Hand, Foot And Mouth Disease Patients Research Articles

Establishment of a rhesus macaque model for Coxsackievirus A6 infection: Pilot study to evaluate infection initiated through the respiratory or digestive track

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.

Epidemiological characterization of hand, foot, and mouth disease among hospitalized children from 2014 to 2023 in a hospital in Henan Province: Longitudinal surveillance study.

Hand, foot, and mouth disease (HFMD) is an acute infectious illness primarily caused by enteroviruses. The present study aimed to describe the epidemiological characteristics of hospitalized HFMD patients in a hospital in Henan Province (Zhengzhou, China), and to predict the future epidemiological parameters. In this study, we conducted a retrospective analysis of general demographic and clinical data on hospitalized children who were diagnosed with HFMD from 2014 to 2023. We used wavelet analysis to determine the periodicity of the disease. We also conducted an analysis of the impact of the COVID-19 epidemic on the detection ratio of severe illness. Additionally, we employed a Seasonal Difference Autoregressive Moving Average (SARIMA) model to forecast characteristics of future newly hospitalized HFMD children. A total of 19 487 HFMD cases were included in the dataset. Among these cases, 1515 (7.8%) were classified as severe. The peak incidence of HFMD typically fell between May and July, exhibiting pronounced seasonality. The emergence of COVID-19 pandemic changed the ratio of severe illness. In addition, the best-fitted seasonal ARIMA model was identified as (2,0,2)(1,0,1)12. The incidence of severe cases decreased significantly following the introduction of the vaccine to the market (χ2 = 109.9, p < 0.05). The number of hospitalized HFMD cases in Henan Province exhibited a seasonal and declining trend from 2014 to 2023. Non-pharmacological interventions implemented during the COVID-19 pandemic have led to a reduction in the incidence of severe illness.

Single nucleotide polymorphism of Notch1 gene rs3124599 allele is associated with the severity of CVA6-related HFMD in the Chinese Han population

BackgroundThere is evidence suggesting that Notch1 signaling pathway contributes to the development of hand, foot, and mouth disease (HFMD); however, the role of Notch1 gene polymorphisms in the severity of coxsackievirus A6 (CVA6)-related HFMD remains unclear. This study aimed to investigate the correlation between Notch1 gene polymorphisms and the severity of CVA6-related HFMD.MethodsA total of 196 patients (Chinese Han population) diagnosed with CVA6-related HFMD through nucleic acid testing were included in this study. Among them, 97 patients were classified as severe cases, while 99 cases were categorized as mild. The mRNA levels of Notch1 in the peripheral blood leukocytes of HFMD patients were detected by quantitative real-time polymerase chain reaction (qRT-PCR), and the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique was utilized for genotyping of rs3124599, rs3124603, and rs3124591.ResultsThe frequencies of rs3124599 alleles were G (39.0%) and A (61.0%), while the frequencies of rs3124599 genotypes were GG (12.2%), GA (53.6%), and AA (34.2%), respectively. In the recessive model, the frequency of rs3124599 AA genotypes significantly increased in severe patients, compared to mild patients (P < 0.05). Due to the low frequency of alleles for rs3124591 and rs3124603 in patients, as well as the absence of any difference in their distribution between the two groups (P > 0.05), no additional statistical analysis was performed. After adjusting for age and sex, patients with rs3124599 AA genotype had a significantly higher risk of severe HFMD in comparison to G allele carriers (GA/GG), with an odds ratio (95% confidence interval) of 2.010 (1.094, 3.691). Meanwhile, the mRNA levels of Notch1 were found to be significantly higher in severe patients compared to mild patients (P < 0.05), and a positive correlation was observed between Notch1 mRNA levels and the peripheral blood monocyte count (r = 0.42, P < 0.001). Additionally, there were significant differences observed in Notch1 mRNA levels and peripheral blood monocyte counts between patients with the AA genotype of rs3124599 and those with the GA genotype or G allele carriers (P < 0.05).ConclusionIn the Chinese Han population, there is a strong correlation between the Notch1 rs3124599 allele and the severity of CVA6-related HFMD. This correlation may be attributed to genetic polymorphism of rs3124599 regulating Notch1 transcription levels. These findings reveal the important role of Notch1 gene polymorphism in CVA6 infection, establishing a scientific foundation for the precise control of severe HFMD.

