16 B1, and CVA6 D3 dated 1993.79, 1982.62, 1995.86, and 2007.31, respectively, suggesting that the viruses were likely introduced and cryptically circulated in Thailand before the HFMD cases were recognized. We demonstrated these viruses' fluctuation and cyclical pattern throughout the two decades of observation. This study provided insight into evolutionary dynamics concerning molecular epidemiology and supported the selection of current genotype-matched vaccines, vaccine development, and implementation., (© 2023. Springer Nature Limited.)

The same viral infection in different hosts may result in varying levels of clinical symptoms, which is related to the genetic background of the host itself. A total of 406 common cases and 452 severe cases of enterovirus 71 (EV71) infection in Yunnan Province were selected as the research subjects, and SNaPshot technology was used to detect genetic polymorphisms for 25 Tag single-nucleotide polymorphisms (TagSNPs) in the selectin P ligand (SELPLG) and scavenger receptor class B member 2 (SCARB2) genes. Our results demonstrate that SCARB2 polymorphisms (rs74719289, rs3733255 and rs17001551) are related to the severity of EV71 infection (A vs G: OR 0.330; 95% CI 0.115 - 0.947; T vs C: OR 0.336; 95% CI 0.118 - 0.958; and A vs G: OR 0.378; 95% CI 0.145 - 0.984). The SELPLG polymorphisms were not significantly different between common cases and severe cases. Therefore, we conclude that the SCARB2 gene has a protective effect on the course of hand, foot and mouth disease caused by EV71 infection and that SCARB2 gene mutations can reduce the severity of the disease.

Objectives: This study aims to investigate molecular epidemiology and clinical characteristics of enterovirus associated hand-foot-mouth disease (HFMD) in Chengdu, China, 2013-2022. Monitoring the molecular epidemiology and clinical features of HFMD for up to 10 years may provide some ideas for future protection and control measures., Methods: We conducted a retrospective analysis of the medical records of all patients with laboratory-confirmed HFMD-related enterovirus infection at the West China Second University Hospital from January 2013 to December 2022. We described the characteristics in serotype, age, sex distribution and hospitalization of enterovirus infection cases using data analysis and graphic description., Results: A total of 29,861 laboratory-confirmed cases of HFMD-related enterovirus infection were reported from 2013 to 2022. There was a significant reduction in the number and proportion of EV-A71 cases after 2016, from 1713 cases (13.60%) in 2013-2015 to 150 cases (1.83%) in 2017-2019. During the COVID-19 pandemic, EV-A71 cases even disappeared. The proportion of CV-A16 cases decreased from 13.96% in 2013-2015 to 10.84% in 2017-2019 and then to 4.54% in 2020-2022. Other (non-EV-A71 and non-CV-A16) serotypes accounted for 95.45% during 2020-2022, with CV-A6 accounting for 50.39% and CV-A10 accounting for 10.81%. Thus, CV-A6 and CV-A10 became the main prevalent serotypes. Furthermore, There was no significant difference in the enterovirus prevalence rate between males and females. The hospitalization rate of EV-A71 patients was higher that of other serotypes. In general, the proportion of HFMD hospitalizations caused by other pathogens except for EV-A71, CV-A16, CV-A10 and CV-A16 was second only to that caused by EV-A71. The proportion of children over 4 years old infected with enterovirus increased., Conclusion: The incidence of HFMD associated with enterovirus infection has decreased significantly and CV-A6 has been the main pathogen of HFMD in Chengdu area in recent years. The potential for additional hospitalizations for other untested enterovirus serotypes suggested that attention should also be paid to the harms of infections with unknown enterovirus serotypes. Children with HFMD were older. The development of new diagnostic reagents and vaccines may play an important role in the prevention and control of enterovirus infection., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Coxsackievirus A16 (CV-A16) is a significant pathogen responsible for causing hand foot and mouth disease (HFMD) and herpangina (HA). This study aimed to investigate the recent evolution and spread of CV-A16 by monitoring HFMD and HA cases in 29 hospitals across 16 districts in Beijing from 2019 to 2021. The first five cases of HFMD and the first five cases of HA each month in each hospital were included in the study. Real-time reverse transcription polymerase chain reaction was used to identify CV-A16, CV-A6, and EV-A71. From each district, two to four CV-A16 positive samples with a relatively long sampling time interval every month were selected for sequencing. A total of 3344 HFMD cases and 2704 HA cases were enrolled in this study, with 76.0% (2541/3344) of HFMD and 45.4% (1227/2704) of HA cases confirmed to be infected by enterovirus. Among the EV-positive samples, CV-A16 virus was detected in 33.61% (854/2541) of HFMD cases and 13.4% (165/1227) of HA cases, with the predominant cluster being B1a. Both B1a and B1b had a co-circulation of local and imported strains, with different origin time (1993 vs. 1995), different global distribution (14 countries vs. 10 countries), and different transmission centers but mainly distributed in the southern and eastern regions of Beijing. Strengthening surveillance of HFMD in southern and eastern regions will improve the prevention and control efficiency of enterovirus infections., (© 2023 Wiley Periodicals LLC.)

Outbreaks of hand, foot and mouth disease (HFMD) have occurred frequently in the Asian-Pacific region over the last two decades, caused mainly by the serotypes in Enterovirus A species. High-quality monoclonal antibodies (mAbs) are needed to improve the accuracy and efficiency of the diagnosis of enteroviruses associated HFMD. In this study, a mAb 1A11 was generated using full particles of CV-A5 as an immunogen. In indirect immunofluorescence and Western blotting assays, 1A11 bound to the viral proteins of CV-A2, CV-A4, CV-A5, CV-A6, CV-A10, CV-A16, and EV-A71 of the Enterovirus A and targeted VP3. It has no cross-reactivity to strains of Enterovirus B and C . By mapping with over-lapped and truncated peptides, a minimal and linear epitope 23 PILPGF 28 was identified, located at the N-terminus of the VP3. A BLAST sequence search of the epitope in the NCBI genus Enterovirus (taxid: 12059) protein database indicates that the epitope sequence is highly conserved among the Enterovirus A species, but not among the other enterovirus species, first reported by us. By mutagenesis analysis, critical residues for 1A11 binding were identified for most serotypes of Enterovirus A . It may be useful for the development of a cost-effective and pan- Enterovirus A antigen detection for surveillance, early diagnosis and differentiation of infections caused by the Enterovirus A species.

