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Coxsackievirus A6 (CVA6) is a primary pathogen associated with hand, foot, and mouth disease (HFMD) and is typified by fever, rashes or herpetic lesions at distinct locations. Although HFMD patients exhibit mild symptoms, a subset of patients may develop severe complications, such as viral encephalitis, myocarditis, pneumonia, and neurological disorders. However, in addition to rodent models, such as the CVA6-infected mouse model, no definitive nonhuman primate animal model or related research or analysis tool is available, which makes the development of suitable nonhuman primate animal models particularly crucial. In this study, 3- to 4-month-old rhesus monkeys were infected via the respiratory or digestive tract, and the pathogenic, pathological, and immunological alterations following CVA6 infection were investigated. The results revealed that the infected rhesus monkeys exhibited symptoms similar to those of patients, including signs of HFMD, blood cell changes, viremia, viral excretion, and inflammatory reactions during the acute phase (1-11 days). Pathological observations revealed inflammatory reactions in the intestinal and lymph node tissues. Notably, the acute symptoms gradually waned in the recovery phase (12-120 days), and a high level of neutralizing antibodies was sustained. Intriguingly, no significant disparity was observed between the infections initiated via the respiratory or digestive tract in terms of clinical symptoms, hemogram results or virus shedding. Overall, this study yielded a comprehensive dataset regarding the physiological, pathological, and immunological outcomes of CVA6 infection in a primate host, enhancing our comprehension of the mechanism of CVA6 infection and providing essential data for related vaccine and drug development., 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 © 2024 Elsevier Inc. All rights reserved.)

Coxsackievirus A10 (CV-A10) infection, a prominent cause of childhood hand-foot-and-mouth disease (HFMD), frequently manifests with the intriguing phenomenon of onychomadesis, characterized by nail shedding. However, the underlying mechanism is elusive. Here, we found that CV-A10 infection in mice could suppress Wnt/β-catenin signaling by restraining LDL receptor-related protein 6 (LRP6) phosphorylation and β-catenin accumulation and lead to onychomadesis. Mechanistically, CV-A10 mimics Dickkopf-related protein 1 (DKK1) to interact with Kringle-containing transmembrane protein 1 (KRM1), the CV-A10 cellular receptor. We further found that Wnt agonist (GSK3β inhibitor) CHIR99021 can restore nail stem cell differentiation and protect against nail shedding. These findings provide novel insights into the pathogenesis of CV-A10 and related viruses in onychomadesis and guide prognosis assessment and clinical treatment of the disease., (© 2024 Cui et al.)

Coxsackievirus B1 (CVB1), an enterovirus with multiple clinical presentations, has been associated with potential long-term consequences, including hand, foot, and mouth disease (HFMD), in some patients. However, the related animal models, transmission dynamics, and long-term tissue tropism of CVB1 have not been systematically characterized. In this study, we established a model of CVB1 respiratory infection in rhesus macaques and evaluated the clinical symptoms, viral load, and immune levels during the acute phase (0-14 days) and long-term recovery phase (15-30 days). We also investigated the distribution, viral clearance, and pathology during the long-term recovery period using 35 postmortem rhesus macaque tissue samples collected at 30 days postinfection (d.p.i.). The results showed that the infected rhesus macaques were susceptible to CVB1 and exhibited HFMD symptoms, viral clearance, altered cytokine levels, and the presence of neutralizing antibodies. Autopsy revealed positive viral loads in the heart, spleen, pancreas, soft palate, and olfactory bulb tissues. HE staining demonstrated pathological damage to the liver, spleen, lung, soft palate, and tracheal epithelium. At 30 d.p.i., viral antigens were detected in visceral, immune, respiratory, and muscle tissues but not in intestinal or neural tissues. Brain tissue examination revealed viral meningitis-like changes, and CVB1 antigen expression was detected in occipital, pontine, cerebellar, and spinal cord tissues at 30 d.p.i. This study provides the first insights into CVB1 pathogenesis in a nonhuman primate model of HFMD and confirms that CVB1 exhibits tissue tropism following long-term infection., (© 2024 The Author(s). Journal of Medical Virology published by Wiley Periodicals LLC.)

Coxsackievirus A6 (CVA6) is currently considered as a predominant pathogen of hand, foot, and mouth disease (HFMD), and is occasionally linked to myocardial injury. We first established a mouse model of CVA6-induced myocardial injury. Next, we analyzed the immune cell phenotypes CVA6-infected mice hearts by fluorescence-activated cell sorting, and found that CVA6 led to massive neutrophils infiltration, suggesting their potential link with the occurrence of myocardial injury. We further used either αGr-1 or αLy6G antibody to deplete neutrophils, and found that neutrophil-depleted animals showed decreased cardiac enzymes, lower degree of pathology in hearts, and reduced inflammatory cytokine production compared to isotype controls. Finally, we confirmed the involvement of neutrophils in myocardial injury of clinical patients with severe HFMD. Our study suggests that excessive neutrophils contribute to myocardial injury caused by CVA6 infection, which provides new insights into myocardial injury during the development of HFMD severity and the outcome of immune cell-mediated therapies., Competing Interests: Potential conflicts of interest. All authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2023. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The majority of solid testicular tumors are treated with orchiectomy given the high risk of malignancy. We present a case of a testicular mass in an adult patient in the setting of recent hand, foot, and mouth disease that was managed conservatively with serial ultrasounds. Even though cases of viral-associated testicular masses are rare, this differential diagnosis should be considered in patients with a new testicular mass in the setting of recent viral infection and negative tumor markers. For these patients, observation may be an option instead of immediate orchiectomy.