Clinical features of hand, foot and mouth disease caused by Coxsackievirus A6 in Xi'an, China, 2013-2019: A multicenter observational study

PurposeTo investigate the clinical features of hand, foot, and mouth disease (HFMD) caused by coxsackievirus A6 (CVA6) and this work may help early diagnose of atypical HFMD. MethodsFrom January 2013 to December 2019, a total of 7,208 patients with a clinical diagnosis of HFMD in Xi'an Children's Hospital, Xi'an Central Hospital, and Xi'an Jiaotong University Second Affiliated Hospital, were included in this observational study. The clinical data, specimens and follow-up results were collected. Real-time RT‒PCR was performed for the detection and typing of enterovirus nucleic acids. ResultsOf the 7,208 clinically diagnosed HFMD patients, 5,622 were positive for enterovirus nucleic acids, and the positive proportions of CVA6, enterovirus 71 (EV-A71), coxsackievirus A16 (CVA16), and other enteroviruses were 31.0% (1,742/5,622), 27.0% (1,518/5,622), 35.0% (1,968/5,622), and 7.0% (394/5,622), respectively. Based on the etiology, patients were divided into CVA6 group, EV-A71group, and CVA16 group. The mean age at onset was significantly higher in the CVA6 group (4.62±2.13 years) than in the EV-A71 group and CVA16 group (3.45±2.25 years and 3.35±2.13 years, respectively; both P < 0.05). The male/female ratio was 1.45 (1,031/711) in the CVA6 group and was not significantly different from the other two groups. The incidence of fever was significantly higher in the CVA6 group [82.5% (1,437/1,742)] than in the EV-A71 group [51.3% (779/1,518)] and the CVA16 group [45.9% (903/1,968)] (P < 0.05). In the CVA6 group, the rashes were more frequently on the trunk and elbows/knees and were significantly different from the other two groups (P < 0.05). The number of patients with two or more rash morphologies was significantly higher in the CVA6 group than in the other two groups (P < 0.05). The incidence of bullous rash in the CVA6 group [20.2%; n = 352] was higher than in the EV-A71 group [0.33%; n = 5] and CVA16 group [0.66%; n = 13] (P < 0.05). The incidence of neurological complications was significantly higher in the EV-A71 group [52.1% (791/1,518)] than in the CVA16 group [5.1% (100/1,968)] and the CVA6 group [0.8% (14/1,742)] (P < 0.05). In the follow-up period, 160 patients (9.2%) with CVA6 HFMD experienced onychomadesis, but no onychomadesis was observed in the EV-A71 and CVA16 groups. The average WBC count was significantly higher in the CVA6 group than in the CVA16 group (P < 0.05). The number of patients with increased CRP was significantly larger in the CVA6 group than in the CVA16 group but was significantly smaller than that in the EV-A71 group (P < 0.05). ConclusionsCVA6 has become one of the main pathogens of HFMD in the Xi'an area during 2013–2019. The main clinical manifestations were slightly different from those of HFMD caused by EV-A71 or CVA16, with a higher frequency of fever, diverse morphologies and diffuse distribution of rashes, fewer neurological complications and some onychomadesis.

A multiplex one-step fluorescence quantitative differential diagnosis method for severe hand, foot and mouth disease caused by coxsackievirus A16

Hand foot and mouth disease (HFMD) is a common childhood infectious disease which is caused by human enterovirus. The objective of this study was to develop a rapid, sensitive, and accurate method for detecting severe HFMD caused by coxsackievirus A16 (CV-A16). A closed-tube sensitive multiplex one-step reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) was applied to detect CV-A16 in the early stage of severe HFMD. This assay targeted the CV-A16 structure protein VP1 to distinguish CV-A16 from other coxsackieviruses The 5’UTR region of enteric viruses was used for detecting the enterovirus and ribonuclease P (RNaseP) was adopted as the internal reference gene. The multiplex MGB probe assay system was used to detect PCR amplicons with different fluorescence reporters in the same system. The limit of detection (LOD) of the RT-qPCR assay for the CV-A16 VP1 gene was 125.893 copies/μl, for the 5′ UTR was 50.1187 copies/μl and for the RNaseP gene was 158.49 copies/μl. Furthermore, specificity analysis showed that the multiplex RT-PCR had no cross-reactivity with the influenza virus, herpangina virus and SARS-COV-2. In correlation analysis, the sensitivity of the multiplex RT-qPCR assay for CV-A16 detection was 100 % (288/288) and the specificity of the multiplex RT-qPCR assay was 99.94 % (3395/3397). The overall agreement between the multiplex RT-qPCR and the results of clinical diagnosis was 99.95 % (3683/3685) and kappa value was 0.996 (p<0.001). The entire procedure, from specimen processing to result reporting, could be completed within 1.5 hours. The one-step multiplex RT-qPCR assay for detecting CV-A16 developed in this study is a good laboratory diagnostic tool for rapid and reliable distinguished detection of CV-A16, especially for severe HFMD patients at an early stage in the disease with low virus load of CV-A16.