Hand, foot and mouth disease (HFMD) is caused by a variety of serotypes in species A of the Enterovirus genus, including recently re-emerged Coxsackievirus A2 (CV-A2), CV-A4 and CV-A5. For development of diagnostic reagents, for surveillance, and the development of multivalent vaccines against HFMD, the antigenicity of HFMD-associated enteroviruses warrants investigation. The purified virions of CV-A4 were inoculated into Balb/c mice and hybridomas were obtained secreting monoclonal antibodies (mAbs) directed against CV-A4 and cross-reacting with other closely related species A enteroviruses. The mAbs were characterized by ELISA, Western blotting and in vitro neutralizing assays. The majority of mAbs was non-neutralizing, with only 2% of the mAbs neutralizing CV-A4 specifically. Most of mAbs bound to linear VP1 epitopes of CV-A4. Interestingly, four types of mAbs were obtained which bound specifically to CV-A4 or were broadly to CV-A4/-A2, CV-A4/-A5 and CV-A4/-A2/-A5, respectively. Mapping with overlapping or single-amino-acid mutant peptides revealed that the four types of mAbs all bound to the first 15 amino acids at the N-terminus of the VP1. This region of picornaviruses is functionally important as it is involved in uncoating and releasing of viral RNA into the cytosol. The binding footprints of four type mAbs are composed of conserved and variable residues and are different from each other. The newly discovered broadly cross-reactive mAbs reflect the high homology of CV-A4/ CV-A2/CV-A5. The results also demonstrate that it is possible and beneficial to develop the diagnostic reagents to detect rapidly the main pathogens of enteroviruses associated with HFMD cause by CV-A4/CV-A2/CV-A5., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Coxsackievirus (CV)-A6 infections cause hand, foot, and mouth disease (HFMD) in children and adults. Despite the serious public health threat presented by CV-A6 infections, our understanding of the mechanisms by which new CV-A6 strains emerge remains limited. This study investigated the molecular epidemiological trends, evolutionary dynamics, and recombination characteristics of CV-A6-associated HFMD in Thailand between 2019 and 2022. In the HFMD patient samples collected during the 4-year study period, we identified enterovirus (EV) RNA in 368 samples (48.7%), of which CV-A6 (23.7%) was the predominant genotype, followed by CV-A4 (6%), EV-A71 (3.7%), and CV-A16 (3.4%). According to the partial viral protein (VP) 1 sequences, all these CV-A6 strains belonged to the D3 clade. Based on the viral-RNA-dependent RNA polymerase (RdRp) gene, four recombinant forms (RFs), RF-A (147, 84.5%), RF-N (11, 6.3%), RF-H (1, 0.6%), and newly RF-Y (15, 8.6%), were identified throughout the study period. Results from the similarity plot and bootscan analyses revealed that the 3D polymerase (3Dpol) region of the D3/RF-Y subclade consists of sequences highly similar to CV-A10. We envisage that the epidemiological and evolutionarily insights presented in this manuscript will contribute to the development of vaccines to prevent the spread of CV-A6 infection.

Three inactivated enterovirus A71 (EV-A71) vaccines have been widely vaccinated among children in the targeted age group in mainland China since mid-2016. However, comprehensive virological surveillance of hand, foot and mouth disease (HFMD) over multiple years after the use of EV-A71 vaccines has rarely been conducted. Using long-term data extracted from the Public Health and Clinical Center of Chengdu, we described the clinical, aetiological, and epidemiological characteristics of HFMD inpatients after the use of EV-A71 vaccines from 2017 through 2022. A total of 5115 patients were selected for analysis with a male-to-female ratio of 1.63:1 and were mostly under 5 years of age (97.6%). Among these cases, 4.3% presented with severe symptoms, and 4.1% of severe cases experienced significant complications. EV-A71 was no longer the major serotype for laboratory-confirmed HFMD, responsible for 15.6% of severe cases and 1.2% of mild cases. A significant downwards trend of EV-A71 infections was observed after the use of EV-A71 vaccines (P for trend < 0.001). Coxsackievirus A6 was the predominant pathogen, accounting for 63.5% of mild cases and 36.2% of severe cases. Coxsackievirus A10 (CV-A10) and A16 were sporadically detected, and an upwards trend was observed in the proportion of CV-A10 infections. This study provides baseline molecular epidemiology for the evaluation of EV-A71 vaccination impact and potential serotype replacement based on HFMD inpatients. Additional nationwide and population-based epidemiologic and serologic studies are essential to elucidate HFMD dynamics after the use of EV-A71 vaccines, and to inform public health authorities to introduce optimized intervention strategies.

Introduction: This study was designed to explore the association between the TMEM173 polymorphism (rs7447927) and the severity of enterovirus 71 (EV71) infection among Chinese children., Methods: The TMEM173 polymorphism was identified in EV71-infected patients (n = 497) and healthy controls (n = 535) using the improved multiplex ligation detection reaction (iMLDR). The interferon-α (IFN-α) serum levels were detected using enzyme linked immunosorbent assay (ELISA)., Results: The frequencies of the GG genotype and G allele of TMEM173 rs7447927 in the mild EV71 infection and severe EV71 infection groups were markedly higher than those in the control group. The GG genotype and G allele frequencies in severely infected EV71 patients were significantly higher than those in mildly infected EV71 patients. Severely infected EV71 patients with the GG genotype had higher white blood cell counts (WBC), and C-reactive proteins (CRP), and blood glucose (BG) levels, longer fever duration, higher vomiting frequency, spirit changes, and electroencephalography (EEG) abnormalities. IFN-α serum concentration in severely infected patients was significantly higher than in the mildly infected group. The IFN-α concentration in the GG genotype was significantly higher compared with those in the GC and CC genotypes in severe cases., Conclusions: The TMEM173 rs7447927 polymorphism was associated with EV71 infection susceptibility and severity. The G allele and GG genotype are susceptibility factors in the development of severe EV71 infection in Chinese children., (© 2022 The Authors. Immunity, Inflammation and Disease published by John Wiley & Sons Ltd.)

Coxsackievirus A16 (CVA16) is well known for causing hand-foot-and-mouth disease (HFMD) and outbreaks were frequently reported in Taiwan in the past twenty years. The epidemiology and genetic variations of CVA16 in Taiwan from 1998 to 2021 were analyzed in this study. CVA16 infections usually occurred in early summer and early winter, and showed increased incidence in 1998, 2000-2003, 2005, 2007-2008, and 2010 in Taiwan. Little or no CVA16 was detected from 2017 to 2021. CVA16 infection was prevalent in patients between 1 to 3 years old. A total of 69 isolates were sequenced. Phylogenetic analysis based on the VP1 region showed that CVA16 subgenotype B1 was dominantly isolated in Taiwan from 1998 to 2019, and B2 was identified only from isolates collected in 1999 and 2000. There was a high frequency of synonymous mutations in the amino acid sequences of the VP1 region among CVA16 isolates, with the exception of position 145 which showed positive selection. The recombination analysis of the whole genome of CVA16 isolates indicated that the 5'-untranslated region and the non-structural protein region of CVA16 subgenotype B1 were recombined with Coxsackievirus A4 (CVA4) and enterovirus A71 (EVA71) genotype A, respectively. The recombination pattern of subgenotype B2 was similar to B1, however, the 3D region was similar to EVA71 genotype B. Cross-neutralization among CVA16 showed that mouse antisera from various subgenotypes viruses can cross-neutralize different genotype with high neutralizing antibody titers. These results suggest that the dominant CVA16 genotype B1 can serve as a vaccine candidate for CVA16.

The C4 sub-genotype of Enterovirus 71 (EV71) has been identified as the most dominant sub-genotype circulating in the Chinese mainland since 1998. The circulation situation of EV71 before 1998 is not well established due to insufficient experimental data. The C1 subgenotype of EV71 has not yet been reported in the Chinese mainland by now. Based on the AFP surveillance system of the mainland of China, this study conducted a retrospective study of AFP cases for 1985-1999: a strain of EV-A71 C1 subgenotype was found. To our knowledge, this strain (SD92-41) is the first C1 sub-genotype reported in the Chinese mainland. This study demonstrates that the C1 gene subtype also appeared in the Chinese mainland, but it is unknown whether it is an imported or a local epidemic strain. With sufficient information known from retrospective studies, the source of the SD92-41 strain will be identified and the prevalence of EV-A71 in the Chinese mainland before 1998 will be clearer., (© 2022. The Author(s).)