Coxsackievirus B3 (CVB3) is the pathogen causing hand, foot and mouth disease (HFMD), which manifests across a spectrum of clinical severity from mild to severe. However, CVB3-infected mouse models mainly demonstrate viral myocarditis and pancreatitis, failing to replicate human HFMD symptoms. Although several enteroviruses have been evaluated in Syrian hamsters and rhesus monkeys, there is no comprehensive data on CVB3. In this study, we have first tested the susceptibility of Syrian hamsters to CVB3 infection via different routes. The results showed that Syrian hamsters were successfully infected with CVB3 by intraperitoneal injection or nasal drip, leading to nasopharyngeal colonization, acute severe pathological injury, and typical HFMD symptoms. Notably, the nasal drip group exhibited a longer viral excretion cycle and more severe pathological damage. In the subsequent study, rhesus monkeys infected with CVB3 through nasal drips also presented signs of HFMD symptoms, viral excretion, serum antibody conversion, viral nucleic acids and antigens, and the specific organ damages, particularly in the heart. Surprisingly, there were no significant differences in myocardial enzyme levels, and the clinical symptoms resembled those often associated with common, mild infections. In summary, the study successfully developed severe Syrian hamsters and mild rhesus monkey models for CVB3-induced HFMD. These models could serve as a basis for understanding the disease pathogenesis, conducting pre-trial prevention and evaluation, and implementing post-exposure intervention., Competing Interests: Conflict of interest The authors of this study declared that they do not have any conflict of interest., (Copyright © 2024 The Authors. Publishing services by Elsevier B.V. All rights reserved.)

Hand, foot and mouth disease (HFMD) caused by Human Enterovirus A71 (HEVA71) infection is typically a benign infection. However, in minority of cases, children can develop severe neuropathology that culminate in fatality. Approximately 36.9% of HEVA71-related hospitalizations develop neurological complications, of which 10.5% are fatal. Yet, the mechanism by which HEVA71 induces these neurological deficits remain unclear. Here, we show that HEVA71-infected astrocytes release CXCL1 which supports viral replication in neurons by activating the CXCR2 receptor-associated ERK1/2 signaling pathway. Elevated CXCL1 levels correlates with disease severity in a HEVA71-infected mice model. In humans infected with HEVA71, high CXCL1 levels are only present in patients presenting neurological complications. CXCL1 release is specifically triggered by VP4 synthesis in HEVA71-infected astrocytes, which then acts via its receptor CXCR2 to enhance viral replication in neurons. Perturbing CXCL1 signaling or VP4 myristylation strongly attenuates viral replication. Treatment with AZD5069, a CXCL1-specific competitor, improves survival and lessens disease severity in infected animals. Collectively, these results highlight the CXCL1-CXCR2 signaling pathway as a potential target against HFMD neuropathogenesis., (© 2022. The Author(s).)

Background: This research aimed to explore the association between the RIG-I-like receptor (RIG-I and MDA5 encoded by DDX58 and IFIH1, respectively) pathways and the risk or severity of hand, foot, and mouth disease caused by enterovirus 71 (EV71-HFMD). In this context, we explored the influence of gene methylation and polymorphism on EV71-HFMD., Methodology/principal Findings: 60 healthy controls and 120 EV71-HFMD patients, including 60 mild EV71-HFMD and 60 severe EV71-HFMD patients, were enrolled. First, MiSeq was performed to explore the methylation of CpG islands in the DDX58 and IFIH1 promoter regions. Then, DDX58 and IFIH1 expression were detected in PBMCs using RT-qPCR. Finally, imLDR was used to detect DDX58 and IFIH1 single-nucleotide polymorphism (SNP) genotypes. Severe EV71-HFMD patients exhibited higher DDX58 promoter methylation levels than healthy controls and mild EV71-HFMD patients. DDX58 promoter methylation was significantly associated with severe HFMD, sex, vomiting, high fever, neutrophil abundance, and lymphocyte abundance. DDX58 expression levels were significantly lower in mild patients than in healthy controls and lower in severe patients than in mild patients. Binary logistic regression analysis revealed statistically significant differences in the genotype frequencies of DDX58 rs3739674 between the mild and severe groups. GeneMANIA revealed that 19 proteins displayed correlations with DDX58, including DHX58, HERC5, MAVS, RAI14, WRNIP1 and ISG15, and 19 proteins displayed correlations with IFIH1, including TKFC, IDE, MAVS, DHX58, NLRC5, TSPAN6, USP3 and DDX58., Conclusions/significance: DDX58 expression and promoter methylation were associated with EV71 infection progression, especially in severe EV71-HFMD patients. The effect of DDX58 in EV71-HFMD is worth further attention., Competing Interests: The authors declare that they have no competing interests.

Hand, foot, and mouth disease (HFMD) caused by enterovirus A71 (EV-A71) is a contagious viral disease, and toll-like receptors (TLRs) play essential roles in resisting the pathogen. The aim of this study was to assess the potential relationship between several TLRs polymorphisms and the HFMD severity in a Chinese children population. A total of 328 Chinese children with HFMD were included in the present study. The polymorphisms of TLR3 (rs3775290, rs3775291, rs3775296, rs1879026, rs5743312, rs5743313, rs5743303, rs13126816, and rs3775292), TLR4 (rs4986790, rs4986791, rs2149356, rs11536889, and rs41426344), TLR7 (rs179009, rs179010, rs179016, rs3853839, rs2302267, rs1634323, and rs5741880), and TLR8 (rs3764880, rs2159377, rs2407992, rs5744080, rs3747414, rs3764879, and rs5744069) genes were selected. The study indicated that individuals with the GG genotype of TLR3 single-nucleotide polymorphism rs1879026 had a higher risk of developing severe cases (GG vs. GT: OR = 1.875; 95% CI, 1.183-2.971; p = .007). Meanwhile, TLR3 rs3775290 CC genotype and C allele were associated with lower disease severity in females (CC vs. CT: OR = 0.350; 95% CI, 0.163-0.751; p = .006; C vs. T: OR = 0.566; 95% CI, 0.332-0.965; p = .036). TLR3 rs3775291 CC genotype showed 2.537 folds higher risk of developing severe cases in females (CC vs. CT: OR = 2.537; 95% CI, 1.108-5.806; p = .026). Moreover, TLR3 rs1879026 GG genotype was found to be related to increased risk of severe cases in males (GG vs. GT: OR = 2.076; 95% CI, 1.144-3.768; p = .016). The current findings show that the genetic variants of TLR3 rs1879026, rs3775290, and rs3775291 are associated with the severity of EV-A71-associated HFMD in a Chinese children population., (© 2021 Wiley Periodicals LLC.)