Evolution, recombination and geographic spreading of global Coxsackievirus A6

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

Epidemiological and Clinical Profile of Hand, Foot, and Mouth Disease in Children in a Tertiary Care Center in Jammu.

Background Hand, foot, and mouth disease (HFMD) is a viral illness commonly seen in children under five years of age, characterized by typical manifestations such as oral lesions and rashes on the hands and feet. Coxsackievirus A-16 (CV-A16) and Enterovirus A-71 (EV-A71) are the major etiological agents of this disease. Over the past two decades, there have been several outbreaks of HFMD all across India. As there is no chemoprophylaxis available for the disease, it becomes even more significant to conduct regular research and surveillance for HFMD. Aim and objective To observe the clinico-epidemiologicalprofile along with constitutional symptoms in HFMD patients attending pediatric OPD. Methods This hospital-based prospective observational study was conducted in the Post Graduate Department of Pediatrics, Acharya Shri Chander College of Medical Sciences and Hospital (ASCOMS & H), Sidra, Jammu and Kashmir, India, oversix months from April to September 2023. A total of 132 children with symptoms of HFMD visited the pediatric OPD. After using inclusive and exclusive criteria, we selected a sample size of 112 children with HFMD. The descriptive data were expressed in terms of percentages and proportions, and their graphical representation was done using MS Excel (Microsoft Corporation, Redmond, Washington, United States). Results Among the 112 HFMD patients examined, the highest peak was seen in August, followed by another one in September. Most of the cases were seen in the age group of zero to three years, and it was observed that there wasa linear fall in the number of cases with the increase in age. Nearly 61% of cases were male, showing a slight male preponderance. Vesiculopapular rash on the hand and foot was the most common clinical characteristic, whereas painful deglutitionwas noted to be the most common constitutional symptom in HFMD patients. About 27% had a positive family history, and nail changes post-recovery were present in 1.79% of cases during their regular follow-ups. Conclusions This study reveals that HFMD cases surged in August and September, with a history of contact in one-fourth of cases. Disease is seen more commonly in children under three years of age, and the incidence of cases decreases with the increase in age. The illness is usually contagious and can spread quickly; therefore, more awareness programs should be done toeducate parents and promote hygiene to prevent contact cases.

Health-Related Quality of Life and Economic Burden Among Hospitalized Children with Hand, Foot, and Mouth Disease: A Multiregional Study in China.

Hand, foot, and mouth disease (HFMD) is an infectious disease with high morbidity and mortality rates among children under 5years old. This study aimed to explore the health-related quality of life (HRQOL), economic burden, and related influencing factors among Chinese HFMD patients. From January to October 2019, a longitudinal cohort study of 296 hospitalized patients (≤5years old) with HFMD and their guardians was conducted using the proxy version of the 5-level EQ-5D-Y (EQ-5D-Y-5L, Y-5L) in face-to-face interviews in Shanghai, Zhengzhou, and Kunming, representing three regions with different economic development levels. Multiple linear regression was used to explore the factors associated with HRQOL and costs. The mean Y-5L health utility score (HUS) (standard deviation, SD), and visual analogue scale (VAS) score (SD) were 0.730 (0.140) and 60.33 (16.52) at admission and increased to 0.920 (0.120) and 89.95 (11.88) at discharge, respectively. The children from Shanghai had the lowest HUSs at admission and had the best health improvement. The mean hospitalization cost and total cost were 4037 CNY and 5157 CNY, respectively. The children from Shanghai had the highest hospitalization cost (4559 CNY) and total cost (5491 CNY). Multiple regression analysis suggested that medical insurance status, type of employment, residence type, and religious status were significantly associated with the baseline HUS and improvement in the HUS after treatment. Region, loss of work time, and length of stay had a significant impact on the hospitalization cost and total cost. Our findings demonstrate that HFMD could lead to poor HRQOL and the economic burden varies in different regions in China. Many pediatric patients still have physical or mental health problems shortly after treatment.