Introduction: This study aimed to explore the association between the IRAK4 polymorphism rs4251545 and the severity of enterovirus 71 (EV71) infection in Chinese children., Methods: We analyzed the IRAK4 polymorphism rs4251545 in 617 EV71-infected patients and 410 controls using the improved multiplex ligation detection reaction. IRAK4 mRNA expression was tested by qRT-PCR. Serum concentrations of IL-6 and NF-κB were detected using ELISA., Results: The frequencies of the GA + AA genotype and A allele in the mild EV71 infection group and in the severe EV71 infection group were significantly higher than those in the normal control group. The frequency of the GA + AA genotype and A allele in severely infected EV71 patients was markedly higher than that in mildly infected EV71 patients. IRAK4 mRNA expression in mildly infected EV71 patients and severely infected patients was significantly higher than that in the control group. IRAK4 mRNA expression in GA + AA genotypes in both mild and severe EV71 infection groups was significantly higher than that in patients with the GG genotype. IL-6 concentration and the ratio of IL-6/NF-κB in severe EV71 cases were significantly lower in patients with the GA + AA genotype than in those with the GG genotype. The ratio of IL-6/NF-κB was distinctly higher in severely infected EV71 patients than in mildly infected and control subjects., Conclusions: The IRAK4 polymorphism rs4251545 was associated with the susceptibility and severity of EV71 infection. The A allele is a susceptible factor in the development of severe EV71 infection in Chinese children., (© 2022 The Authors. Immunity, Inflammation and Disease published by John Wiley & Sons Ltd.)

Recombination plays important roles in the genetic diversity and evolution of Enterovirus A71 (EV-A71). The phylogenetics of EV-A71 in mainland China found that one strain DL71 formed a new subgenotype C6 with unknown origin. This study investigated the detailed genetic characteristics of the new variant. DL71 formed a distinct cluster within genotype C based on the genome and individual genes (5'UTR, VP4, VP1, 2A, 2B, 2C, 3D, and 3'UTR). The average genetic distances of the genome and individual genes (VP3, 2A, 2B, 2C, 3A, 3C, and 3D) between DL71 and reference strains were greater than 0.1. Nine recombination events involving smaller fragments along DL71 genome were detected. The strains Fuyang-0805a (C4) and Tainan/5746/98 (C2) were identified as the parental strains of DL71. In the non-recombination regions, DL71 had higher identities with Fuyang-0805a than Tainan/5746/98, and located in the cluster with C4 strains. However, in the recombination regions, DL71 had higher identities with Tainan/5746/98 than Fuyang-0805a, and located in the cluster with C2 strains. Thus, DL71 was a novel multiple inter-subgenotype recombinant derived from the dominant subgenotype C4 and the sporadic subgenotype C2 strains. Monitoring the emergence of new variants by the whole-genome sequencing remains essential for preventing disease outbreaks and developing new vaccines., (© 2022. The Author(s).)

Enterovirus A71 (EV-A71) inactivated vaccines have been widely inoculated among children in Kunming City after it was approved. However, there was a large-scale outbreak of Enteroviruses (EVs) infection in Kunming, 2018. The epidemiological characteristics of HFMD and EVs were analysed during 2008-2018, which are before and three years after EV-A71 vaccine starting to use. The changes in infection spectrum were also investigated, especially for severe HFMD in 2018. The incidence of EV-A71 decreased dramatically after the EV-A71 vaccine starting use. The proportion of non-CV-A16/EV-A71 EVs positive patients raised to 77.17-85.82%, while, EV-A71 and CV-A16 only accounted for 3.41-7.24% and 6.94-19.42% in 2017 and 2018, respectively. CV-A6 was the most important causative agent in all clinical symptoms (severe HFMD, HFMD, Herpangina and fever), accounting from 42.13% to 62.33%. EV-A71 only account for 0.36-2.05%. In severe HFMD, CV-A6 (62.33%), CV-A10 (11.64%), and CV-A16 (10.96%) were the major causative agent in 2018. EV-A71 inactivated vaccine has a good protective effect against EV-A71 and induced EVs infection spectrum changefully. EV-A71 vaccine has no or insignificant cross-protection effect on CV-A6, CV-A10, and CV-A16. Herein, developing 4-valent combined vaccines is urgently needed.

Background: The incidence of hand foot and mouth disease (HFMD) has increased in recent years, making it a very common childhood illness worldwide. The relationship between different enterovirus genotypes and disease severity is not clearly understood. Given that enteroviruses are transmitted through the gastrointestinal tract, we hypothesized that variation in intestinal microorganisms of the host might play a role in the prognosis of HFMD., Methods: We carried out a meta-transcriptomic-wide association study of fecal samples obtained from a cohort of children (254 patients, 227 tested positive for enterovirus, including 16 patients co-infectied with 2 kinds of enterovirus) with mild and severe HFMD and healthy controls., Results: We found there was no significant difference in the amount of each virus type between the mild and severe cases. Genes of enterovirus 71 (EV71) and coxsackievirus A (CV-A) from the severe and mild cases did not show significant clustering. Clostridium sp. L2-50 and Bacteroides stercoris ATCC 43183 were enriched in the guts of children with severe HFMD and KEGG enrichment was found between mild and severe cases., Conclusions: Intestinal microorganisms appear to interact with enterovirus to determine the progression of HFMD. Genes of Bacteroides and Clostridium may be used as predictive markers for a more efficient prognosis and intervention. The enrichment of intestinal bacteria genes with functions may facilitate the development of severe symptoms for HFMD patients., (© 2021. The Author(s).)

Coxsackievirus A6 (CVA6) is a key pathogen causing hand, foot and mouth disease (HFMD). However, there are currently no specific antiviral drugs or vaccines for treating infections caused by CVA6. In this study, human rhabdomyosarcoma (RD), African green monkey kidney (Vero), and human embryonic lung diploid fibroblast (KMB17) cells were used to isolate CVA6 from 327 anal swab and fecal samples obtained during HFMD monitoring between 2009 and 2017. The VP1 genes of the isolates were sequenced and genotyped, and the biological characteristics of the representative CVA6 strains were analyzed. A total of 37 CVA6 strains of the D3 gene subtypes were isolated from RD cells, all of which belonged to the epidemic strains in mainland China. Using the adaptive culture method, 10 KMB17 cell-adapted strains were obtained; however, no Vero cell-adapted strains were acquired. Among the KMB17 cell-adapted strains, only KYN-A1205 caused disease or partial death in suckling mice, and its virulence was stronger than its RD cell-adapted strain. The pathogenic KYN-A1205 strain caused strong tropism to the muscle tissue and led to pathological changes, including muscle necrosis and nuclear fragmentation in the forelimb and hindlimb. Sequence analysis demonstrated that the KYN-A1205 strain exhibited multiple amino acid mutations after KMB17 cell adaptation. Moreover, it showed strong pathogenicity, good immunogenicity and genetic stability, and could be used as an experimental CVA6 vaccine candidate., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Liu, Zhang, Feng, Cong, Xu, Sun, Yang and Ma.)

