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Coxsackievirus A6 (CV-A6) has emerged as the predominant causative agent of hand, foot, and mouth disease (HFMD) in young children. Since the declaration of coronavirus disease 2019 (COVID-19) as a global pandemic, the incidence of infectious diseases, including HFMD, has decreased markedly. When social mitigation was relaxed during the COVID-19 pandemic in 2022, the re-emergence of HFMD was observed in Gwangju, South Korea, and seasonal characteristics of the disease appeared to have changed. To investigate the molecular characteristics of enterovirus (EV) associated with HFMD during 2022, 277 specimens were collected. Children aged younger than 5 years accounted for the majority of affected individuals. EV detection and genotyping were performed using real-time RT-PCR and nested RT-PCR followed by sequence analysis. The EV detection rate was found to be 82.3%, and the main genotype identified was CV-A6. Sixteen CV-A6 samples were selected for whole genome sequencing. According to phylogenetic analysis, all CV-A6 strains from this study belonged to the sub-genotype D3 clade based on VP1 sequences. Analysis of 3D polymerase phylogeny showed that only the recombinant RF-A group was identified. In conclusion, circulating EV types should be continuously monitored to understand pathogen emergence and evolution during the post-pandemic era.

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.)

An increasing number of studies have reported that atypical hand, foot, and mouth disease (HFMD) is becoming a new concern for children's health. At present, there is no official definition for atypical HFMD, but some studies have defined that it occurs at anatomic sites not listed in the definition of HFMD issued by the World Health Organization. Several pathogens have been reported to cause atypical HFMD, such as Coxsackievirus (CV)A6. As one of the most prevalent enteroviruses in the world, CVA6 seems to affect a wider range of children and causes more severe and prolonged illness than other enteroviruses. The early lesions of atypical HFMD are very similar to the clinical presentations of other diseases, such as eczema, which poses a challenge for clinicians aiming to identify and diagnose HFMD in a timely manner. Here, we report on six atypical HFMD patients caused by recombinant CVA6 variants, and the atypical manifestations include eczema coxsackium, large herpes, rice-like red papules and herpes, purpuric rash, and onychomadesis, as well as and large red herpes on scalp, perianal, testicles, shoulders and neck, and other atypical eruption sites, hoping to draw the attention of other pediatricians. This study will provide scientific guidance for timely diagnosis of HFMD to prevent serious complications., (© 2023 Wiley Periodicals LLC.)

Objective: To investigate the effect of heterogeneous nuclear ribonucleoprotein A2B1 (HNRNPA2B1) on the replication of enterovirus 71 (EV-71) in SK-N-SH cells., Methods: The mRNA and protein expression of HNRNPA2B1 in SK-N-SH cells were detected by real-time quantitative PCR (qRT-PCR) and western blotting (WB), respectively. WB was used to detect HNRNPA2B1 protein expression in the nucleus and cytosol. The localization of HNRNPA2B1 protein in the nucleus and cytosol was detected by immunofluorescence (IF). The expression of HNRNPA2B1 was inhibited by small interfering RNA (si-HNRNPA2B1). Viral RNA, viral structural protein VP1, and viral titer were detected by qRT-PCR, WB, and viral dilution counting, respectively., Results: EV-71 infection significantly upregulates the expression of HNRNPA2B1 in SK-N-SH cells. EV-71 infection promotes HNRNPA2B1 nucleus-cytoplasm redistribution. Down-regulation of HNRNPA2B1 expression significantly inhibited EV-71 replication., Conclusion: HNRNPA2B1 protein redistributed from nucleus to cytoplasm and is highly expressed in the cytoplasm during EV-71 infection. Inhibition of HNRNPA2B1 levels effectively inhibits EV-71 replication in SK-N-SH cells., 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 The Author(s). Published by Elsevier B.V. All rights reserved.)

Hand, foot and mouth disease (HFMD) is a common childhood infectious disease, caused by enteroviruses (EVs), which can present with typical or atypical lesions. The illness is self-limiting, but it can also have serious complications. Since 1997, HFMD infections have become endemic and have increased to epidemic proportions across the Asia Pacific region, including India. Coxsackievirus-A16 (CV-A16) outbreaks occurred in India from 2005 onwards, although the clinical symptoms were noticeably different during this period. Understanding the population dynamics of enteroviruses that cause HFMD is crucial in the post-polio era because one of the circulating strain may replace another as the dominant strain. The aim of this study is to describe the genetic features of the CV-A16 strains isolated from hand, foot and mouth disease (HFMD) patients in India. Reverse transcription PCR (RT-PCR) and cell-culture-based isolation of CV-A16 was done from the 55 clinical samples. The entire genome of the CV-A16 isolate was performed from the seven isolates. After the sequences were analysed, a phylogenetic tree was created using bioinformatics tools. The total genomic length obtained was 7411 base pairs (bp). Nucleotide similarity across various regions, including 5'UTR, P1, P2 and 3'UTR, ranged from 87.0-97.9 %, 77.0-95.4 %, 80.3-96.9 %, and 77.9-96.2 %, respectively. Correspondingly, similarities in the VP1 region's nucleotide and amino acid sequences were 91.4-96.4 % and 99.3-99.7 %, respectively. Phylogenetic analysis highlighted that CV-A16 strains identified in Pune, Maharashtra, were grouped within the same cluster. The analysed CV-A16 isolates in this study aligned with subgenotype B1c. These findings have far-reaching implications for the surveillance, prevention and management of HFMD and CV-A16. Monitoring the dynamics of CV-A16 strains, informed by the genetic characteristics identified here, will significantly impact strategies aimed at tackling HFMD and its associated public health challenges.

Coxsackievirus A6 (CVA6) is emerging as the dominant serotype among enteroviruses (EVs) responsible for hand, foot, and mouth disease (HFMD) outbreaks in multiple countries. However, details regarding this serotype in the Philippines are limited. In this study, we investigated the epidemiological and molecular characteristics of laboratory-confirmed CVA6 HFMD cases in the Philippines between 2012 and 2017. Data collected from case report forms submitted to the National Reference Laboratory for Poliovirus and other Enteroviruses were used to determine the distribution and clinical findings of laboratory-confirmed CVA6 HFMD. Phylogenetic analyses of the complete viral protein 1 (VP1) and partial 3D polymerase (3D pol ) gene sequences were performed to determine the genotype and recombinant (RF) form of the selected samples. An increase in the detection rate of CVA6 among enterovirus-positive HFMD cases was observed from 61.9% (140/226) in 2012 to 88.1% (482/587) in 2017, with most cases coming from the Luzon island group. Among the detected cases, the majority were children, with a median age of 2 years old (interquartile range: 1.17-3.40). Respiratory-related morbidities were the commonly reported complications (7.9%; 72/907). Based on the VP1 and 3D pol gene sequence analysis, the CVA6 strains in this study were classified as genotype D3b and RF-A group, respectively. This study elucidated that CVA6 was the most prevalent enterovirus serotype causing HFMD in the Philippines in 2012-2017, with genotype D3b/RF-A circulating within this period. This study highlights the importance of viral surveillance and molecular epidemiological analysis to broaden our understanding of HFMD in the Philippines., 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 The Authors. Published by Elsevier B.V. All rights reserved.)