High mobility group box 1 knockdown inhibits EV71 replication and attenuates cell pyroptosis through TLR4/NF-κB/NLRP3 axis.

Enterovirus 71 (EV71) is a major causative agent of hand, foot, and mouth disease (HFMD) in children. Nowadays, there are still no effective antiviral drugs for EV71 infection. High mobility group box 1 (HMGB1) is reported to be highly expressed in HFMD patients. However, therole and underlying mechanism of HMGB1 in EV71-associated HFMD are still unclear. HMGB1 expression was detected using RT-qPCR and western blot assays. Loss- and gain-function experiments were performed to evaluate the effects of HMGB1 on EV71-infected cells. The virus titer was examined by TCID50. CCK-8 and flow cytometry assays were applied to detect the cell viability and cell cycle. Oxidative stress was determined by relative commercial kits. HMGB1 level was elevated in the serum of EV71-infected patients with HFMD and EV71-induced RD cells. EV71 infection induced the transfer of HMGB1 from the nucleus into the cytoplasm. HMGB1 knockdown inhibited virus replication,viral protein (VP1) expression and promoted antiviral factor expression. In addition, the inhibition of HMGB1 improved cell viability, protected against S phase arrest, and inhibited EV71-induced cell injury and oxidative stress, whereasHMGB1 overexpression showed the opposite effects. In terms of mechanism, HMGB1 overexpression activated the TLR4/NF-κB/NLRP3 signaling pathway and promoted cell pyroptosis. The inhibition of TLR4 and NF-κB reversed the effects of HMGB1 overexpression on virus replication, oxidative stress, and pyroptosis. In conclusion, HMGB1 knockdown inhibits EV71 replication and attenuates pyroptosis through TLR4/NF-κB/NLRP3 axis.

Emerging concerns of atypical hand foot and mouth disease caused by recombinant Coxsackievirus A6 variants in Henan, China.

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.

Potential relevance of salivary legumain for the clinical diagnostic of hand, foot, and mouth disease.

The fight against hand, foot, and mouth disease (HFMD) remains an arduous challenge without existing point-of-care (POC) diagnostic platforms for accurate diagnosis and prompt case quarantine. Hence, the purpose of this salivary biomarker discovery study is to set the fundamentals for the realization of POC diagnostics for HFMD. Whole salivary proteome profiling was performed on the saliva obtained from children with HFMD and healthy children, using a reductive dimethylation chemical labeling method coupled with high-resolution mass spectrometry-based quantitative proteomics technology. We identified 19 upregulated (fold change = 1.5-5.8) and 51 downregulated proteins (fold change = 0.1-0.6) in the saliva samples of HFMD patients in comparison to that of healthy volunteers. Four upregulated protein candidates were selected for dot blot-based validation assay, based on novelty as biomarkers and exclusions in oral diseases and cancers. Salivary legumain was validated in the Singapore (n = 43 healthy, 28 HFMD cases) and Taiwan (n = 60 healthy, 47 HFMD cases) cohorts with an area under the receiver operating characteristic curve of 0.7583 and 0.8028, respectively. This study demonstrates the feasibility of a broad-spectrum HFMD POC diagnostic test based on legumain, a virus-specific host systemic signature, in saliva.

Antibody signatures in hospitalized hand, foot and mouth disease patients with acute enterovirus A71 infection.