Objective: To study the genomic sequence of Coxsackievirus A8 (CV-A8) associated with hand, foot and mouth disease (HFMD) from 2013 to 2018 in China and to analyze the genetic evolution of each coding region of the full-length genome. Methods: The genome sequences of 11 CV-A8 strains isolated from patients with HFMD in different regions of China from 2013 to 2018 were determined. Sequence alignment and genetic evolution analysis were performed by Sequencher 5.0 and MEGA 7.0 software, etc. Results: Sequence alignment showed that the genome length of 11 CV-A8 strains ranged from 7 393 bp to 7 400 bp. There was no base insertion or deletion in the coding region compared with the prototype strain, but there were individual base insertion or deletion in the non-coding region. The nucleotide and amino acid similarities in the VP1 region of 11 CV-A8 strains were 78.3%-98.6% and 92.6%-99.7%, respectively, and the nucleotide and amino acid sequences identities with the CV-A8 prototype strain were 78.3%-98.2% and 92.6%-99.7%, respectively. Based on the phylogenetic analysis of VP1 region sequences, the CV-A8 can be divided into five genotypes: A, B, C, D and E. The 11 CV-A8 strains in this study belonged to genotypes C (1 strain), D (2 strains) and E (8 strains). The nucleotide and amino acid similarities of 11 CV-A8 full-length genomes were 81.3%-98.8% and 95.9%-99.5%, respectively. The phylogenetic tree of the P2 region showed that the eight E genotypes CV-A8 had the closest evolutionary distance with CV-A4, CV-A14, and CV-A16. The phylogenetic tree of the P3 region showed that the eight E genotypes CV-A8 had a close evolutionary distance with CV-A5, CV-A16, CV-A14 and CV-A4. Conclusions: The 11 CV-A8 stains in this study showed significant intra-genotype diversity in capsid region and recombinant diversity in non-capsid region which indicated that CV-A8 quasispecies were still undergoing dynamics variation. CV-A8 may become an important pathogen of HFMD and the monitoring of CV-A8 needs to be further strengthened.

Enterovirus A71 (EV-A71) is an important pathogen of severe hand, foot, and mouth disease (HFMD) in young children. This study aimed to retrospectively analyze the molecular epidemiology and recombination of EV-A71 in mainland China during 1987-2017. Phylogenetic tree showed that besides the previously reported subgenotypes A, B5, C0, C2, C3, and C4, a new subgenotype C6 emerged in mainland China. Recombination analysis indicated that C4 EV-A71 was derived from a common ancestor as a "double-recombinant" virus by intertypic recombination between C EV-A71 and CVA4, CVA5, CVA14, and CVA16 strains in P3 region and intratypic recombination between C and B EV-A71 strains in P2 region. The B5 EV-A71 shared high similarity with C EV-A71 in P1 region while it contained an unidentified sequence in P2 and P3 regions with two possible recombination patterns: one occurred between C4 EV-A71 and CVA3, CVA5, CVA6, CVA10, and CVA12 stains with one breakpoint in 3C, and the other occurred between C1, C2, C3, and C5 EV-A71 and CVA4, CVA5, CVA14, and CVA16 strains with two breakpoints in the 2A/2B junction and 3C. The C2 EV-A71 was probably a recombinant virus between C4 EV-A71 and CVA8 strains with two breakpoints located in the 5'UTR and 2A/2B junction. Moreover, an incredible recombination of C6 EV-A71 occurred between C4 and C2 EV-A71 with multiple breakpoints. Thus, continuous studies on EV-A71 genome characteristics are still useful and essential for monitoring emergence of new viruses and preventing HFMD outbreaks., (© 2021. The Author(s), under exclusive licence to Springer Nature Switzerland AG part of Springer Nature.)

Enterovirus 71 (EV71) has caused large hand, foot, and mouth disease (HFMD) epidemics among young children, and EV71 infection is the leading cause of severe HFMD cases and deaths. In mainland China, the prevalence and risk factors of non-C4 EV71 strains are still unclear. In this study, we monitored non-C4 strains over a 10-year HFMD epidemiological surveillance period in Xiamen. The 5'UTR and VP1 coding region of EV71 strains were amplified by RT-nested PCR and sequenced. Thirty-two non-C4 EV71 strains were identified during 2009-2018. This study provides important information about the prevalence of EV71 in China that will be applicable for development of vaccines and diagnostic reagents as well as establishment of policies for HFMD prevention and control., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Background: Enterovirus A71 (EVA71) is one of the main pathogens causing hand, foot and mouth disease (HFMD). In China, the proportion of cases of HFMD caused by EVA71 is known to be significantly lower following EVA71 vaccination; however, infection with EVA71 can still occur after vaccination., Methods: The complete genomic sequences of EVA71-KM18A and KM18B (from two rare cases of EVA71 infection following vaccination) were obtained. Phylogenetic analysis, nucleotide mutation analysis, recombinant analysis and comparative analysis of amino acid mutations were performed., Results: Phylogenetic analysis determined that the EVA71 strains belonged to the C4a subgenotype. The KM18A and KM18B strains were highly similar to the vaccine strains. For the KM18B strain, there were some obvious homologous recombination signals in the 5'non-coding region, region 2A, region 2C and region 3D. Amino acid mutations were observed in the SP55 (position 729) and 71-6 (position 500) conformational neutralizing epitopes of the KM18A and KM18B strains., Conclusions: These amino acid mutations may affect the SP55 and 71-6 conformational neutralizing epitopes and change their spatial conformation, thereby weakening vaccine effectiveness., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: To investigate the relationship between polymorphism of scavenger receptor class B member 2 (SCARB2) gene and clinical severity of enterovirus (EV)-71 associated hand-foot-mouth disease (HFMD)., Methods: Among the 100 recruited cases, 56 were in the severe HFMD group (case group) and 44 were in the general HFMD group (control group). By screening functional single nucleotide polymorphisms (SNPs) and hot SNPs, and performing SNP site optimization, some SNP sites of SCARB2 gene were selected for analysis. Genotyping was performed using a MassArray platform. PLINK software was used for statistical processing and analysis of the correlation differences between the mutant genotypes in the severe and general HFMD groups. The relationship between the SNPs and clinical severity of enterovirus (EV)-71 associated HFMD was assessed., Results: 28 SNPs in SCARB2 were selected by site optimization. Then three loci were not in agreement with the minor allele frequency (MAF) in the 1000 Han Chinese in Beijing (CHB) dataset. Another three loci could not be detected. Nine loci were not suitable for further analysis (MAF < 0.01 and Hardy-Weinberg [HWE] P < 0.001). A total of 13 sites were subsequently analyzed. Through Fisher analysis, the frequency of the rs6812193 T allele was 0.134 and 0.034 in the severe and general HFMD groups, respectively (P 0.023 < 0.05, odds ratio [OR] 4.381 > 1). Logistic regression analysis of rs6812193 T alleles between the severe and general HFMD groups, respectively (P 0.023 < 0.05, OR 4.412 > 1, L95 1.210 > 1). Genotype logistic regression analysis of the rs6812193 alleles CT + TT versus CC gave an OR of 4.56 (95% confidence interval [95% CI] 1.22-17.04, P = 0.012)., Conclusion: The rs6812193 T allele was a susceptibility SNP for SHFMD, and the rs6812193 polymorphism might be significantly associated with the susceptibility to EV-71 infection.