Enteroviruses are pathogens responsible for several diseases, being enterovirus A71 (EVA71) the second leading cause of hand, foot, and mouth disease (HFMD), especially in Asia-Pacific countries. HFMD is mostly common in infants and children, with mild symptoms. However, the disease can result in severe nervous system disorders in children as well as in immunosuppressed adults. The virus is highly contagious, and its transmission occurs via fecal-oral, oropharyngeal secretions, and fomites. The EVA71 burdens the healthy systems and economies around the world, however, up to date, there is no antiviral approved to treat infected individuals and the existent vaccines are not available or approved to be used worldwide. In this context, an extensive literature research was conducted to describe and summarize the recent advances in natural and/or synthetic compounds with antiviral activity against EVA71. The summarized data presented here might simply encourage the future studies in EVA71 antiviral development, by encouraging further research encompassing these compounds or even the application of the techniques and technologies to improve or produce new antiviral molecules., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Outbreaks of HFMD in children aged <5 years have been reported worldwide and the major causative agents are Coxsackievirus (CV) A16, enterovirus (EV)-A71 and recently CVA6. In India, HFMD is a disease that is not commonly reported. The purpose of the study was to identify the enterovirus type(s) associated with large outbreak of Hand, foot, and mouth disease during COVID-19 pandemic in 2022. Four hundred and twenty five clinical samples from 196-suspected cases were collected from different parts of the country. This finding indicated the emergence of CVA6 in HFMD along with CVA16, soon after the gradual easing of non-pharmaceutical interventions during-pandemic COVID-19 and the relevance of continued surveillance of circulating enterovirus types in the post-COVID pandemic era., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Enterovirus 71 (EV71) infection mainly causes hand, foot, and mouth disease (HFMD) and remains a serious public health problem to the children under the age of 5. Until now, there is no specific drug to treat HFMD in clinical and there is an urgent to explore the new target and the new drug to address clinical challenges. At present, we found histone deacetylase 11 (HDAC11) involves in supporting EV71 replication. We also used HDAC11 siRNA and an HDAC11 inhibitor FT895 to downregulate HDAC11 expression and found that targeting HDAC11 could significantly restrict EV71 replication in vitro and in vivo. Our results revealed the new role of HDAC11 participating in EV71 replication and broadened our knowledge regarding the functions of HDAC11 and the roles of HDACs in the epigenetic regulation of viral infectious diseases. Our results for the first time identified FT895 as an effective inhibitor of EV71 in vitro and in vivo, which may contribute to be a potential drug to treat HFMD., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflicts of interest to this work, (Copyright © 2023. Published by Elsevier B.V.)

Hand, foot and mouth disease (HFMD) is a kind of infectious disease caused by enterovirus infection. In this study we analysed the epidemiological characteristics and time trends of HFMD, vaccination status and vaccine protection effect assessment of EV71 vaccine from 2011 to 2021 in Huangpu District, Shanghai, China. HFMD cases showed a decreasing trend year by year from 2011 to 2021, from 122 cases reported in 2012 to 7 cases in 2020, and 12 cases in 2021. Etiological diagnosis was CV-A6 in 185 cases (29.8%), CV-A16 in 209 cases (33.7%), EV-A71 in 118 cases (19.0%) and other enteroviruses in 109 cases (17.6%). After the launch of EV71 vaccine, a total of 32,221 doses of EV71 vaccine were administered between 2016 and 2021. The case-control results showed that there was no evidence to support the effectiveness of EV71 vaccine, OR (95% CI) =0.52 (0.12 ~ 2.3), p  = 0.37. The epidemic strains have changed. Surveillance and management of HFMD remain very important in the future and EV71 vaccine is considered to be included in National Immunization Program., 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 © 2023 Wang and Zhang.)

Coxsackievirus A10 (CVA10) causes hand, foot, and mouth disease (HFMD) and herpangina, which can result in severe neurological symptoms in children. CVA10 does not use the common enterovirus 71 (EV71) receptor, human SCARB2 (hSCARB2, scavenger receptor class B, member 2), for infection but instead uses another receptor, such as KREMEN1. Our research has shown that CVA10 can infect and replicate in mouse cells expressing human SCARB2 (3T3-SCARB2) but not in the parental NIH3T3 cells, which do not express hSCARB2 for CVA10 entry. Knocking down endogenous hSCARB2 and KREMEN1 with specific siRNAs inhibited CVA10 infection in human cells. Co-immunoprecipitation confirmed that VP1, a main capsid protein where virus receptors for attaching to the host cells, could physically interact with hSCARB2 and KREMEN1 during CVA10 infection. It is the efficient virus replication following virus attachment to its cellular receptor. It resulted in severe limb paralysis and a high mortality rate in 12-day-old transgenic mice challenged with CVA10 but not in wild-type mice of the same age. Massive amounts of CVA10 accumulated in the muscles, spinal cords, and brains of the transgenic mice. Formalin inactivated CVA10 vaccine-induced protective immunity against lethal CVA10 challenge and reduced the severity of disease and tissue viral loads. This is the first report to show that hSCARB2 serves as an associate to aid CVA10 infection. hSCARB2-transgenic mice could be useful in evaluating anti-CVA10 medications and studying the pathogenesis induced by CVA10.

Hand, foot and mouth disease, a childhood disorder caused by enteroviruses, is intermittently endemic in the Asia-Pacific region and endangers the lives of many infants and young children. Coxsackievirus A16 (CV-A16) is one of the major pathogens causing hand, foot, and mouth disease on occasion, resulting in catastrophic neurological sequelae and patient death. Currently, no clinical interventions are available that completely block the CV-A16 infection. Therefore, research on anti-CV-A16 treatment continues to be a significant focus of interest. This report provides a detailed background on and an introduction to CV-A16; a description of the viral gene and protein structures and a summary of the current advances in pharmaceutical targets, drug research and other related areas.

Hand, foot, and mouth disease (HFMD) is a highly contagious disease in children caused by a group of enteroviruses. HFMD currently presents a major threat to infants and young children because of a lack of antiviral drugs in clinical practice. Drug repositioning is an attractive drug discovery strategy aimed at identifying and developing new drugs for diseases. Notably, repositioning of well-characterized therapeutics, including either approved or investigational drugs, is becoming a potential strategy to identify new treatments for virus infections. Various types of drugs, including antibacterial, cardiovascular, and anticancer agents, have been studied in relation to their therapeutic potential to treat HFMD. In this review, we summarize the major outbreaks of HFMD and the progress in drug repositioning to treat this disease. We also discuss the structural features and mode of action of these repositioned drugs and highlight the opportunities and challenges of drug repositioning for HFMD.

Hand-foot-and-mouth disease (HFMD) is an infectious disease of children caused by more than 20 types of enteroviruses, with most cases recovering spontaneously within approximately one week. Severe HFMD in individual children develops rapidly, leading to death, and is associated with other complications such as viral myocarditis and type I diabetes mellitus. The approval and marketing of three inactivated EV-A71 vaccines in China in 2016 have provided a powerful tool to curb the HFMD epidemic but are limited in cross-protecting against other HFMD-associated enteroviruses. This review focuses on the epidemiological analysis of HFMD-associated enteroviruses since the inactivated EV-A71 vaccine has been marketed, collates the progress in the development of multivalent enteroviruses vaccines in different technical routes reported in recent studies, and discusses issues that need to be investigated for safe and effective HFMD multivalent vaccines.