Enterovirus A71 (EV-A71) infection is a major cause of severe hand, foot and mouth disease (HFMD) in young children. The characteristics of EV-A71 neutralizing antibodies in HFMD patients are not well understood. In this study, we identified and cloned EV-A71-neutralizing antibodies by single cell RNA and B cell receptor sequencing of peripheral blood mononuclear cells. From 145 plasmablasts, we identified two IgG1 monoclonal antibodies (mAbs) and six IgM mAbs that neutralized EV-A71. Four of the IgM mAbs harbor germline variable sequences and neutralize EV-A71 potently. Two genetically similar IgM antibodies from two patients have recurrent heavy chain variable domain gene usage and similar complementarity-determining region 3 sequences. We mapped the residues of EV-A71 critical for neutralization through selection of virus variants resistant to antibody neutralization in the presence of neutralizing mAbs. The residues critical for neutralization are conserved among EV-A71 genotypes. Epitopes for the two genetically similar antibodies overlap with the SCARB2 binding site of EV-A71. We used escape variants to measure the epitope-specific antibody response in acute phase serum samples from EV-A71 infected HFMD patients. We found that these epitopes are immunogenic and contributed to the neutralizing antibody response against the virus. Our findings advance understanding of antibody response to EV-A71 infection in young children and have translational potential: the IgM mAbs could potentially be used for prevention or treatment of EV-A71 infections.

Gut Microbiota in Children with Hand Foot and Mouth Disease on 16S rRNA Gene Sequencing.

Hand foot and mouth disease (HFMD) is a contagious and seasonal viral disease in children. The gut microbiota of HFMD children is not clear now. The study aimed to explore the gut microbiota of HFMD children. The 16S rRNA gene of the gut microbiota of ten HFMD patients and ten healthy children were sequenced on the NovaSeq and PacBio platforms respectively. There were significant differences in gut microbiota between the patients and healthy children. The diversity and abundance of gut microbiota in HFMD patients were lower than that in healthy children. The species Roseburia inulinivorans and Romboutsia timonensis were more abundant in healthy children than those in HFMD patients, which suggests that the two species may be used as probiotics for adjusting the gut microbiota of HFMD patients. Meanwhile, the results of 16S rRNA gene sequences from the two platforms were different. The NovaSeq platform identified more microbiota and has the characteristics of high throughput, short time and low price. However, the NovaSeq platform has low resolution at the species level. The PacBio platform has high resolution based on its long reads length, which is more suitable for species-level analysis. But, the shortcomings of the high price and low throughput of PacBio still need to be overcome. With the development of sequencing technology, the reduction in sequencing price and the increase in throughput will promote the third-generation sequencing technology used in the study of gut microbes.

Genomic Epidemiology and Transmission Dynamics of Global Coxsackievirus B4.

The aim of this study was to determine the global genetic diversity and transmission dynamics of coxsackievirus B4 (CVB4) and to propose future directions for disease surveillance. Next-generation sequencing was performed to obtain the complete genome sequence of CVB4, and the genetic diversity and transmission dynamics of CVB4 worldwide were analyzed using bioinformatics methods such as phylogenetic analysis, evolutionary dynamics, and phylogeographic analysis. Forty complete genomes of CVB4 were identified from asymptomatic infected individuals and hand, foot, and mouth disease (HFMD) patients. Frequent recombination between CVB4 and EV-B multiple serotypes in the 3Dpol region was found and formed 12 recombinant patterns (A-L). Among these, the CVB4 isolated from asymptomatic infected persons and HFMD patients belonged to lineages H and I, respectively. Transmission dynamics analysis based on the VP1 region revealed that CVB4 epidemics in countries outside China were dominated by the D genotype, whereas the E genotype was dominant in China, and both genotypes evolved at a rate of > 6.50 × 10-3 substitutions/site/year. CVB4 spreads through the population unseen, with the risk of disease outbreaks persisting as susceptible individuals accumulate. Our findings add to publicly available CVB4 genomic sequence data and deepen our understanding of CVB4 molecular epidemiology.

The upregulation of peripheral blood polyamine metabolites spermidine and spermine in children with hand, foot, mouth disease is related to enterovirus 71 capsid protein VP1, but not VP4.