Sporadic occurrences and outbreaks of hand, foot, and mouth disease (HFMD) caused by Coxsackievirus A2 (CVA2) have frequently reported worldwide recently, which pose a great challenge to public health. Epidemiological studies have suggested that the main cause of death in critical patients is pulmonary edema. However, the pathogenesis of this underlying comorbidity remains unclear. In this study, we utilized the 5-day-old BALB/c mouse model of lethal CVA2 infection to evaluate lung damage. We found that the permeability of lung microvascular was significantly increased after CVA2 infection. We also observed the direct infection and apoptosis of lung endothelial cells as well as the destruction of tight junctions between endothelial cells. CVA2 infection led to the degradation of tight junction proteins (e.g., ZO-1, claudin-5, and occludin). The gene transcription levels of von Willebrand factor (vWF), endothelin (ET), thrombomodulin (THBD), granular membrane protein 140 (GMP140), and intercellular cell adhesion molecule-1 (ICAM-1) related to endothelial dysfunction were all significantly increased. Additionally, CVA2 infection induced the increased expression of inflammatory cytokines (IL-6, IL-1β, and MCP-1) and the activation of p38 mitogen-activated protein kinase (MAPK). In conclusion, the disruption of the endothelial barrier contributes to acute lung injury induced by CVA2 infection; targeting p38-MAPK signaling may provide a therapeutic approach for pulmonary edema in critical infections of HFMD.

Severe hand, foot, and mouth disease (HFMD) caused by enterovirus 71 (EV71) infection is associated with high mortality and disability. DC-SIGN, a receptor for EV71, is widely distributed in dendritic cells and may influence the severity of HFMD caused by EV71 infection. This observational study attempts to explore whether single-nucleotide polymorphisms (SNPs) in DC-SIGN are related to the severity of EV71-associated HFMD. Based on linkage disequilibrium and functional predictions, two DC-SIGN SNPs were selected and tested to explore their potential association with the severity of HFMD caused by EV71 infection. Two hundred sixteen Han Chinese children with HFMD caused by EV71 were enrolled to obtain clinical data, including the severity of HFMD, serum DC-SIGN levels, and DC-SIGN SNPs. We found a significant association between the rs7248637 polymorphism (A vs. G: OR = 0.644, 95% CI = 0.515-0.806) and the severity of HFMD caused by EV71 infection, as well as the rs4804800 polymorphism (A vs. G: OR = 1.539, 95% CI =1.229-1.928). These two DC-SIGN SNPs may have an effect on the severity of HFMD caused by EV71 infection.

Children with HFMD due to EV71 infection are more likely to suffer from neurogenic complications, leading to higher morbidity and mortality. ILCs play crucial roles in the initiation of host immunity. However, the contribution of ILCs to the occurrence and development of HFMD due to EV71 infection remains to be explored. The results of our study showed that the levels of peripheral ILC1s and Th1 cells were increased in children with severe HFMD compared to healthy children, as were ILC1- and Th1-related cytokines and transcription factors. Furthermore, HFMD children with a higher frequency of circulating ILC1s exhibited a 2.9-fold greater risk of severity when HFMD was accompanied by VEM. Our study is the first to show that ILC1 abnormalities contribute to the pathogenesis of the severity of HFMD, in which ILC1s are aberrant increased and affect the cellular and humoral immunity. ILC1s could be used in the diagnosis of HFMD., (Copyright © 2020. Published by Elsevier Inc.)

Enterovirus 71 (EV-A71) and coxsackievirus A16 (CV-A16) are the major pathogens responsible for hand, foot and mouth disease (HFMD), but the mechanism by which these viruses cause disease remains unclear. In this study, we used transcriptome sequencing technology to investigate changes in the transcriptome profiles after infection with EV-A71 and CV-A16 in human bronchial epithelial (16HBE) cells. Using systematic bioinformatics analysis, we then searched for useful clues regarding the pathogenesis of HFMD. As a result, a total of 111 common differentially expressed genes were present in both EV-A71- and CV-A16-infected cells. A trend analysis of these 111 genes showed that 91 of them displayed the same trend in EV-A71 and CV-A16 infection, including 49 upregulated genes and 42 downregulated genes. These 91 genes were further used to conduct GO, pathway, and coexpression network analysis. It was discovered that enriched GO terms (such as histone acetylation and positive regulation of phosphorylation) and pathways (such as glycosylphosphatidylinositol (GPI)-anchor biosynthesis and DNA replication) might be closely associated with the pathogenic mechanism of these two viruses, and key genes (such as TBCK and GPC) might be involved in the progression of HFMD. Finally, we randomly selected 10 differentially expressed genes for qRT-PCR to validate the transcriptome sequencing data. The experimental qRT-PCR results were roughly in agreement with the results of transcriptome sequencing. Collectively, our results provide clues to the mechanism of pathogenesis of HFMD induced by EV-A71 and CV-A16.

Hand, foot, and mouth disease (HFMD) is most frequently caused by several serotypes of human enterovirus (EV) including Enterovirus 71 (EV-A71), coxsackievirus A16 (CV-A16), or other types of EV. The aim of this study was to determine the epidemiological characteristics of HFMD and to describe the epidemiologic characteristics of HFMD among severe and mild cases. We collected 4760 HFMD cases in Hangzhou from 2016 to 2018. Specimens from these cases were collected and tested for EV-A71, CV-A16, CV-A6, CV-A10, CV-A2, and CV-A5 by reverse transcriptase polymerase chain reaction. From 2016 to 2018, the prevalence of HFMD was seasonal each year. Among the 4760 probable HFMD cases, 3559 cases were confirmed (74.8%), including 426 cases of EV-A71 infections (8.9%), 249 cases of CV-A16 infections (5.2%), and 2884 cases of other EV infections (60.6%). The percentage of other EV infections was more than 80%, which increased year by year. Random selection of samples for detection of other EV infections in 2017 and 2018, among the 1297 cases, showed there were 835 (64.4%) cases of CV-A6 infections, 177 (13.6%) cases of CV-A10 infections, 100 (7.7%) cases of CV-A2 infections, 40 (3.1%) cases of CV-A5 infections, 3 (0.02 %) cases of mixed infections, and 11.0% untyped EV infections. Preschool children were still the primary population susceptible to HFMD. In severe cases, EV-A71 infection was the main cause. Characterizing the epidemiology and the relationship between severe and common cases of HFMD would provide relevant evidences for the prevention and treatment of HFMD.