Background: Hand, foot, and mouth disease (HFMD) caused by enterovirus A71 (EV-A71) poses a serious threat to children's health. Kinetics of the neutralising antibody (NAb) response in EV-A71 infected HFMD patients remains unclear. The ideal sampling time of paired serum samples for serological diagnosis of EV-A71 infection is not well defined., Methods: HFMD inpatients admitted to Henan Children's Hospital between February 15, 2017 and February 15, 2018 were enrolled. Serial serum samples collected during hospitalisation and up to 1.5 years after discharge were tested for NAb against EV-A71. Random intercept modelling with B-spline was conducted to characterize the kinetics of the EV-A71 NAb response over time after illness onset., Findings: A total of 524 serum samples from 264 EV-A71 RNA positive HFMD inpatients were collected. NAb titres of EV-A71 infected patients were estimated to increase from 40 (95% CI: 9-180) at the day of onset to the peak of 2417 (95% CI: 1859-3143) at day 13, then remained above 1240 until 26 months. For serological diagnosis of EV-A71 infection, if at least a 4-fold rise in titre was used as the criteria, the acute phase serum should be collected at 0-4 days, the corresponding convalescent serum should be collected 14.9 days (95% CI: 9.1-23.8) after illness onset., Interpretation: EV-A71 infection induced a strong and persistent humoral immune response in HFMD patients. The findings provide a scientific support for determining the collection time of paired serum samples for serological diagnosis of EV-A71 infected HFMD patients., Funding: National Science Fund for Distinguished Young Scholars., Competing Interests: Declaration of Competing Interest H Yu has received research funding from Sanofi Pasteur, GlaxoSmithKline, Yichang HEC Changjiang Pharmaceutical Company, and Shanghai Roche Pharmaceutical Company, outside this study. All authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

Surveillance of recombinant enterovirus 71 (EV71) and subgenotype replacement is vital for preventing and controlling hand, foot, and mouth disease (HFMD) outbreaks. Despite this, data on recombinant variants and phylogeny of circulating EV71 strains in mainland China are limited. In this study, recombinant variants of EV71 were identified in mainland China from 2009 to 2018. Phylogenetic analysis indicated that except for individual strains (CQ2014-86/CQ/CHN/2014 and EV71/Xiamen/2009 (B5)), almost all of the EV71 strains in mainland China belonged to the subgenotype C4a. Analysing complete genome sequences of 196 EV71 isolates, 3 intertypic recombination strains (VR1432, 30-2/2015/BJ, and Guangdong-2009) and 5 intratypic recombination strains (EV71/P1034/2013, VR1432, Henan-ZMD/CHN/2012, Hubei-WH/CHN/2012, and EV71/P868/2013/China) were identified among naturally circulating EV71. The breakpoints of these recombinant strains were located within the P1, P2, and P3 encoding regions. Notably, a double recombinant (VR1432) resulting from recombination between EV71 subgenotype C4a and C4b strain SHZH98 and a CA8 strain Donovan was identified. This study reports these specific intertypic and intratypic recombination events for the first time highlighting the importance of genetic recombination in the emergence of new enterovirus variants.

To understand the prevalence and etiological characteristics of hand, foot and mouth disease(HFMD) in Yangzhou, so as to provide scientific basis for prevention and control of HFMD. HFMD cases from six sentinel hospitals in Yangzhou from January 2015 to December 2019 were taken as the subject of study. The epidemiological data of HFMD were analyzed by descriptive epidemiology method, the enterovirus were detected using RT-PCR method and the etiological characteristics were analyzed. The data were collected by Excel 2007 and statistically analyzed by SPSS22.0 software. A total of 1 151 positive cases were detected from all 2 129 HFMD clinical specimens collected in Yangzhou from 2015 to 2019, with a total positive rate of 54.06%, including 148 cases of EV71(6.95%), 382 cases of CA16(17.94%) and 621 cases of other enterovirus(29.17%). The difference of positive rate in different years( χ 2 =99.28, P <0.05), different months( χ 2 =92.09, P <0.05) and different districts( χ 2 =71.39, P <0.05)was statistically significant. Each subtype of enterovirus showed alternating prevalence in different years. The peak period of detection rate was from April to September (720 cases, 62.55%). The reported incidence for males was higher than females, with the male-female ratio of 1.58∶1 and children under six (971 cases, 84.36%) as the major attacked population. There were obvious seasonal, regional and population characteristics of HFMD in Yangzhou City from 2015 to 2019. It reminds us that surveillance of HFMD should be continually implemented, more attention should be paid to the prevention and control of key population in high-risk seasons, and the booming HFMD cases caused by other enterovirus should be emphasized especially.

Background: EV-A71 is a common causative agent of hand foot and mouth disease. In mainland China, EV-A71 subgenotype C4 has been the sole circulating genotype since 2008, and was used in the production of multiple licensed vaccines. Here, we report the first detection EV-A71 C1 strains in China., Methods: Full genomic sequence were obtained. The origin of the EV-A71 C1 strains were tracked down by Bayesian inferences. Recombination was analyzed using Simplot program. And the antigenicity were tested using the microneutralization test., Results: The C1-GD2019 shared high identity with the C1-like lineage recently identified in Europe and was introduced into Guangdong in 2018-2019. Close genetic relatedness between the C1-GD2019 and Europe C1-like strains were observed except for the 3D-3'UTR region. The late showed high similarity with CVA genomes. Antigenic variance was found. The C1-GD2019 could not be effectively neutralized by EV-A71 C4a neutralizing antibody positive samples., Conclusion: This is the first report of EV-A71 subgenotype C1 isolated in China. It is a recombinant strain originating from C1-like strains recently identified in Europe and CVA strains. The different antigenicity between the C1 strains and C4a vaccine strains highlighted the importance on closely monitoring the EV-A71 C1 strains in China., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2020. Published by Elsevier Ltd.)

Background: To analyze the prevalence of latent infection of pathogens of hand, foot, and mouth disease (HFMD) in Chinese healthy population and its influencing factors, so as to provide reference for the prevention and control of HFMD., Methods: A systematic literature searching about the incidence of latent infection of HFMD was conducted in Chinese and English databases. The inclusion and exclusion criteria of the retrieved literature were established. The qualified literatures were screened and the data were extracted. The pooled rate and its 95% confidence interval was used to assess the latent infection rate of HFMD pathogens in healthy Chinese population, and subgroup analysis was conducted based on gender and age. All statistical analyses were performed using the STATA version 12.0 software., Results: A total of 31 literatures were included in this meta-analysis. The recessive infection rate of HFMD pathogens reported in the literature of Chinese healthy people ranged from 4.59% to 44.12%. The results of meta-analysis showed that the latent infection rate of human enteroviruses (HEVs) in healthy Chinese population was 17.5% (14.9-20.1%), among which, the latent infection rates of EV-A71, CV-A16, and other HEVs were 3.3% (2.2-4.4%), 1.7% (1.0-2.5%), and 15.1% (11.1-17.1%), respectively. The latent infection rates of HEVs in healthy men and women in China were 16.7% (12.9-20.4%) and 14.4% (10.8-18.0%), respectively. The latent infection rates of HEVs in the healthy population aged 0 to 5 years and over 5 years were 24.4% (20.4-28.5%) and 9.4% (6.5-12.2%), respectively. Meta regression showed that the factors affecting the latent infection rate of HEVs in Chinese healthy population included sampling period, sampling area, and study population., Conclusion: The latent infection rate of HEVs is high in healthy people in China, but it is mainly caused by other enteroviruses. The latent infection rate of HEVs in male was higher than that of female and was greater in people aged 0 to 5 than that of aged over 5 years. Limited by the quantity and quality of the included studies, more high-quality studies are needed for further verification in the future., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2021 the Author(s). Published by Wolters Kluwer Health, Inc.)