CVA6 is one of Enteroviruses causing worldwide epidemics of HFMD with neurological and systemic complications. A suitable animal model is necessary for studying the pathogenesis of CVA6 and evaluating antiviral and vaccine efficacy. In this study, we generated a mouse-adapted CVA6 strain that successfully infected 10-day-old ICR mice via oral route. All infected mice were paralyzed and died within 11 dpi. Analysis of pathological changes and virus loads in fourteen tissues showed that CVA6 triggered systematic damage similar to i.p. inoculation route. Unlike i.p. route, we detected oral and gastrointestinal lesions with the presence of viral antigens. Both specific anti-CVA6 serum and inactivated vaccines successfully generated immune protection in mice. Meanwhile, we also established a successful infection of CVA6 via i.p. and i.m. route in 10-day-old mice. After infection, mice developed remarkably neurological signs and systemic manifestations such as emaciation, polypnea, quadriplegia, depilation and even death. Through i.p. inoculation, pathological examination showed brain and spinal cord damage caused by the virus infection with neuronal reduction, apoptosis, astrocyte activation, and recruitment of neutrophils and monocytes. Following neurological manifestation, the CVA6 infection became systemic, and high viral loads were detected in multiple organs along with morphological changes and inflammation. Moreover, analysis of spleen cells by FACS indicated that CVA6 led to immune system activation, which further contributed to systemic inflammation. Taken together, our novel murine model of CVA6 provides a useful tool for studying the pathogenesis and evaluating antiviral and vaccine efficacy.

Object: To investigate the evolution and genetic characteristics of Coxsackievirus A6 (CVA6) which acted as the predominant pathogen of hand, foot and mouth disease (HFMD) in children in Beijing, China, 2017-2019., Methods: Throat swab specimens were collected for general Enterovirus (EV), enterovirus A71 (EV-A71) and CVA16 detection by Real-time PCR. These general EV-positive samples were identified by semi-nested RT-PCR method and sequencing. The CVA6 VP1 gene and genome sequences were amplified and sequenced. The phylogenetic, variation and recombination analyses were performed., Results: A total of 1721 HFMD patients were enrolled in this study, with the male to female ratio of 1.62:1. The majority of cases were less than five years, which accounted for 73.50%. The overall detection rate of EV was 88.32% (1520/1721). A total of 8 EV types were identified, including CVA6 (55.86%), CVA16 (26.32%), EV-A71 (2.24%), CVA10 (2.04%), CVA4 (1.05%), CVA5 (0.59%), CVA2 (0.33%), and CVA8 (0.07%), while 175 (11.51%) EV were untyped. The main pathogen of HFMD was CVA6 from 2017 to 2018, while CVA6 and CVA16 were the main causative pathogens in 2019. The nucleotide and amino acid sequence identities of the 120 CVA6 complete VP1 gene sequences in this study were 91.2%-100.0% and 97.7%-100.0%, respectively. Compared with the prototype strain (Gdula) of CVA6, the nucleotide and amino acid sequence identities were 81.7%-84% and 94.7%-96.3%, respectively. The phylogenetic tree indicated that all 120 CVA6 sequences belonged to sub-genotype D3, while 119 CVA6 sequences belonged to evolutionary branch D3a, except one from 2017 belonged to D3b. Recombination analysis based on the complete genome sequences showed that potential multiple recombination may have occurred in 2B and 3D protein coding regions with EV-A114., Conclusions: The main pathogens of HFMD were CVA6 and CVA16 in Beijing, China, 2017-2019; while these CVA6, as recombination strains, belonged to the D3a evolutionary branch., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Objectives: This study aims to explore the interaction of different pathogens in Hand, foot and mouth disease (HFMD) by using a mathematical epidemiological model and the reported data in five regions of China., Methods: A cross-regional dataset of reported HFMD cases was built from four provinces (Fujian Province, Jiangsu province, Hunan Province, and Jilin Province) and one municipality (Chongqing Municipality) in China. The subtypes of the pathogens of HFMD, including Coxsackievirus A16 (CV-A16), enteroviruses A71 (EV-A71), and other enteroviruses (Others), were included in the data. A mathematical model was developed to fit the data. The effective reproduction number ( R eff ) was calculated to quantify the transmissibility of the pathogens., Results: In total, 3,336,482 HFMD cases were collected in the five regions. In Fujian Province, the R eff between CV-A16 and EV-A71&CV-A16, and between CV-A16 and CV-A16&Others showed statistically significant differences ( P < 0.05). In Jiangsu Province, there was a significant difference in R eff ( P < 0.05) between the CV-A16 and Total. In Hunan Province, the R eff between CV-A16 and EV-A71&CV-A16, between CV-A16 and Total were significant ( P < 0.05). In Chongqing Municipality, we found significant differences of the R eff ( P < 0.05) between CV-A16 and CV-A16&Others, and between Others and CV-A16&Others. In Jilin Province, significant differences of the R eff ( P < 0.05) were found between EV-A71 and Total, and between Others and Total., Conclusion: The major pathogens of HFMD have changed annually, and the incidence of HFMD caused by others and CV-A16 has surpassed that of EV-A71 in recent years. Cross-regional differences were observed in the interactions between the pathogens., 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 © 2022 Yang, Rui, Qi, Ye, Niu, Luo, Deng, Zhang, Yu, Liu, Li, Wang, Wei, Zhang, Huang, Zuo, Zhang, Zhang, Yang, Guo, Zhao, Wu, Li, Chen and Chen.)

The main pathogen causing severe and neurotrophic hand, foot and mouth disease (HFMD) is enterovirus A71 (EV71). EV71 infection is among the major cause of serious public health burden and economic loss especially in the Asia-pacific region. Yet, no specific anti-viral treatment against this life-threatening infection is currently available. Thus, the best way to control EV71 infection is by vaccination with an effective and safe vaccine. Several strategies are being employed to develop vaccines against EV71. These include conventional and modern recombinant vaccine strategies. Conventional vaccines such as inactivated EV71 vaccines are the most studied and advanced vaccines against HFMD. Recombinant HFMD vaccines developed based on the recombinant DNA technology have been employed but are mostly at early or late preclinical development stage. In this article, we discuss the recent progress and advances in modern recombinant strategies of EV71 vaccine development including subunit, VLP, epitope-based, DNA, and vector-based vaccines, as well as conventional approaches, focusing on their various prospects, advantages and disadvantages., Competing Interests: Declaration of competing interest The authors have no conflict of interest to declare., (Copyright © 2022 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.)