Enterovirus 71 (EV71) is a causative agent of hand, foot, and mouth disease (HFMD). However, this infection is sometimes associated with severe neurological complications. Identification of neurovirulence determinants is important to understand the pathogenesis of EV71. One of the problems in evaluating EV71 virulence is that its genome sequence changes rapidly during replication in cultured cells. The factors that induce rapid mutations in the EV71 genome in cultured cells are unclear. Here, we illustrate the population dynamics during adaptation to RD-A cells using EV71 strains isolated from HFMD patients. We identified a reproducible amino acid substitution from glutamic acid (E) to glycine (G) or glutamine (Q) in residue 145 of the VP1 protein (VP1-145) after adaptation to RD-A cells, which was associated with attenuation in human scavenger receptor B2 transgenic (hSCARB2 tg) mice. Because previous reports demonstrated that VP1-145G and Q mutants efficiently infect cultured cells by binding to heparan sulfate (HS), we hypothesized that HS expressed on the cell surface is a major factor for this selection. Supporting this hypothesis, selection of the VP1-145 mutant was prevented by depletion of HS and overexpression of hSCARB2 in RD-A cells. In addition, this mutation promotes the acquisition of secondary amino acid substitutions at various positions of the EV71 capsid to increase its fitness in cultured cells. These results indicate that attachment receptors, especially HS, are important factors for selection of VP1-145 mutants and subsequent capsid mutations. Moreover, we offer an efficient method for isolation and propagation of EV71 virulent strains with minimal selection pressure for attenuation., Competing Interests: The authors have declared that no competing interests exist.

Enterovirus 71 (EV71) is a causative agent of hand-foot-mouth disease, and it sometimes causes severe neurological disease. Development of effective vaccines and animal models to evaluate vaccine candidates are needed. However, the animal models currently used for vaccine efficacy testing, monkeys and neonatal mice, have economic, ethical, and practical drawbacks. In addition, EV71 strains prepared for lethal challenge often develop decreased virulence during propagation in cell culture. To overcome these problems, we used a mouse model expressing human scavenger receptor B2 (hSCARB2) that showed lifelong susceptibility to EV71. We selected virulent EV71 strains belonging to the subgenogroups B4, B5, C1, C2, and C4 and propagated them using a culture method for EV71 without an apparent reduction in virulence. Here, we describe a novel EV71 vaccine efficacy test based on these hSCARB2 transgenic (Tg) mice and these virulent viruses. Adult Tg mice were immunized subcutaneously with formalin-inactivated EV71. The vaccine elicited sufficient levels of neutralizing antibodies in the immunized mice. The mice were subjected to lethal challenge with virulent viruses via intravenous injection. Survival, clinical signs, and body weight changes were observed for 2 weeks. Most immunized mice survived without clinical signs or histopathological lesions. The viral replication in immunized mice was much lower than that in nonimmunized mice. Mice immunized with the EV71 vaccine were only partially protected against lethal challenge with coxsackievirus A16. These results indicate that this new model is useful for in vivo EV71 vaccine efficacy testing. IMPORTANCE The development of new vaccines for EV71 relies on the availability of small animal models suitable for in vivo efficacy testing. Monkeys and neonatal mice have been used, but the use of these animals has several drawbacks, including high costs, limited susceptibility, and poor experimental reproducibility. In addition, the related ethical issues are considerable. The new efficacy test based on hSCARB2 Tg mice and virulent EV71 strains propagated in genetically modified cell lines presented here can overcome these disadvantages and is expected to accelerate the development of new EV71 vaccines., (Copyright © 2020 American Society for Microbiology.)

In recent years, outbreaks of hand-foot-mouth disease (HFMD) in China, Singapore and other Western Pacific Region, involving millions of children, have become a big threat to public health. This study aimed to quantitatively assess all qualified studies and identify the risk factors for HFMD death. A systematic search of the databases PubMed, Medline, Embase and the Cochrane Library was performed. Study heterogeneity and publication bias were estimated. Seven case-control studies involving 1641 participants (634 died and 1007 survived) were included in the meta-analysis. Human enterovirus 71 infection, male, age ⩽3 years, vomiting, cyanosis, convulsion, duration of fever ⩾3 days, atypical rashes and abdominal distention were not significantly related to HFMD death (P ⩽ 0.05). Lethargy (odds ratio (OR) = 6.62; 95% CI 3.61-12.14; I2 = 0%; P < 0.0001), pneumonoedema/pneumorrhagia (OR = 4.09; 95% CI 2.44-6.87; I2 = 0%; P < 0.0001), seizures (OR = 6.85; 95% CI 2.37-19.74; I2 = 0%; P = 0.0004), dyspnoea (OR = 8.24; 95% CI 2.05-33.19; I2 = 83%; P = 0.003) and coma (OR = 3.76; 95% CI 1.85-7.67; I2 = 0%; P = 0.0003) were significantly associated with HFMD death, which were risk factors for HFMD death.