Development of multivalent hand, foot, and mouth disease (HFMD) vaccines against enterovirus A71 (EV-A71) and several non-EV-A71 enteroviruses is needed for this life-threatening disease with a huge economic burden in Asia-Pacific countries. Comprehensive studies on the molecular epidemiology and genetic and antigenic characterization of major causative enteroviruses will provide information for rational vaccine design. Compared with molecular studies on EV-A71, that for non-EV-A71 enteroviruses remain few and limited in Vietnam. Therefore, we conducted a 10-year study on the circulation and genetic characterization of coxsackievirus A16 (CV-A16) and CV-A6 isolated from patients with HFMD in Northern Vietnam between 2008 and 2017. Enteroviruses were detected in 2228 of 3212 enrolled patients. Of the 42 serotypes assigned, 28.4% and 22.4% accounted for CV-A6 and CV-A16, being the second and the third dominant serotypes after EV-A71 (31.7%), respectively. The circulation of CV-A16 and CV-A6 showed a wide geographic distribution and distinct periodicity. Phylogenetic analyses revealed that the majority of Vietnamese CV-A6 and CV-A16 strains were located within the largest sub-genotypes or sub-genogroups. These comprised strains isolated from patients with HFMD worldwide during the past decade and the Vietnamese strains have been evolving in a manner similar to the strains circulating worldwide. Amino acid sequences of the putative functional loops on VP1 and other VPs among Vietnamese CV-A6 and CV-A16 isolates were highly conserved. Moreover, the functional loop patterns of VP1 were similar to the dominant patterns found worldwide, except for the T164K substitution on the EF loop in Vietnamese CV-A16. The findings suggest that the development of a universal HFMD vaccine, at least in Vietnam, must target CV-A6 and CV-A16 as two of the three major HFMD-causing serotypes. Vietnamese isolates or their genome sequences can be considered for rational vaccine design., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Hand, foot, and mouth disease (HFMD) caused by enterovirus 71 (EV71) can lead to high morbidity and mortality, and genetic background plays an important role during the disease process. We investigated the association between the single-nucleotide polymorphism (SNP) rs2564978 of the CD55 gene and susceptibility to and severity of HFMD using the SNPs can multiple SNP typing methods. Soluble CD55 (sCD55) expression was significantly lower in the EV71 HFMD group than in the control group and lower in severe cases than in mild cases (P < .001). Moreover, CD55 rs2564978 (C vs T OR = 1.300, 95% CI, 1.120-1.509) was associated with the risk of EV71 infection, and genotype TC was related to the severity of the infection (TC vs TT OR = 4.523, 95% CI, 2.033-10.066). Our results suggest that sCD55 expression and the CD55 polymorphism rs2564978 may influence the susceptibility to and severity of EV71 infection., (© 2020 Wiley Periodicals LLC.)

Coxsackievirus A8 (CV-A8) is one of the pathogens associated with hand, foot and mouth disease (HFMD) and herpangina (HA), occasionally leading to severe neurological disorders such as acute flaccid paralysis (AFP). Only one study aimed at CV-A8 has been published to date, and only 12 whole-genome sequences are publicly available. In this study, complete genome sequences from 11 CV-A8 strains isolated from HFMD patients in extensive regions from China between 2013 and 2018 were determined, and all sequences from GenBank were retrieved. A phylogenetic analysis based on a total of 34 complete VP1 sequences of CV-A8 revealed five genotypes: A, B, C, D and E. The newly emerging genotype E presented a highly phylogenetic divergence compared with the other genotypes and was composed of the majority of the strains sequenced in this study. Markov chain Monte Carlo (MCMC) analysis revealed that genotype E has been evolving for nearly a century and somehow arose in approximately 2010. The Bayesian skyline plot showed that the population size of CV-A8 has experienced three dynamic fluctuations since 2001. Amino acid residues of VP1 100N , 103Y , 240T and 241V , which were embedded in the potential capsid loops of genotype E, might enhance genotype E adaption to the human hosts. The CV-A8 whole genomes displayed significant intra-genotypic genetic diversity in the non-capsid region, and a total of six recombinant lineages were detected. The Chinese viruses from genotype E might have emerged recently from recombining with European CV-A6 strains. CV-A8 is a less important HFMD pathogen, and the capsid gene diversity and non-capsid recombination variety observed in CV-A8 strains indicated that the constant generation of deleterious genomes and a constant selection pressure against these deleterious mutations is still ongoing within CV-A8 quasispecies. It is possible that CV-A8 could become an important pathogen in the HFMD spectrum in the future. Further surveillance of CV-A8 is greatly needed.

To evaluate the epigenetic regulation of the VDR gene in enterovirus 71 (EV71)-associated severe hand, foot, and mouth disease (HFMD), a total of 116 patients with EV71-HFMD, including 58 with mild EV71-HFMD and 58 with severe EV71-HFMD, as well as 60 healthy controls, were enrolled in this study. Quantitative real-time PCR was used to measure the relative levels of VDR mRNA expression, and the methylation status of the VDR promoter was assessed using a MethylTarget™ assay. The DNA methylation levels of the VDR promoter in children with EV71-associated severe HFMD were lower than those in the healthy controls and in children with mild HFMD (P < 0.05). Hypomethylation at CpG site 133 and hypermethylation at the CpG 42 sites and 68 downregulated VDR expression. Moreover, the methylation level of VDR could be used for differential diagnosis of mild and severe EV71-associated HFMD (AUC 56 , 0.73; AUC 68 , 0.699; AUC 42 , 0.694; AUC 66 , 0.693). VDR expression and promoter methylation were associated with the progression of EV71 infection. Determining the VDR promoter status might help clinicians initiate the appropriate strategy for treatment of EV71-associated HFMD.

Enterovirus A71 (EV-A71) infection is known to cause hand, foot, and mouth disease (HFMD). Last year, an inactivated EV-A71 whole virus vaccine was used to prevent this disease in Yunnan, China. To obtain a viral genetic background for evaluating vaccine protection and monitor the adaptive evolution of the virus after the vaccination, a 5-year molecular epidemiology survey was performed before the vaccination. Twenty-six EV-A71 strains were separated from 561 stool specimens of patients with serious HFMD. The whole-genomic sequences of these strains were sequenced. Phylogenetic trees were constructed, and the mutation spectra were analyzed based on these viral sequences. There was no obvious mutation for the circular EV-A71 strains of the same year. Pathogenic EV-A71 strains may arise from a "subgroup" randomly each year. Whole-genomic analyses showed that a hotspot nonsynonymous substitution potentially affecting the immunogenicity of vaccines was found in the 2A gene, but not in genes of the viral capsid proteins, and the genetic diversity of whole viral genomes associated with the incidence of HFMD. Therefore, it will be valuable to monitor the genome-wide changes of EV-A71 to detect the adaptive mutations affecting immunogenicity or perform investigations using genetic diversity as a parameter., (© 2019 Wiley Periodicals, Inc.)