Group B coxsackievirus is an enterovirus that can cause a variety of diseases, including myocarditis, aseptic meningitis, and hand, foot, and mouth disease. Currently, there is no effective antiviral drug against this virus. In this study, we used a cytopathic effect-based viral inhibition assay to screen an FDA-approved drug library and found that doxepin hydrochloride had potential antiviral activity. Doxepin hydrochloride exhibited strong antiviral activity against coxsackievirus B types 1-3 with a 50% inhibitory concentration of 10.12 ± 0.85 μM. Moreover, doxepin hydrochloride did not exert antiviral activity against other enteroviruses, including enterovirus A71 (subtypes BrCr/C4) and coxsackievirus A (subtypes 6/10/16). Furthermore, doxepin hydrochloride inhibited virus replication in the early stage of the infection cycle rather than affecting the entry or assembly process. In addition, a few mechanism-related pharmacophores were discovered through gene association network analysis. These findings identify a possible lead compound for treating coxsackievirus B infection and simultaneously offer valuable clues for drug repositioning., (Copyright © 2022. Published by Elsevier B.V.)

Enterovirus A ( EV-A ) species cause hand, foot and mouth disease (HFMD), threatening the health of young children. Understanding the mutual codon usage pattern of the virus and its host(s) has fundamental and applied values. Here, through examining multiple codon usage parameters, we found that the codon usage bias among EV-A strains varies and is clade-specific. EVA76, EVA89, EVA90, EVA91 and EVA92, the unconventional clade of EV-A strains, show unique codon usage pattern relative to the two conventional clades, including EVA71, CVA16, CVA6 and CVA10, etc. Analyses of Effective Number of Codon (ENC), Correspondence Analysis (COA) and Parity Rule 2 (PR2), etc. , revealed that the codon usage patterns of EV-A strains are shaped by mutation pressure and natural selection. Based on the neutrality analysis, we determined the dominant role of natural selection in the formation of the codon usage bias of EV-A . In addition, we have determined the codon usage compatibility of potential hosts for EV-A strains using codon adaptation index (CAI), relative codon deoptimization index (RCDI) and similarity index (SiD) analyses, and found that EV-A showed host-specific codon adaptation patterns in different clades. Finally, we confirmed that the unique codon usage pattern of the unconventional clade affected protein expression level in human cell lines. In conclusion, we identified novel characteristics of codon usage bias in distinct EV-A clades associated with their host range, transmission and pathogenicity., 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 © 2022 Zeng, Chen, Wang, Zhu, Yan, Zhang, Xu and Zhang.)

Enterovirus 71 (EV71) is an infectious virus affecting all age groups of people around the world. It is one of the major aetiologic agents for HFMD (hand, foot and mouth disease) identified globally. It has led to many outbreaks and epidemics in Asian countries. Infection caused by this virus that can lead to serious psychological problems, heart diseases and respiratory issues in children younger than 10 years of age. Many studies are being carried out on the pathogenesis of the virus, but little is known. The host immune response and other molecular responses against the virus are also not clearly determined. This review deals with the interaction between the host and the EV71 virus. We discuss how the virus makes use of its proteins to affect the host's immunity and how the viral proteins help their replication. Additionally, we describe other useful resources that enable the virus to evade the host's immune responses. The knowledge of the viral structure and its interactions with host cells has led to the discovery of various drug targets for the treatment of the virus. Additionally, this review focusses on the antiviral drugs and vaccines developed by targeting various viral surface molecules during their infectious period. Furthermore, it is asserted that the improvement of prevailing vaccines will be the simplest method to manage EV71 infection swiftly. Therefore, we summarise numerous vaccines candidate for the EV71, such as the use of an inactivated complete virus, recombinant VP1 protein, artificial peptides, VLPs (viral-like particles) and live attenuated vaccines for combating the viral outbreaks promptly., (© 2022 John Wiley & Sons Ltd.)

Hand, foot and mouth disease (HFMD) is a common infectious disease in western Asia area and the full range of the long-term sequelae of HFMD remains poorly described. We conducted a retrospective hospital-based cohort study of HFMD patients with central nervous system (CNS) complications caused by EV-A71 or CV-A16 between 2010 and 2016. Patients were classified into three groups, including CNS only, autonomic nervous system (ANS) dysregulation, and cardiorespiratory failure. Neurologic examination, neurodevelopmental assessments, Magnetic Resonance Imaging (MRI) and lung function, were performed at follow up. Of the 176 patients followed up, 24 suffered CNS only, 133 ANS dysregulation, and 19 cardiorespiratory failure. Median follow-up period was 4.3 years (range [1.4-8.3]). The rate of neurological abnormalities was 25% (43 of 171) at discharge and 10% (17 of 171) at follow-up. The rates of poor outcome were significantly different between the three groups of complications in motor (28%, 38%, 71%) domain (p=0.020), but not for cognitive (20%, 24%, 35%), language (25%, 36%, 41%) and adaptive (24%, 16%, 26%) domains ( p  = 0.537, p  = 0.551, p  = 0.403). For children with ventilated during hospitalization, 41% patients (14 of 34) had an obstructive ventilatory defect, and one patient with scoliosis had mixed ventilatory dysfunction. Persistent abnormalities on brain MRI were 0% (0 of 7), 9% (2 of 23) and 57% (4 of 7) in CNS, ANS and cardiorespiratory failure group separately. Patients with HFMD may have abnormalities in neurological, motor, language, cognition, adaptive behaviour and respiratory function. Long-term follow-up programmes for children's neurodevelopmental and respiratory function may be warranted.

Although epidemics of hand, foot, and mouth disease (HFMD) caused by enterovirus A71 (EV-A71) have occurred worldwide, the Asia-Pacific region has seen large sporadic outbreaks with many severe neurological cases. This suggests that the virulence of the circulating viruses fluctuates in each epidemic and that HFMD outbreaks with many severe cases occur when highly virulent viruses are circulating predominantly, which has not been experimentally verified. Here, we analyzed 32 clinically isolated strains obtained in Japan from 2002 to 2013, along with 27 Vietnamese strains obtained from 2015 to 2016 that we characterized previously using human SCARB2 transgenic mice. Phylogenetic analysis of the P1 region classified them into five clades belonging to subgenogroup B5 (B5-I to B5-V) and five clades belonging to subgenogroup C4 (C4-I to C4-V) according to the epidemic year and region. Interestingly, clades B5-I and B5-II were very virulent, while clades B5-III, B5-IV, and B5-V were less virulent. Clades C4-II, C4-III, C4-IV, and C4-V were virulent, while clade C4-I was not. The result experimentally showed for the first time that several clades with different virulence levels emerged one after another. The experimental virulence evaluation of circulating viruses using SCARB2 transgenic mice is helpful to assess potential risks of circulating viruses. These results also suggest that a minor nucleotide or amino acid substitution in the EV-A71 genome during circulation causes fluctuations in virulence. The data presented here may increase our understanding of the dynamics of viral virulence during epidemics. IMPORTANCE Outbreaks of hand, foot, and mouth disease (HFMD) with severe enterovirus A71 (EV-A71) cases have occurred repeatedly, mainly in Asia. In severe cases, central nervous system complications can lead to death, making it an infectious disease of importance to public health. An unanswered question about this disease is why outbreaks of HFMD with many severe cases sometimes occur. Here, we collected EV-A71 strains that were prevalent in Japan and Vietnam over the past 20 years and evaluated their virulence in a mouse model of EV-A71 infection. This method clearly revealed that viruses belonging to different clades have different virulence, indicating that the method is powerful to assess the potential risks of the circulating viruses. The results also suggested that factors in the virus genome cause an outbreak with many severe cases and that further studies facilitate the prediction of large epidemics of EV-A71 in the future.