Enterovirus 71 (EV-A71) is one of the major causative agents of hand, foot, and mouth disease. EV-A71 infection is sometimes associated with severe neurological diseases such as acute encephalitis, acute flaccid paralysis, and cardiopulmonary failure. Therefore, EV-A71 is a serious public health concern. Scavenger receptor class B, member 2 (SCARB2) is a type III transmembrane protein that belongs to the CD36 family and is a major receptor for EV-A71. SCARB2 supports attachment and internalization of the virus and initiates conformational changes that lead to uncoating of viral RNA in the cytoplasm. The three-dimensional structure of the virus-receptor complex was elucidated by cryo-electron microscopy. Two α-helices in the head domain of SCARB2 bind to the G-H loop of VP1 and the E-F loop of VP2 capsid proteins of EV-A71. Uncoating takes place in a SCARB2- and low pH-dependent manner. In addition to SCARB2, other molecules support cell surface binding of EV-A71. Heparan sulfate proteoglycans, P-selectin glycoprotein ligand-1, sialylated glycan, annexin II, vimentin, fibronectin, and prohibitin enhance viral infection by retaining the virus on the cell surface. These molecules are known as "attachment receptors" because they cannot initiate uncoating. In vivo, SCARB2 expression was observed in EV-A71 antigen-positive neurons and epithelial cells in the crypts of the palatine tonsils in patients that died of EV-A71 infection. Adult mice are not susceptible to infection by EV-A71, but transgenic mice that express human SCARB2 become susceptible to EV-A71 infection and develop neurological diseases similar to those observed in humans. Attachment receptors may also be involved in EV-A71 infection in vivo. Although heparan sulfate proteoglycans are expressed by many cultured cell lines and enhance infection by a subset of EV-A71 strains, they are not expressed by cells that express SCARB2 at high levels in vivo. Thus, heparan sulfate-positive cells merely adsorb the virus and do not contribute to replication or dissemination of the virus in vivo. In addition to these attachment receptors, cyclophilin A and human tryptophanyl aminoacyl-tRNA synthetase act as an uncoating regulator and an entry mediator that can confer susceptibility to non-susceptibile cells in the absence of SCARB2, respectively. The roles of attachment receptors and other molecules in EV-A71 pathogenesis remain to be elucidated.

An atypical variant of hand-foot-mouth disease (HFMD) has sporadically been reported in recent years, with outbreaks in Europe, Asia, the USA and South America. A new lineage of Coxsackie virus A6 has been identified as the causative agent, a virus-type belonging to the group of enteroviruses. HFMD is transmitted through droplet infection or through faecal-oral transmission. The disease may begin with a prodromal stage and is often accompanied by fever and malaise. Typical skin findings include a papular and vesiculobullous exanthema that might be accompanied by confluent blisters (bullae), crusting, and ulceration. In contrast to "classic" HFMD, predilection sites include the dorsal aspects of the hands and feet, forearms, lower legs, neck and trunk. Oral lesions may be present, but are less often seen compared to "classic" HFMD. The course of the disease is self-limiting, with complete resolution usually within 7-14 days after disease onset. The treatment of atypical HFMD is usually symptomatic. A diagnosis of atypical HFMD might be challenging due to the polymorphous presentation of the disease. This review describes a rarely reported but more frequently diagnosed viral condition.

Hand-foot-mouth disease (HFMD) is an acute intestinal virus infectious disease which is one of major public health problems in mainland China. Previous studies indicated that HFMD was significantly influenced by climatic factors, but the associated factors were different in different areas and few study on HFMD forecast models was conducted. Here, we analyzed epidemiological characteristics of HFMD in Yiwu City, Zhejiang Province and constructed three forecast models. Overall, a total of 32554 HFMD cases were reported and 12 cases deceased in Yiwu City, Zhejiang Province. The incidence of HFMD peaked every other year and the curve of HFMD incidence had an approximately W-shape. The majority of HFMD cases were children and 95.76% cases aged ≤5 years old from 2008 to 2016. Furthermore, we constructed and compared three forecast models using autoregressive integrated moving average (ARIMA) model, negative binomial regression model (NBM), and quasi-Poisson generalized additive model (GAM). All the three models had high agreements between predicted values and observed values, while GAM fitted best. The exposure-response curve of monthly mean temperature and HFMD was approximately V-shaped. Our study explored epidemiological characteristics of HFMD in Yiwu City and provided accurate methods for early warning which would be great importance for the control and prevention of HFMD.

An atypical clinical variant of hand-foot-and-mouth disease (HFMD) with more extensive lesions and affecting adults has emerged during the past years, usually associated to the Coxsackievirus serotype A6 (CV-A6). We present a 19-year-old woman with a 3-day evolution eruption of papulovesicular lesions, which first appeared around the mouth and frontal area and rapidly spread. In addition, we present a 61-year-old man with a 4-day evolution asymptomatic eruption of papulovesicular lesions in both the hands and feet after suffering a cold 1 week before. Skin biopsies of both patients showed intraepidermal vesicles with spongiosis and ballooning, leading to reticular degeneration, apoptotic keratinocytes, and epidermal necrosis of the upper layers with neutrophil sloughing. Immunohistochemical studies for Coxsackie, Enterovirus, herpes virus, adenovirus, and measles were all negative. Cultures of blister fluid, reverse transcription polymerase chain reaction of skin biopsies, blood tests and serologies for exanthematic virus, and serum viral arrays were also negative. Only reverse transcription polymerase chain reaction of blister fluid confirmed Cocksakie A6. In conclusion, immunohistochemical studies with the commercially available viral antibodies do not seem to be useful in atypical HFMD cases. In these cases, to determine the typical histopathological features in HE is the fastest diagnostic aid.

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 most cases, the cause of hand, foot, and mouth disease (HFMD) is coxsackievirus A type 16. The infection can also be caused by other strains of coxsackievirus, spreading mainly by the oral-fecal route, while it is less likely to be transmitted through secretions. HFMD occurs mainly in summer and is more common in children under ten. Skin lesions develop during the disease but rarely become necrotic. When present, they are a severe complication requiring hospitalization. This paper reports the case of a patient with HFMD who developed necrotic mucocutaneous lesions that responded favorably to intravenous acyclovir, fluids, and electrolyte support therapy.

Hand, foot and mouth disease (HFMD) is an infectious disease caused by a variety of enterovirus infections, and the most common types of virus infections are the newenterovirus71 (EV71) and coxsackievirus A group 16 (CoxA16). A small fraction of HFMD will cause further severe HFMD. A rapid and accurate diagnosis biomarker of severe HFMD is important for the timely treatment. In the study, we conducted a clinical biomarker discovery study using iTRAQ combined with MS. Serum proteome alterations in severe HFMD group (n = 32) and health control group (n = 32) were analyzed. 47 proteins were upregulated (fold change > 1.5) between the severe HFMD group and HC group. The identified proteins were classified into different groups according to the molecular function, biology processes, cellular component. During the up-regulated proteins, serum amyloid A (SAA) and human β-actin (ACTB), were confirmed in the serum of the severe HFMD and HC by ELISA assay. SAA and ACTB levels were significantly higher in the sever HFMD patients (P < .01), consistent with iTRAQ-LC-MS/MS analysis. In summary, Our results showed that SAA and human β-actin (ACTB) may be served as a potential biomarker of the clinical diagnosis of severe HFMD., (Copyright © 2019. Published by Elsevier Inc.)