Enterovirus 71 (EV71) is one of the major causative agents for hand, foot and mouth disease (HFMD) in children. Although there are three inactivated virus-based HFMD vaccines licensed in China, alternative approaches have been taken to produce an effective and safer vaccine that is easier to manufacture in large scale. Among these, a virus-like particles (VLPs) based EV71 vaccine is under active development. For this purpose, an efficient methodology for the production of EV71-VLPs by recombinant technology is needed. We here report the construction and expression of the P1 and 3C genes of EV71 in Pichia pastoris for producing VLP-based EV71 vaccine antigen with a high yield and simple manufacturing process. Based on codon-optimized P1 and 3C genes, EV71-VLPs were efficiently expressed in Pichia pastoris system, and the expression level reached 270 mg/L. Biochemical and biophysical analyses showed that the produced EV71-VLPs consisted of processed VP0, VP1, and VP3 present as ~35nm spherical particles. The immune response as a function of EV71-VLPs and adjuvant dose ratio was investigated for vaccine development. Immunization with EV71-VLPs of 1-5 µg/dose and adjuvant of 225 µg/dose induced robust neutralizing antibody responses in mice and provided effective protection against lethal challenge in both maternally transferred antibody and passive transfer protection mouse models. Therefore, the yeast produced EV71-VLPs antigen is a promising candidate for the development of a vaccine against HFMD.

Objectives: To better understand the spectrums of pathogens causing herpangina and circulation of Coxsackievirus A4 in Yancheng, China., Methods: Stool samples from herpangina and HFMD cases were collected. Real Time PCR Kits was used to identify Enterovirus 71, CV-A16 and CV-A6, and nested reverse transcription PCR (nRT-PCR) to detect the other enterovirus types. Complete VP1 and genome sequence of CV-A4 were amplified by using nRT-PCR. Genetic, phylogenetic and recombination analysis were performed., Results: Co-circulation of three recombinant CV-A4 groups, including one novel (C2 lineage), was identified in Yancheng, China, 2016 and 2018. One was the major causative agent of herpangina, and another two were responsible for HFMD. Phylogenetic and recombination analysis indicated that the non-structural region of their genome originated from the same ancestry and subsequently adaptation. C2 lineage of CV-A4 group may be introduced from countries outside China and its genome occurred recombination in China., Conclusion: Novel recombinant CV-A4 was mainly associated with herpanginain in Yancheng, 2018, China. C2 lineage of CV-A4 group with recombinant non-structural region was also identified in HFMD patients., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Human enteroviruses (EVs), including coxsackieviruses, the numbered enteroviruses, and echoviruses, cause a wide range of diseases, such as hand, foot, and mouth disease (HFMD), encephalitis, myocarditis, acute flaccid myelitis (AFM), pneumonia, and bronchiolitis. Therefore, broad-spectrum anti-EV drugs are urgently needed to treat EV infection. Here, we demonstrate that FNC (2'-deoxy-2'-β-fluoro-4'-azidocytidine), a small nucleoside analog inhibitor that has been demonstrated to be a potent inhibitor of HIV and entered into a clinical phase II trial in China, potently inhibits the viral replication of a multitude of EVs, including enterovirus 71 (EV71), coxsackievirus A16 (CA16), CA6, EVD68, and coxsackievirus B3 (CVB3), at the nanomolar level. The antiviral mechanism of FNC involves mainly positive- and negative-strand RNA synthesis inhibition by targeting and competitively inhibiting the activity of EV71 viral RNA-dependent RNA polymerase (3D pol ), as demonstrated through quantitative real-time reverse transcription-PCR (RT-qPCR), in vitro 3D pol activity, and isothermal titration calorimetry (ITC) experiments. We further demonstrated that FNC treatment every 2 days with 1 mg/kg of body weight in EV71 and CA16 infection neonatal mouse models successfully protected mice from lethal challenge with EV71 and CA16 viruses and reduced the viral load in various tissues. These findings provide important information for the clinical development of FNC as a broad-spectrum inhibitor of human EV pathogens. IMPORTANCE Human enterovirus (EV) pathogens cause various contagious diseases such as hand, foot, and mouth disease, encephalitis, myocarditis, acute flaccid myelitis, pneumonia, and bronchiolitis, which have become serious health threats. However, except for the EV71 vaccine on the market, there are no effective strategies to prevent and treat other EV pathogen infections. Therefore, broad-spectrum anti-EV drugs are urgently needed. In this study, we demonstrated that FNC, a small nucleoside analog inhibitor that has been demonstrated to be a potent inhibitor of HIV and entered into a clinical phase II trial in China, potently inhibits the viral replication of a multitude of EVs at the nanomolar level. Further investigation revealed that FNC inhibits positive- and negative-strand RNA synthesis of EVs by interacting and interfering with the activity of EV71 viral RNA-dependent RNA polymerase (3D pol ). Our findings demonstrate for the first time that FNC is an effective broad-spectrum inhibitor for human EV pathogens., (Copyright © 2020 Xu et al.)

Background: Hand, foot, and mouth disease (HFMD) is a common infectious disease occurring in children under 5 years of age worldwide, and Enterovirus A71 (EV-A71) and Coxsackievirus A16 (CVA-16) are identified as the predominant pathogens. In recent years, Coxsackievirus A6 (CVA-6) and Coxsackievirus A10 (CVA-10) have played more and more important role in a series of HFMD outbreaks. This study aimed to understand the epidemic characteristics associated with HFMD outbreak in Guangzhou, 2018., Methods: The clinical and laboratory data of 1220 enterovirus-associated HFMD patients in 2018 were analysed in this study. Molecular diagnostic methods were performed to identify its serotypes. Phylogenetic analyses were depicted based on the complete VP1 gene., Results: There were 21 enterovirus serotypes detected in Guangzhou in 2018. Three serotypes of enterovirus, CVA-6 (364/1220, 29.8%), CVA-10 (305/1220, 25.0%), and CVA-16 (397/1220, 32.5%), were identified as the causative pathogens and accounted for 87.3% among all 1220 HFMD patients. In different seasons, CVA-6 was the predominant pathogen of HFMD during autumn, and CVA-10 as well as CVA-16 were more prevalent in summer. Patients infected by CVA-6, CVA-10 or CVA-16 showed similar clinical features and laboratory characteristics, and the ratios of severe HFMD were 5.8, 5.9, and 1.5% in the three serotypes. Phylogenetic analyses of VP1 sequences showed that the CVA-6, CVA-10, and CVA-16 sequences belonged to the sub-genogroup E2, genogroup E, and genogroup B1, respectively., Conclusions: CVA-6, CVA-10, and CVA-16 were the predominant and co-circulated serotypes in Guangzhou China, 2018, which should be the new target for prevention and control of HFMD. Our findings provide useful information for diagnosis, treatment, and prevention of HFMD.