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.)

Three inactive monovalent EV-A71 vaccines have been launched in China since 2016, which may change the HFMD pathogen spectrum and epidemiological trend. Using notifications from the national surveillance system, we analyzed the epidemiological character profiles and the possible pathogen replacement. The proportion of HFMD cases aged 0-12 months decreased from 23.0% to 15.3% between 2013-2015 and 2017-2019 (p < 0.01). EV-A71 among laboratory-confirmed severe cases in 2013-2015 (62.8%) transformed to other EVs (67.2%) in 2017-2019. The age distribution of EV-A71 infection shifted to the older. The cumulative coverage of the EV-A71 vaccine for children aged six months to five years in Guangxi has increased, while in severe cases, the positive rate declined. After gradually expanded vaccination, EV-A71 associated incidence rate, case-severity rate has decreased, and other serotypes are becoming dominant. Thus, bivalent even polyvalent vaccines are urgently needed to control HFMD., 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 © 2021 Elsevier Ltd. All rights reserved.)

Background: Enteroviruses (EVs) occur frequently worldwide and are known to be associated with a broad spectrum of clinical manifestations from mild syndromes to neurological disease. To understand the epidemiology of EV in Korea, we characterized EV-infected cases during 2012-2019 based on national surveillance., Methods: We collected specimens from patients with suspected EV infections and analyzed the data using real-time reverse-transcription polymerase chain reaction and VP1 gene sequencing., Results: Among the 18 261 specimens collected, EVs were detected in 6258 (34.3%) cases. Although the most common EV types changed annually, EV-A71, echovirus 30, coxsackievirus B5, coxsackievirus A6, and coxsackievirus A10 were commonly identified. Among the human EVs, the case numbers associated with the 2 major epidemic species (EV-A and EV-B) peaked in the summer. While EV-A species affected 1-year-old children and were associated with herpangina and hand, foot, and mouth disease, EV-B species were mostly associated with neurologic manifestations. The highest incidence of EV-B species was observed in infants aged <12 months. Feces and respiratory specimens were the most predictive of EV infection. Specimens collected within 5 days of symptom onset allowed for timely virus detection., Conclusions: EV-A and EV-B species co-circulating in Korea presented different epidemiologic trends in clinical presentation, affected subjects, and seasonality trends. This study could provide information for the characterization of EVs circulating in Korea to aid the development of EV antivirals and vaccines, as well as public health measures to control enteroviral diseases., (© The Author(s) 2020. Published by Oxford University Press on behalf of The Journal of the Pediatric Infectious Diseases Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Human hand, foot, and mouth disease (HFMD), an important infectious disease in children, is caused mainly by enterovirus 71 (EV71) and coxsackievirus A16 (CA16). In this study, a bivalent inactivated EV71/CA16 vaccine is developed and evaluated in immunized BALB/c mice injected through the intradermal route. Q-RT-PCR detection of the mRNA of immune signal molecules in local epithelial tissues inoculated with the vaccine indicates activation of innate immunity, which includes upregulation of immune-related chemokines, interferons and CD molecules. Further, the finding that neutralizing antibodies and specific T cellular responses were elicited in adult mice after two immunizations with the vaccine at a 28-day interval, which endowed offspring mice to defend a viral challenge, suggests the successful induction of specific protective antiviral immunity. All these data suggest that immunization with this bivalent EV71/CA16 vaccine via the intradermal route elicits effective immunity against EV71 and CA16 infection., 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.)

This report provides the findings of a retrospective surveillance study on the emergence and circulation of enteroviruses with their associated clinical symptoms over a nine-year period detected at the National Enterovirus Reference Laboratory in Hungary between 2010-2018.Enterovirus (EV) detection and genotyping were performed directly from clinical samples. From 4,080 clinical specimens 25 EV types were identified with a median age of patients of 5 years and 68% of all cases affected children aged 10 years or younger, although infections occurred in all age-groups. In 130 cases neurological symptoms were recorded, in 123 cases the infection presented in skin related signs including hand, foot, and mouth disease (HFMD), herpangina and rash. In 2010 EV-A71 was found to cause the majority of diagnosed EV infections while in 2011 and from 2014-2018, Coxsackievirus (CV)-A6 was identified most often. Echovirus E6 accounted for the most cases in 2012 and Echovirus 30 dominated in 2013. EV-D68 was identified only in 2010 and 2013.Widespread circulation of several EV-A and EV-B viruses with occasional occurrence of EV-C and EV-D was detected. The ability of EVs to cause severe infections in sporadic cases and regular outbreaks highlight the importance of continued monitoring of circulating EV types.

Background: Examining associations between viral genomic loads of enteroviruses and clinical severity is important for promoting and improving development of antiviral drugs related to hand, foot and mouth disease (HFMD)., Methods: Throat swabs were collected from HFMD cases at acute phase of illness using a standardized technique in a prospective study. The viral genomic load was categorized into low, medium, and high groups using parameters of real-time reverse transcription-polymerase chain reaction. The clinical severities were assessed with four indicators, respectively., Findings: We analysed 1109 HFMD cases, including 538 children with CV-A6, 231 with CV-A16, 156 with EV-A71, 78 with CV-A10, 59 with CV-A4, and 47 with CV-A2. EV-A71 genomic load categories were associated with risks of diagnoses of CNS complications (p = 0.016), requiring systemic corticosteroids or IVIG (p = 0.011), intensive care unit admission (p = 0.002) and length of hospital stay over 5 days (p = 0.048). In the multivariate analyses, point estimates of adjusted odds ratio (OR) tended to increase with viral genomic loads for all four severe outcomes and ORs of highest viral genomic load were all significantly larger than one for EV-A71., Interpretation: HFMD clinical severities positively associate with viral genomic loads of EV-A71 in throat swabs. Specific antiviral drugs should be developed to reduce enterovirus load and to alleviate the clinical severities for HFMD cases., Funding: National Science Fund for Distinguished Young Scholars., Competing Interests: Declaration of Competing Interests HJY has received research funding from Sanofi Pasteur, GlaxoSmithKline, Yichang HEC Changjiang Pharmaceutical Company and Shanghai Roche Pharmaceutical Company, outside the submitted work. BJC has received honoraria from Roche and Sanofi Pasteur, outside the submitted work. All other authors declare no competing interests., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

To clarify crucial key micro-RNAs and mRNAs associated with hand, foot, and mouth disease (HFMD) virus infection, we conducted this bioinformatics analysis from four GEO datasets. The following datasets were used for the analysis: GSE85829, GSE94551, GSE52780, and GSE45589. Differentially expressed genes (DEGs) were acquired, and the analysis of functional and pathway enrichment and the relative regulatory network were conducted. After screening common differentially expressed miRNAs (DE-miRNAs), five key miRNAs were acquired: miR-100-3p, miR-125a-3p, miR-1273g-3p, miR-5585-3p, and miR-671-5p. There were three common enriched GO terms between miRNA-derived prediction and mRNA-derived analysis: biosynthetic process, cytosol, and nucleoplasm. There was one common KEGG pathway, i.e., cell cycle shared between miRNA-based and mRNA-based enrichment. Using TarBase V8 in DIANA tools, we acquired 1,520 potential targets (mRNA) from the five key DE-miRNAs, among which the159 DE-mRNAs also included 11 DEGs. These common DEGs showed a PPI network mainly connected by SMC1A, SMARCC1, SF3B3, LIG1, and BRMS1L. Together, changes in five key miRNAs and 11 key mRNAs may play crucial roles in HFMD progression. A combination of these roles may benefit the early diagnosis and treatment of HFMD., Competing Interests: Conflict of interest The authors do not report any financial or personal connections with other persons or organizations, which might negatively affect the contents of this publication and/or claim authorship rights to this publication., (© 2020 Sheng Lin et al.)