Hand, foot, and mouth disease (HFMD) is a common infectious disease caused by enteroviruses (EVs). In this study, a total of 341 children with serious HFMD were admitted to a pediatric hospital in Yunnan, China in 2012 to 2016. EVs were detected in 283 specimens (83.0%) and were assigned to 17 EV types. Enterovirus A71 (EV-A71) was predominant, accounting for 41.6%, and was followed by coxsackievirus A16 (CV-A16; 18.8%), CV-A6 (9.1%), CV-A10 and E-9 (2.9%), CV-B5 (1.8%), CV-A9 (1.2%), E-30 (0.9%), E-18, CV-A4, C-B3, and CV-A2 (0.6%) and other EV types such as CV-A8, CV-A14, E-14, E-11, and CV-B4 (0.3%). All of the EV-A71 isolates belonged to C4a; the CV-A16 belonged to B1b or B1a, although the B1b strains were predominant; and CV-A6 belonged to D3. In 2012 to 2014, E-9 was the third most frequent serotype (8.2%, 5.0%, and 6.5%, respectively). E-9 was not detected in 2015 and 2016. CV-A6 was not detected in 2012 but was the second most frequent serotype (25.3%) in 2015. Active etiological surveillance of HFMD makes it necessary to be aware of these emerging pathogens., (© 2019 Wiley Periodicals, Inc.)

Enterovirus A71 (EV-A71) is known for its manifestation as hand foot and mouth disease (HFMD), which has caused countless large-scale epidemic outbreaks throughout the world. However, the molecular pathogenesis of EV-A71 infection is still elusive. Previous studies found that the biological characteristics of a mild EV-A71 strain (SDLY1) and a severe EV-A71 strain (SDLY107) are significantly different, and sequence analysis showed that there are several differences in nucleotide sites of UTRs (88 nt, 123 nt, 143 nt, 154 nt, 187 nt, 241 nt, 243 nt, 253 nt, 291 nt, 438 nt, 440 nt, 571 nt, 579 nt, 602 nt, 658 nt, 664 nt, 690 nt, 696 nt, 7328 nt, 7335 nt, 7367 nt, and 7395 nt). The aim of this study was to determine whether these amino sites in UTRs are associated with the pathogenesis of EV-A71 and are responsible for different clinical manifestations. Based on the reverse genetics technology, we rescued two chimeric viruses SDLY107(1-5'UTR) and SDLY107(1-3'UTR) by replacing 5'UTR/3'UTR gene fragments of an infectious cDNA clone. Replication kinetics and cytotoxicity assays showed that the virulence of the two chimeric strains significantly changed in vitro. The viral loads of the two chimeric strains in infected ICR mice were reduced and pathological damage in the brains, lungs, intestinal tissues, and muscles were lightened. Our findings suggest that some nucleotide sites in UTRs may have a function in the pathogenicity and virulence of EV-A71., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Objective: Early recognition of children with severe Hand-Foot-and-Mouth disease (HFMD) is especially important, as severe cases are associated with poor prognosis. To accomplish this, authors designed a quantitative assessment tool to build a nomogram to assist in clinical diagnosis., Methods: A total of 2332 HFMD patients were enrolled in this study; 1750 cases in the mild group and 582 cases in the severe group. Analysis of all of the data was performed using R software version 3.4.3. Multivariate logistic regression was utilized to screen predictors to construct a nomogram model. Finally, predictive performance of the model was evaluated using a receiver operating characteristic (ROC) curve and classifier calibration plot., Results: A nomogram was constructed with five variables: age, peak temperature, fever duration, pathogen, and vomiting. For the nomogram, the area under the curve was 0.87, and the model prediction accuracy rate was 85.2%. Depending upon the comparison of the area under the ROC curve, the nomogram model was superior to the traditional pediatric clinical illness score (PCIS). With the help of the Hosmer-Lemeshow test and resampling model calibration curve, the fitting performance of the nomogram was stable., Conclusions: With advantages such as simplicity, intuitiveness, and practicality, the nomogram (including age, peak temperature, fever duration, pathogen, and vomiting) is capable of predicting severe HFMD and has certain auxiliary value in clinical applications.

Severe hand, foot, and mouth disease (HFMD) is sometimes associated with critical complications that can cause substantial child mortality. Activity of the vitamin D receptor (VDR) may influence the outcomes of enterovirus 71 (EV71) infection. This case-control study aimed to assess the association of single-nucleotide polymorphisms (SNPs) in the gene encoding the VDR with the severity of EV71-associated HFMD. We selected four VDR SNPs based on linkage disequilibrium and functional prediction, and we tested them using the SNPscan multiple SNP typing method for potential association with severity of EV71-associated HFMD. We found a significant association in the case of rs11574129 (G vs A: odds ratio (OR), 0.3439; 95% confidence intervals (CI), 0.1778-0.6653) and rs739837 (T vs G: OR, 0.5580; 95%CI, 0.3352-0.9291). Our results suggest that these two SNPs may influence the severity of EV71-associated HFMD., (© 2018 Wiley Periodicals, Inc.)