Objectives: Enterovirus 71 (EV71) and coxsackievirus A16 (CA16) were responsible for 43.3% (235 123/543 243) and 24.8% (134 607/543 243) of all laboratory-confirmed hand, foot and mouth disease (HFMD) cases during 2010-2015 in China. Three monovalent EV71 vaccines have been licensed in China while bivalent EV71/CA16 vaccines are under development. A comparative cost-effectiveness analysis of bivalent EV71/CA16 versus monovalent EV71 vaccination would be useful for informing the additional value of bivalent HFMD vaccines in China., Methods: We used a static model parameterized with the national HFMD surveillance data during 2010-2013, virological HFMD surveillance records from all 31 provinces in mainland China during 2010-2013 and caregiver survey data of costs and health quality of life during 2012-2013. We estimated the threshold vaccine cost (TVC), defined as the maximum additional cost that could be paid for a cost-effective bivalent EV71/CA16 vaccine over a monovalent EV71 vaccine, as the outcome. The base case analysis was performed from a societal perspective. Several sensitivity analyses were conducted by varying assumptions governing HFMD risk, costs, discounting and vaccine efficacy., Results: In the base case, choosing the bivalent EV71/CA16 over monovalent EV71 vaccination would be cost-effective only if the additional cost of the bivalent EV71/CA16 compared with the monovalent EV71 vaccine is less than €4.7 (95% CI 4.2-5.2). Compared with the TVC in the base case, TVC increased by up to €8.9 if all the test-negative cases were CA16-HFMD; decreased by €1.1 with an annual discount rate of 6% and exclusion of the productivity loss; and increased by €0.14 and €0.3 with every 1% increase in bivalent vaccine efficacy against CA16-HFMD and differential vaccine efficacy against EV71-HFMD, respectively., Conclusions: Bivalent EV71/CA16 vaccines can be cost-effective compared with monovalent EV71 vaccines, if suitably priced. Our study provides further evidence for determining the optimal use of HFMD vaccines in routine paediatric vaccination programme in China., (Copyright © 2019 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background: The most recent documented Australian outbreak of enterovirus A71 (EV-A71) occurred in Sydney from 2012 to 2013. Over a four-month period more than 100 children presented to four paediatric hospitals with encephalitic presentations including fever and myoclonic jerks. The heterogeneous presentations included typical encephalomyelitis, and cardiopulmonary complications., Objectives: To characterise the genomes of enterovirus strains circulating during the 2013 Sydney EV-A71 outbreak and determine their phylogeny, phylogeography and association between genome and clinical phenotype., Study Design: We performed an analysis of enterovirus (EV) positive specimens from children presenting to hospitals in the greater Sydney region of Australia during the 2013 outbreak. We amplified near full-length genomes of EV, and used next generation sequencing technology to sequence the virus. We used phylogenetic/phylogeographic analysis to characterize the outbreak viruses., Results: We amplified and sequenced 23/63 (37 %) genomes, and identified the majority (61 %) as EV-A71. The EV-A71 sequences showed high level sequence homology to C4a genogroups of EV-A71 circulating in China and Vietnam during 2012-13. Phylogenetic analysis showed EV-A71 strains associated with more severe symptoms, including encephalitis or cardiopulmonary failure, grouped together more closely than those from patients with hand, foot and mouth disease. Amongst the non-EV-A71 sequences were five other EV subtypes (representing enterovirus subtypes A and B), reflecting the diversity of EV co-circulation within the community., Conclusions: This is the first Australian study investigating the near full-length genome of EV strains identified during a known outbreak of EV-A71. EV-A71 sequences were very similar to strains circulating in Asia during the same time period. Whole genome sequencing offers additional information over routine diagnostic testing such as characterisation of emerging recombinant strains and inform vaccine design., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background: Hand, foot and mouth disease (HFMD) is usually caused by EVA71 and CVA16 except for a few cases that are caused by non-EVA71 non-CAV16 enteroviruses. Coxsackievirus B3 (CVB3) is mostly associated with myocarditis, occasionally with HFMD., Methods: The partial VP1 gene of enteroviruses were amplified and sequenced from 610 throat swabs from clinically confirmed HFMD children. All available CVB3 near full-length genomic and VP1 sequences were downloaded from GenBank. Phylogenetic and distance analyses were performed using MEGA 7.0., Results: A total of 238 partial VP1 sequences were obtained, including 93 EVA71 (39%), 79 CAV16 (33%), 29 CVB3 (12%), 24 CVA6 (10%), and 13 other enterovirus serotypes (5.5%). CVB3 is classified into seven genotypes A-G according to phylogenetic and distance analyses. All CVB3 strains from Zhenjiang belonged to genotype A. In contrast to other genotypes that are prevalent in Europe and other regions of China, and often associated with aseptic meningitis and myocarditis, CVB3 genotype A strains identified in Zhenjiang were only detected among HFMD patients., Conclusions: This high prevalence of CVB3 genotype A among HFMD children has never been reported. This phenomenon has revealed a new epidemic trend of CVB3 among HFMD in China, and it has epidemiological implications for monitoring the epidemic risk of CVB3., (Copyright © 2019 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Neurovirulent enterovirus 71 (EV-A71) caused a massive epidemic in China in 2008-2011. While subgenotype C4 was the major causative agent, a few isolates were almost identical to the prototype EV-A71 strain and belonged to genotype A. This variant was allegedly extinct since 1970, and its identification in this epidemic suggests reintroduction of the archive virus. Regression analysis of genetic distances (TempEst software) was of moderate utility due to the low resolution of classical phylogenetic methods. Bayesian phylogenetic analysis (BEAST software) suggested artificial introduction event based on highly aberrant phylogenetic tree branch rates that differed by over three standard deviations from the mean substitution rate for EV71. Manual nucleotide-level analysis was used to further explore the virus spread pattern after introduction into circulation. Upon reintroduction, the virus accumulated up to seven substitutions in VP1, most of them non-synonymous and located within the capsid's canyon or at its rims, compatible with readaptation of a lab strain to natural circulation., Competing Interests: The authors declare no conflicts of interest.

Sporadic outbreaks caused by coxsackievirus A4 (CV-A4) have been reported worldwide. To further elucidate the detailed genetic characteristics and evolutionary recombination events of CV-A4, virus samples from nationwide hand, foot and mouth disease (HFMD) surveillance, encompassing 27 out of the 31 provinces in China, were investigated. Comprehensive and systematic phylogenetic analyses were performed by using 29 complete genomes, 142 complete CV-A4 VP1 sequences. Four genotypes (A, B, C and D) and five sub-genotypes (C1-C5) were re-identified based on the complete VP1 sequences. C2 is the predominant sub-genotype of CV-A4 associated with HFMD and has evolved into 3 clusters. Cluster 1 is a major cluster that has been persistently and extensively circulating in China since 2006 and has been associated with all severe cases. All the sequences showed high homology with the CV-A4 prototype in the P1 region, while higher identities with CV-A5, CV-14 and CV-16 in the P2 and P3 regions. Recombination analysis revealed that C2 had two specific genetic recombination patterns with other EV-A prototypes in the 5'-UTR and 3D region compared with C5. These recombination patterns might be associated with the increased transmissibility of C2 viruses, which were obtained due to their persistent and extensive circulation in populations.

Hand, foot, and mouth disease (HFMD) is endemic in the Pacific region, especially in mainland China. The case-fatality ratio of HFMD is increasing steadily. Knowledge of the changing epidemiology of HFMD in different regions is necessary for implementing appropriate intervention strategies. In this study, we describe the clinical and epidemiological characteristics of HFMD in Hunan Children's Hospital between 2013 and 2017. A total of 7203 patients with HFMD were admitted, with complication and mortality rates of 35.62% and 0.78%, respectively. The total number of children with HFMD, proportion of severely ill children, and HFMD mortality rate were the highest in 2014. The number of cases caused by EV-A71 and CV-A16 decreased continuously, while the number of cases caused by 'other enteroviruses' increased yearly since 2014, suggesting that other enteric viruses will gradually replace EV-A71 and CV-A16 as the main pathogenic HFMD agents. Furthermore, EV-A71 and mixed infections accounted for the high case fatality rates in children with severe HFMD, among whom EV-A71 infection resulted in the highest complication and mortality rates; the mild form of the disease was dominated by 'other enteroviruses'. In conclusion, the changing etiological pattern highlights the need to improve pathogen surveillance and vaccine strategies for HFMD control.