Hand, foot and mouth disease (HFMD) is mainly caused by EV-A71 and CV-A16. An increasing number of cases have been found to be caused by CV-A10, CV-A6, CV-B3 and the outbreaks are becoming increasingly more complex, often accompanied by the prevalence of a variety of enteroviruses. Based on the principle of synthetic peptide vaccines, we applied immune-informatics to design a highly efficient and safe multivalent epitope-based vaccine against EV-A71, CV-A16, CV-A10, CV-A6 and CV-B3. By screening B-cells, HTL and CTL cell antigen epitopes with high conservativity and immunogenicity that have no toxic effect on the host, further analysis confirmed that the vaccine built was IFN-γ inductive and IL-4 non-inductive HTL cell epitopes and had population coverage corresponding to MHC molecular alleles associated with T-cell phenotype. The multivalent enterovirus vaccine was constructed to connect the 50 s ribosomal protein L7/L12 adjuvant and candidate epitopes sequentially through appropriate linkers. Then, the antigenic, allergen and physical properties of the vaccine were evaluated, followed by a secondary structure analysis and tertiary structure modeling, disulfide engineering, refinement and validation. Moreover, the conformational B cell epitope of the vaccine was analyzed. The stability of the TLR4/MD2/Vaccine complex and details at atomic level were investigated by docking and molecular dynamics simulation. Finally, in silico immune simulation and in vivo immune experiments were done. This study provides a high cost-effective method of designing a multivalent enterovirus vaccine protect against a wide range of enterovirus pathogens., 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) is an emerging infection with pandemic potential. Knowledge of neutralizing antibody responses among its pathogens is essential to inform vaccine development and epidemiologic research. We used 120 paired-plasma samples collected at enrollment and >7 days after the onset of illness from HFMD patients infected with enterovirus A71 (EV-A71), coxsackievirus A (CVA) 6, CVA10, and CVA16 to study cross neutralization. For homotypic viruses, seropositivity increased from <60% at enrollment to 97%-100% at follow-up, corresponding to seroconversion rates of 57%-93%. Seroconversion for heterotypic viruses was recorded in only 3%-23% of patients. All plasma samples from patients infected with EV-A71 subgenogroup B5 could neutralize the emerging EV-A71 subgenogroup C4. Collectively, our results support previous reports about the potential benefit of EV-A71 vaccine but highlight the necessity of multivalent vaccines to control HFMD.

IRES-driven translation plays an essential role in picornavirus infection. However, there are seldom reports of compounds targeting this pathway with effective protection in animal models. Here, we identified emetine, an antiprotozoal drug, which inhibits EV-A71 with an EC 50 value of 0.04 μM and a CC 50 value of 10 μM in RD cell culture. Interestingly, emetine exhibits activities against a series of human enteroviruses, including CV-A16, CV-B1, EV-D68, Echov-6, etc., at the nanomolar level. When orally administered at 0.20 mg/kg twice a day in an EV-A71 mouse model, emetine reduced viral loads in various organs and completely prevented diseases and death. A mechanistic study demonstrated that emetine suppressed EV-A71 by inhibiting viral IRES-driven translation. Taken together, these data indicate emetine as a promising candidate to treat picornavirus infection., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The present study highlights pathogenesis and molecular aspects of Coxsackie virus A-16 (CVA-16) strains isolated from hand, foot, and mouth disease (HFMD) cases from India using a neonatal mice model. ICR mice were intraperitoneally inoculated with CVA-16/311 strain isolated from HFMD cases. Mice developed hind and forelimb paralysis on day 3 of post infection. Histopathological observations of hind limb muscles showed necrosis, dissolution of muscle fiber cells, infiltration of inflammatory cells, marked dilated ventricle, hemorrhages, and neuronal degeneration in the brain. Immunohistochemical studies revealed high expression of CVA-16/311-specific viral antigen in limb muscles, brain, heart from day 3 till day 7 of post-infection. VP1 gene-based reverse transcription polymerase chain reaction conducted in RNA samples of different tissue organs of infected mice followed by sequencing of the positive amplimers revealed presence of CVA-16/311-specific viral sequences. Phylogenetic analysis based on the VP1 gene showed the presence of B1c sub genotype of CVA-16/311 strain in targeted tissue organs. Sequence analysis revealed major genetic changes in heart, skeletal muscle tissues at the nucleotide and amino acid levels. Genetic changes occurred in organs of mice might predict some potential targets and might act as markers of virulence for neuronal tropism. Pathogenesis and molecular studies of CVA-16 strains isolated from HFMD cases using neonatal mice model was conducted for the first time from India., (© 2019 Wiley Periodicals, Inc.)

In order to access the influence of different age groups on the spread of hand, foot and mouth disease (HFMD), we established the multi-group model with migration following the epidemiology of HFMD. The basic reproduction number of the HFMD epidemic model was calculated by the next generation operator method. According to China's national surveillance data on HFMD, we fitted the model parameters and estimated the transmission rates among different age groups. Besides, we carried out sensitivity analysis for the basic reproduction number to find some valuable regulatory measures. Our findings showed that the children under three years of age were indeed at high risk and adult group who had more contacts with children had a crucial influence on the spread of HFMD.

In China, a rapid expansion of hand, foot, and mouth disease (HFMD) outbreaks has occurred since 2004, and HFMD has become an important issue in China. There are more than 20 types of enterovirus causing HFMD, of which coxsackievirus A16 (CA16) and enterovirus 71 (EV71) are the most common. This study aimed to analyze the epidemiological characteristics of HFMD caused by EV71 and CA16 in Linyi, Shandong province, China, from 2009 to 2017. The stool specimens and throat samples of 5,324 patients with HFMD were obtained for nucleic acid detection of enterovirus. A total of 4,040 HFMD cases were caused by viral pathogens. Of these, 1,706 cases were positive for EV71 and 1,266 were positive for CA16. These 2 virus strains appeared alternately in Linyi city. The incidence of EV71-positive and CA16-positive cases was highest in children aged 0-5 years, with male patients being predominant. This outbreak of HMFD caused by EV71 and CA16 mainly occurred between April and July and appeared alternately between the years 2011 and 2017. These results demonstrated that the epidemiological analysis of EV71 and CA16 can provide a scientific basis for the prevention and treatment of the disease.