Background: Hand-foot-mouth disease (HFMD) is a common contagious viral infection usually affecting infants and children. Recently, literature on HFMD in adults is increasing. It has been reported that adults often present with unusual exanthems with similarities to erythema multiforme (EM). No study has so far compared the histologic features of HFMD with those of EM., Methods: Histopathologic features in 7 biopsies of 6 adult patients with HFMD are compared with biopsies from 9 patients with EM to identify the best criteria for differentiation., Results: HFMD and EM both have a perivascular lymphocytic infiltrate together with epidermal necrosis, spongiosis, ballooning, and reticular alteration. However, large numbers of neutrophils in parakeratosis (P < 0.05), in the viable epidermis (P < 001), or neutrophils forming intraepidermal collections (P < 0.05) are significantly more common in HFMD. In HFMD, necrotic keratinocytes are emphasized in the upper third of the epidermis (P < 0.05), whereas the lower third is typically involved more in EM. Neutrophils are significantly more common and more numerous in the dermal infiltrate of HFMD compared with EM (P < 0.01) Extravasated erythrocytes in the epidermis are more common and more numerous in HFMD (P < 0.05)., Conclusions: Lesions of EM and HFMD can be differentiated based on the amount of neutrophils in the epidermis and in the dermal infiltrate, which are significantly more numerous in HFMD. In addition, necrosis is emphasized in the upper part of the epidermis in HFMD and in the lower part of it in EM.

Coxsackievirus A16 (CV-A16) commonly causes mild symptoms, but severe diseases, such as aseptic meningitis, encephalitis, and even fatal cases, have been reported. Thirteen CV-A16 strains were isolated from patients with severe hand, foot, and mouth disease in Yunnan, Southwest China, from 2009 to 2015. Subgenotype B1a and B1b of CV-A16 were predominantly circulating the region with B1b the predominant strain in recent years. The mean rate of nucleotide substitution based on the VP1 gene sequence was 4.545 × 10 -3  substitution per site per year from 2009 to 2015. These results may help in understanding the genetic diversity of CV-A16 and develop a CV-A16 vaccine., (© 2018 Wiley Periodicals, Inc.)

Rationale: Enterovirus 71 (EV71) is identified as the primary cause of hand, foot, and mouth disease (HFMD) and mainly infects the young infants. Though some fatal cases have been reported, the underlying mechanisms of EV71 infection remain elusive and more further pathologic and molecular studies of EV71 infection are needed., Patient Concerns: A 26-month-old girl with a history of fever and lethargy for 3 days and intermittent seizures for 2 hours associated with rash on 4 limbs was brought to a hospital., Diagnoses: The autopsy was performed to identify the cause of death for a medical dispute. The results of histologic examination, immunohistochemistry (IHC), nested reverse transcription polymerase chain reaction (RT-PCR), and viral isolation confirmed that this patient died of EV71 infection., Interventions: The patient was transferred to neonatal intensive care unit and was intubated and mechanically ventilated. The other treatment included cardiopulmonary resuscitation and intravenous injection of adrenaline., Outcomes: The patient presented persistent coma and intermittent seizures and suddenly developed respiratory arrest and died 16 hours after admission., Lessons: Our results suggest that EV71 might invade into the central nervous system (CNS) through peripheral nerves which control the digestive tract in the early stage of infection. In addition, we successfully isolated one EV71 strain. Phylogenetic analysis showed that the isolated strain clustered in the C4a of C4 subgenotype. This case also highlights that rapid deterioration in HFMD cases is still a challenge to physicians and they must pay special attention to the infants with HFMD symptoms, particularly in EV71 epidemic areas for early diagnosis and treatment.

Enterovirus A71 (EV-A71) infection can cause hand, foot and mouth disease (HFMD), and even fatal meningoencephalitis. Unfortunately, there is currently no effective treatment for EV-A71 infection due to the lack of understanding of the mechanism of neurological diseases. In this study, we employed SH-SY5Y human neuroblastoma cells to explore the roles of caspase-1 in neuropathogenesis. The expression and activity of caspase-1 were analyzed. The potential immuneconsequences mediated by caspase-1 including cell death, lysis, DNA degradation, and secretion of pro-inflammatory were also examined. We found the gene expression levels of caspase-1, IL-1β, IL-18 and active caspase-1 were markedly increased in the SH-SY5Y cells at 48 h post EV-A71 infection. The cell death, lysis, and DNA degradation were also increased during infection, which could be significantly alleviated by caspase-1 inhibition. These observations provided additional experimental evidence supporting caspase-1-mediated pyroptosis as a novel pathway of inflammatory programmed cell death., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Enterovirus 71 (EV71) is the main pathogen of hand-foot-mouth disease (HFMD) and causes several neurological complications. As new strains of EV71 are constantly discovered, it is important to understand the genomic characteristics of the viruses and the mechanism of virulence. Herein, we isolated five strains of EV71 from HFMD patients with or without neurovirulence and sequenced their whole genomes. We then performed whole genome sequence analysis of totally 36 EV71 strains. The phylogenetic analysis of the VP1 region revealed all five isolated strains are clustered into C4a of C4 subgenotype. In addition, by comparing the complete genome sequences of 36 strains, 253 variable amino acid positions were found, 14 of which were identified to be associated with neurovirulence (P < 0.05). Moreover, a similar pattern of amino acid variants combination was identified in four strains without neurovirulence, indicating this type of variant pattern might be associated with avirulence. The strains with neurovirulence appeared to be distinguished from those without neurovirulence by the variants in VP1 and P2 regions, implying VP1 and P2 are the important regions associated with neurovirulence. Indeed, 3-D modeling of VP1 and P2 regions of non-neurovirulent and neurovirulent strains revealed that the different variants resulted in different protein structures and amino acid composition of ligand binding site, which might account for their difference in neurovirulence. In summary, our study reveals 14 variable amino acid positions of VP1, P2 and P3 regions are related to the virulence and that mutations in the capsid proteins of EV71 might contribute to neurovirulence., (© 2018 Wiley Periodicals, Inc.)

Enterovirus 71 (EV-A71) is a major etiological agent of hand, foot and mouth disease (HFMD) that mainly affects young children less than five years old. The onset of severe HFMD is due to neurological complications bringing about acute flaccid paralysis and pulmonary oedema. In this review, we address how genetic events such as recombination and spontaneous mutations could change the genomic organization of EV-A71, leading to an impact on viral virulence. An understanding of the recombination mechanism of the poliovirus and non-polio enteroviruses will provide further evidence of the emergence of novel strains responsible for fatal HFMD outbreaks. We aim to see if the virulence of EV-A71 is contributed solely by the presence of fatal strains or is due to the co-operation of quasispecies within a viral population. The phenomenon of quasispecies within the poliovirus is discussed to reflect viral fitness, virulence and its implications for EV-A71. Ultimately, this review gives an insight into the evolution patterns of EV-A71 by looking into its recombination history and how spontaneous mutations would affect its virulence.

Coxsackievirus A6 (CV-A6) is an emerging pathogen associated with hand, foot, and mouth disease (HFMD). Its genetic characterization and pathogenic properties are largely unknown. Here, we report 39 circulating CV-A6 strains isolated in 2013 from HFMD patients in northeast China. Three major clusters of CV-A6 were identified and related to CV-A6, mostly from Shanghai, indicating that domestic CV-A6 strains were responsible for HFMD emerging in northeast China. Four full-length CV-A6 genomes representing each cluster were sequenced and analyzed further. Bootscanning tests indicated that all four CV-A6-Changchun strains were most likely recombinants between the CV-A6 prototype Gdula and prototype CV-A4 or CV-A4-related viruses, while the recombination pattern was related to, yet distinct from, the strains isolated from other regions of China. Furthermore, different CV-A6 strains showed different capabilities of viral replication, release, and pathogenesis in a mouse model. Further analyses indicated that viral protein 2C contributed to the diverse pathogenic abilities of CV-A6 by causing autophagy and inducing cell death. To our knowledge, this study is the first to report lethal and nonlethal strains of CV-A6 associated with HFMD. The 2C protein region may play a key role in the pathogenicity of CV-A6 strains. IMPORTANCE Hand, foot, and mouth disease (HFMD) is a major and persistent threat to infants and children. Besides the most common pathogens, such as enterovirus A71 (EV-A71) and coxsackievirus A16 (CV-A16), other enteroviruses are increasingly contributing to HFMD. The present study focused on the recently emerged CV-A6 strain. We found that CV-A6 strains isolated in Changchun City in northeast China were associated with domestic origins. These Changchun viruses were novel recombinants of the CV-A6 prototype Gdula and CV-A4. Our results imply that measures to control CV-A6 transmission are urgently needed. Further analyses revealed differing pathogenicities in strains isolated in a neonatal mouse model. One of the possible causes has been narrowed down to the viral protein 2C, using phylogenetic studies, viral sequences, and direct tests on cultured human cells. Thus, the viral 2C protein is a promising target for antiviral drugs to prevent CV-A6-induced tissue damage., (Copyright © 2018 American Society for Microbiology.)

Human enterovirus 71 (EV71) is the second most common cause of hand, foot, and mouth disease (HFMD), which can occur as a severe epidemic especially among children under 5-years old. New and improved treatment strategies to control EV71 infection are therefore urgently required. The heterocyclic compound GS-9620, a potent and selective agonist of Toll-like receptor 7 (TLR7), has been reported to activate plasmacytoid dendritic cells (pDCs), and suppress HBV as well as HIV replication. In this study, we indicated that GS-9620 also could inhibit EV71 replication in the mouse model of EV71 infection. With three-days treatment after EV71 infection, the levels of proinflammatory cytokines/chemokines, like IFN-α, IFN-γ and MCP-1, were sharply reduced in serum compared to those without treatment. Furthermore, GS-9620 activated TLR7 in the limb muscle cells, which stimulated the NF-κB and PI3K/AKT signaling pathways. When NF-κB or PI3K/AKT inhibitors were used, the antiviral effect of the GS-9620 was impacted. Overall, our data implied GS-9620 probably activates NF-κB and PI3K/AKT signaling pathways to clear the virus., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The management of hand-foot-and-mouth disease(HFMD) epidemic is difficult due to the frequent emergence of non-EV71 and non-CVA16 enteroviruses and some cases testing negative for HFMD-associated causative agents. To clarify the virus spectrum of mild and severe HFMD, a comprehensive virome analysis of 238 samples was performed using next-generation sequencing (NGS). The data revealed total thirteen mammalian- and plant- virus families and diverse viral populations including enteroviruses, common respiratory viruses, diarrhea-related viruses, plant viruses and anelloviruses. A total of 18 viruses from 7 virus families were identified in severe cases, versus 37 viruses from 12 virus families in mild cases. Moreover, complicated mixed-infections of enteroviruses with common respiratory viruses were mainly found in severe cases(P = 0.013), while diarrhea-related viruses were mainly found in mild cases(P < 0.001). This study provides the preliminary understanding of viromes both in mild and severe cases, which may benefit the detection of etiologic agents and prevention of HFMD., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Enterovirus 71 (EV-A71) and coxsackievirus A16 (CV-A16) remain the predominant etiological agents of hand, foot, and mouth disease (HFMD). The clinical manifestations caused by the two viruses are obviously different. CV-A16 usually triggers a repeated infection, and airway epithelial integrity is often the potential causative factor of respiratory repeated infections. Our previous studies have demonstrated that there were some differentially expressed miRNAs involved in the regulation of adhesion function of epithelial barrier in EV-A71 and CV-A16 infections. In this study, we compared the differences between EV-A71 and CV-A16 infections on the airway epithelial barrier function in human bronchial epithelial (16HBE) cells and further screened the key miRNA which leaded to the formation of these differences. Our results showed that more rapid proliferation, more serious destruction of 16HBE cells permeability, more apoptosis and disruption of intercellular adhesion-associated molecules were found in CV-A16 infection as compared to EV-A71 infection. Furthermore, we also identified that microRNA-4516 (miR-4516), which presented down-regulation in EV-A71 infection and up-regulation in CV-A16 infection was an important regulator of intercellular junctions by targeting Poliovirus receptor related protein 1(PVRL1). The expressions of PVRL1, claudin4, ZO-1 and E-cadherin in CV-A16-infected cells were significantly less than those in EV-A71-infected cells, while the expressions of these proteins were subverted when pre-treated with miR-4516-overexpression plasmid in EV-A71 infected and miR-4516-knockdown plasmid in CV-A16 infected 16HBE cells. Thus, these data suggested that the opposite expression of miR-4516 in EV-A71 and CV-A16 infections might be the initial steps leading to different epithelial impairments of 16HBE cells by destroying intercellular adhesion, which finally resulted in different outcomes of EV-A71 and CV-A16 infections.