Purpose: Enteroviruses (EV) 71 and coxsackievirus A (CVA) 16 are the most prevalent EV serotypes responsible for hand, foot and mouth disease (HFMD). Nevertheless, CVA6 was found to be the leading cause of HFMD in the Nanjing area, of China in 2013. This study aims to provide insights into the occurrence of the emergent recombinant CVA6 through examination of the evolutionary history and the involved recombination events., Methodology: The viral protein1 (VP1) and non-structural (NS) 2C and 3D of 28 Nanjing CVA6 strains were aligned, among which the full-length sequences of eight strains were further characterized., Results: We revealed the co-existence of two recombinant forms (RFs), RF-A and RF-J, in the local area. RF-J is a novel RF group, comprising a proportion of local and Shanghai CVA6 strains from 2013. The appearance of RF-J CVA6 strains was most likely the result of two recombination events, with the co-circulating CVA4 and CVA8 providing the regions beyond positions 4001~4045 and 4866~4873, respectively. Evolutionary history analysis showed that the VP1 sequences of RF-J derived from RF-A, which was also probably the ancestor of several other RF groups. The 3D region of RF-J was closely related to CVA8. The point in time of emergence of the most recent common ancestor (tMRCA) of RF-J in China was estimated to be around 2011 in both terms of VP1 and 3D region., Conclusion: The emerging recombinant CVA6 variants belong to a novel RF-J group which was most likely formed by at least two recombination events. Continued monitoring on the geographical distribution of various CVA6 RFs is essential.

Hand, foot and mouth disease (HFMD) is a pediatric disease associated with infection by enterovirus (EV) genotypes. The major HFMD EV pathogens are enterovirus A71 (EVA71) and coxsackievirus A16 (CVA16); however, recently, coxsackievirus A6 (CVA6) and coxsackievirus A10 (CVA10) have also emerged. EV genotypes cannot be distinguished on clinical grounds and a new methodology for the rapid detection of the four major HFMD EV genotypes is urgently required. In the present study, a multiplex real-time PCR assay was established for the simultaneous detection of CVA6, CVA10, CVA16 and EVA71. The specificity and sensitivity of the assay was determined on a validation panel of clinical samples, comprising cerebrospinal fluid (n = 51), blood (n = 39), feces (n = 58) and throat swabs (n = 29). The results showed that the multiplex real-time PCR exhibited high specificity, no cross-reactivity with other EV genotypes, lower limits of detection for CVA6, CVA10, CVA16 and EVA71 were 4 × 10 3 , 4 × 10 2 , 5 × 10 2 , and 3 × 10 3 copies/μL, respectively and had comparable sensitivity to singleplex assays testing clinical samples. The multiplex real-time PCR methodology established in this study can be employed for the rapid detection of the four most prevalent HFMD-associated EVs, for epidemiologic surveillance of circulating EV genotypes and for assessing treatment responses and vaccine studies., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Hand-foot-mouth disease (HFMD) is an acute infectious disease caused by serotypes of the enterovirus (EV) family. HFMD reinfection occurs commonly in lack of cross-protection between different EV serotypes. In this study, we investigated the hazards of HFMD reinfection using Cox-proportional hazard model. Retrospective data of 95 209 HFMD cases in Wuhan during 2008-2015 was used. Kaplan-Meier survival methods and Cox-proportional hazard model were used to estimate the hazard probabilities. Of the all HFMD cases, about 2% experienced reinfection (1842/95 209). Kaplan-Meier curves revealed the reinfection risk sharply increased before 40 months from first infection. Higher hazards of reinfection were detected among those who were males, aged 3 years and below, scattered children, belonging to urban areas and first infected with coxsackievirus (CV)-A16 compared with their respective counterparts. Cox-proportional hazard model suggested that gender, age, group, living area and serotypes of first infection had significant effect on reinfection even after adjusting for potential confounding effects of other selected factors considered in the study. These results indicate that boys aged 3 years and below, especially those living in urban areas and first infected with CV-A16 are more prone to reinfection. Interventions should be imposed on these high-risk populations.

Introduction: Hand, foot and mouth disease (HFMD) is usually caused by several serotypes from human enterovirus A species, including enterovirus 71 (EV-A71) and coxsackievirus A16 (CV-A16). Two inactivated monovalent EV-A71 vaccines have been recently licensed in China and monovalent CV-A16 vaccine and bivalent EV-A71 and CV-A16 vaccine are under development., Methods: Using notifications from the national surveillance system, we describe the epidemiology and dynamics of HFMD in the country, before the introduction of EV-A71 vaccination, from 2008 through 2015., Results: Laboratory-identified serotype categories, i.e. CV-A16, EV-A71 and other enteroviruses, circulated annually. EV-A71 remained the most virulent serotype and was the major serotype for fatal cases (range: 88.5-95.4%) and severe cases (range: 50.7-82.3%) across years. Except for 2013 and 2015, when other enteroviruses were more frequently found in mild HFMD (48.8% and 52.5%), EV-A71 was more frequently detected from mild cases in the rest of the years covered by the study (range: 39.4-52.6%). The incidence rates and severity risks of HFMD associated with all serotype categories were the highest for children aged 1 year and younger, and decreased with increasing age., Discussion/conclusion: This study provides baseline epidemiology for evaluation of vaccine impact and potential serotype replacement.

Coxsackievirus A10 (CVA10) is one of the major pathogens associated with hand, foot, and mouth disease (HFMD). CVA10 infection can cause herpangina and viral pneumonia, which can be complicated by severe neurological sequelae. The morbidity and mortality of CVA10-associated HFMD have been increasing in recent years, particularly in the pan-Pacific region. There are limited studies, however, on the pathogenesis and immunology of CVA10-associated HFMD infections, and few antiviral drugs or vaccines have been reported. In the present study, a cell-adapted CVA10 strain was employed to inoculate intramuscularly 5-day-old ICR mice, which developed significant clinical signs, including reduced mobility, lower weight gain, and quadriplegia, with significant pathology in the brain, hind limb skeletal muscles, and lungs of infected mice in the moribund state. The severity of illness was associated with abnormally high expression of the proinflammatory cytokine interleukin 6 (IL-6). Antiviral assays demonstrated that ribavirin and gamma interferon administration could significantly inhibit CVA10 replication both in vitro and in vivo In addition, formaldehyde-inactivated CVA10 whole-virus vaccines induced immune responses in adult mice, and maternal neutralizing antibodies could be transmitted to neonatal mice, providing protection against CVA10 clinical strains. Furthermore, high-titer antisera were effective against CVA10 and could relieve early clinical symptoms and improve the survival rates of CVA10-challenged neonatal mice. In summary, we present a novel murine model to study CVA10 pathology that will be extremely useful in developing effective antivirals and vaccines to diminish the burden of HFMD-associated disease. IMPORTANCE Hand, foot, and mouth disease cases in infancy, arising from coxsackievirus A10 (CVA10) infections, are typically benign, resolving without any significant adverse events. Severe disease and fatalities, however, can occur in some children, necessitating the development of vaccines and antiviral therapies. The present study has established a newborn-mouse model of CVA10 that, importantly, recapitulates many aspects of human disease with respect to the neuropathology and skeletal muscle pathology. We found that high levels of the proinflammatory cytokine interleukin 6 correlated with disease severity and that ribavirin and gamma interferon could decrease viral titers in vitro and in vivo Whole-virus vaccines produced immune responses in adult mice, and immunized mothers conferred protection on neonates against challenge from CVA10 clinical strains. Passive immunization with high-titer antisera could also improve survival rates in newborn animals., (Copyright © 2017 American Society for Microbiology.)

Coxsackievirus A16 (CV-A16), CV-A6, and enterovirus D68 (EV-D68) belong to the Picornaviridae family and are major causes of hand, foot, and mouth disease (HFMD) and pediatric respiratory disease worldwide. The biological characteristics of these viruses, especially their interplay with the host innate immune system, have not been well investigated. In this study, we discovered that the 3C pro proteins from CV-A16, CV-A6, and EV-D68 bind melanoma differentiation-associated gene 5 (MDA5) and inhibit its interaction with MAVS. Consequently, MDA5-triggered type I interferon (IFN) signaling in the retinoic acid-inducible gene I-like receptor (RLR) pathway was blocked by the CV-A16, CV-A6, and EV-D68 3C pro proteins. Furthermore, the CV-A16, CV-A6, and EV-D68 3C pro proteins all cleave transforming growth factor β-activated kinase 1 (TAK1), resulting in the inhibition of NF-κB activation, a host response also critical for Toll-like receptor (TLR)-mediated signaling. Thus, our data demonstrate that circulating HFMD-associated CV-A16 and CV-A6, as well as severe respiratory disease-associated EV-D68, have developed novel mechanisms to subvert host innate immune responses by targeting key factors in the RLR and TLR pathways. Blocking the ability of 3C pro proteins from diverse enteroviruses and coxsackieviruses to interfere with type I IFN induction should restore IFN antiviral function, offering a potential novel antiviral strategy. IMPORTANCE CV-A16, CV-A6, and EV-D68 are emerging pathogens associated with hand, foot, and mouth disease and pediatric respiratory disease worldwide. The pathogenic mechanisms of these viruses are largely unknown. Here we demonstrate that the CV-A16, CV-A6, and EV-D68 3C pro proteins block MDA5-triggered type I IFN induction. The 3C pro proteins of these viruses bind MDA5 and inhibit its interaction with MAVS. In addition, the CV-A16, CV-A6, and EV-D68 3C pro proteins cleave TAK1 to inhibit the NF-κB response. Thus, our data demonstrate that circulating HFMD-associated CV-A16 and CV-A6, as well as severe respiratory disease-associated EV-D68, have developed a mechanism to subvert host innate immune responses by simultaneously targeting key factors in the RLR and TLR pathways. These findings indicate the potential merit of targeting the CV-A16, CV-A6, and EV-D68 3C pro proteins as an antiviral strategy., (Copyright © 2017 American Society for Microbiology.)

Enterovirus 96 (EV-96) is a recently described member of the species Enterovirus C and is associated with paralysis and myelitis. In this study, using metagenomic sequencing, we identified a new enterovirus 96 strain (EV-96-SZ/GD/CHN/2014) as the sole pathogen causing hand, foot, and mouth disease (HFMD). A genomic comparison showed that EV-96-SZ/GD/CHN/2014 is most similar to the EV-96-05517 strain (85% identity), which has also been detected in Guangdong Province. This is the first time that metagenomic sequencing has been used to identify an EV-96 strain shown to be associated with HFMD.

Hand, foot, and mouth disease (HFMD) is highly prevalent in China, and more efficient methods of epidemic detection and early warning need to be developed to augment traditional surveillance systems. In this paper, a method that uses Baidu search queries to track and predict HFMD epidemics is presented, and the outbreaks of HFMD in China during the 60-month period from January 2011 to December 2015 are predicted. The Pearson correlation coefficient (R) of the predictive model and the mean absolute percentage errors between observed HFMD case counts and the predicted number show that our predictive model gives excellent fit to the data. This implies that Baidu search queries can be used in China to track and reliably predict HFMD epidemics, and can serve as a supplement to official systems for HFMD epidemic surveillance.

Hand, foot, and mouth disease (HFMD), which has led to millions of attacks and several outbreaks across the world and become more predominant in Asia-Pacific Region, especially in Mainland China, is caused by several Human Enteroviruses including new enterovirus, coxsakievirus and echovirus. In recent years, much research has focused on the epidemiological characteristics of HFMD. In this article, multiple characteristics of HFMD such as basic epidemiology, etiology and molecular epidemiology; influencing factors; detection; and surveillance are reviewed, as these can be help protect high risks groups, prevalence prediction and policy making for disease prevention.

Atypical hand, foot, and mouth disease (aHFMD) outbreaks have been frequently reported worldwide in recent years. It is believed that coxsackievirus A6 (CA6) is the major pathogen for aHFMD. Studies regarding CA6 infection are limited and the genetic mechanism for the high pathogenicity of some new CA6 variants is still unclear. Infectious clones are powerful tools for studying the genetic mechanisms of RNA viruses. In this study, we describe the construction of a full-length cDNA clone of CA6 strain TW-2007-00141. The whole genome of CA6 was amplified in a single step and ligated into a plasmid vector through an efficient cloning method, Gibson assembly. The whole genome sequence of CA6 strain TW-2007-00141 was determined and phylogenetic analysis indicated that it shared a high degree of similarity (≥94%) with the CA6 strains found in Taiwan in 2009. The infectious clone of CA6 viruses were recovered by transfection into 293FT cells and showed similar biological properties to the parental virus. Viral particles were purified by CsCl isopycnic centrifugation, and two types of viral particles were observed under transmission electron microscopy. The rescued virus showed high virulence in one-day-old suckling mice. This clone may be useful for establishing animal models for the evaluation of CA6 vaccine efficiency in future., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Background: Rapid detection and identification of viruses are important for early diagnosis and effective surveillance of hand, foot, and mouth disease (HFMD). We described a novel assay using multilocus PCR and reverse transcription-PCR coupled with electrospray ionization mass spectrometry (RT-PCR/ESI-MS) to simultaneously detect and identify human enterovirus A-D, adenovirus A-F, human herpesvirus 1-8, parvovirus B19 and polyomavirus., Objectives: To evaluate the accuracy and efficacy of the RT-PCR/ESI-MS method, to detect and type enterovirus from specimens of clinical diagnosed HFMD patients., Study Design: In this study, 152 specimens of clinically diagnosed HFMD patients were studied by the assay using RT-PCR/ESI-MS method. The detection and typing of enterovirus by RT-PCR/ESI-MS were compared with results from reference molecular methods., Results: The assay detected enteroviruses in 97 (63.8%) specimens, resulting in a sensitivity of 93.8% (95% CI: 91.8-95.7%) and a specificity of 87.5% (95% CI: 84.8-90.2%) compared with a reference clinical diagnostic test. Most enterovirus genotypes (65/84; 77%) determined by the platform were consistent with 5' UTR sequence analysis, and most misidentifications resulted from the virus library, which could be further improved by updating the enterovirus database. In addition to enteroviruses, herpesviruses, polyomaviruses, adenoviruses and human rhinoviruses were detected and identified in 55 (36%) HFMD specimens by RT-PCR/ESI-MS., Conclusion: With the capability of high throughput and detection and typing of multiple clinically relevant viruses simultaneously, RT-PCR/ESI-MS can be a rapid and robust laboratory tool for identifying viral pathogens., (Copyright © 2013 Elsevier B.V. All rights reserved.)