Hand, foot, and mouth disease (HFMD) is a common viral childhood illness caused most commonly by enterovirus 71 (EV71) and coxsackievirus A16. The pathogenesis of EV71 has been extensively studied, and the regulation of the host immune response is suspected to aggravate the serious complications induced by EV71. Our previous research showed that EV71 infection significantly increased the release of circulating interleukin (IL)-6, IL-10, IL-13, and IL-27. Notably, these cytokines are related to the EV71 infection risk and clinical stage. Polyamines are compounds that are ubiquitous in mammalian cells and play a key role in various cellular processes. Several studies have shown that targeting polyamine metabolic pathways can reduce infections caused by viruses. However, the significance of polyamine metabolism in EV71 infection remains largely unknown. Serum samples from 82 children with HFMD and 70 healthy volunteers (HVs) were collected to determine the polyamine metabolites spermidine (SPD) and spermine (SPM), and IL-6 levels. In addition, peripheral blood mononuclear cells (PBMCs) were treated with EV71 viral protein 1 (VP1) and EV71 VP4, and the cells and supernatant were then collected to analyze the expression of polyamine metabolism-related enzymes by western blot. The data were analyzed using GraphPad Prism 7.0 software (USA). The serum polyamine metabolites SPD and SPM were elevated in the HFMD patients, especially in the EV71-infected children. Further, a positive correlation was found between serum SPD and IL-6 levels in the EV71-infected children. We also found that the upregulation of peripheral blood polyamine metabolites in the EV71-infected HFMD children was related to EV71 capsid protein VP1, but not VP4. VP1 may promote the expression of polyamine metabolism-related enzymes and promote the production of polyamine metabolites, thereby upregulating the SPD/nuclear factor kappa B/IL-6 signaling pathway. However, VP4 has the opposite effect in this process. Our results suggest that EV71 capsid protein may regulate the polyamine metabolic pathways of infected cells in a variety of ways. This study provides insights into the mechanism of EV71 infection and polyamine metabolism and has good reference value for the development of EV71 vaccine.

A large scale 16S ribosomal RNA gene amplicon dataset of hand, foot and mouth patients and healthy individuals

There is evidence linking hand, foot and mouth disease (HFMD) to gut microbiota dysbiosis, and this relationship was corroborated in a large HFMD patient population in our previous study. Here, we present a bacterial 16S rRNA gene dataset from faecal samples of 713 individuals (254 HFMD patients, 459 healthy controls) aged 2 to 7 years residing in Heyuan and Jiangmen counties, Guangdong Province, southern China. Microbiome analysis indicated a significant increase in genus Prevotella, Cetobacterium, and Megamonas was observed in patients with HFMD, whereas a large increase in genus Bacteroides, Ruminococcus, and Faecalibacterium were seen in the control group. We also share the bioinformatic analytical pipeline for this analysis, from data preprocessing to data filtering and amplicon sequence variant (ASV) table generation. We expect that the dataset will be reprocessed, evaluated and fully analysed with various analysis methods to further elucidate the role of the gut microbiota in HFMD development.

Local immune dysregulation and subsequent inflammatory response contribute to pulmonary edema caused by Enterovirus infection in mice.

Pulmonary edema that comes on suddenly is the leading cause of mortality in hand-foot-and-mouth disease (HFMD) patients; however, its pathogenesis is still largely unclear. A range of research suggest immunopathogenesis during the occurrence of pulmonary edema in severe HFMD patients. Herein, to investigate the potential mechanism of immune dysregulation in the development of pulmonary edema upon Enterovirus (EV) infection, we established mouse infection models for Enteroviruses (EVs) including Coxsackievirus (CV) A6, Enterovirus A71 (EVA71), and CVA2 exhibiting a high incidence of pulmonary edema. We found that EVs infection induced an immune system disorder by reducing the numbers of pulmonary and circulatory T cells, B cells, macrophages, and monocytes and increasing the numbers of lung neutrophils, myeloid-derived suppressor cells (MDSCs), and activated T cells. In addition, the concentrations of C-X-C motif chemokine ligand 1 (CXCL-1), tumor necrosis factor-alpha, monocyte chemoattractant protein-1, and interleukin 6 were increased in EV-infected lungs. Moreover, we found that EVs replication in mice lungs lead to apoptosis of lung cells and degradation of tight junction proteins. In conclusion, EVs infection likely triggered a complexed immune defense mechanism and caused dysregulation of innate immune cells (MDSCs, neutrophils, monocytes, and macrophages) and adaptive cellular immunity (B cells, T cells). This dysregulation increased the release of cytokines and other inflammatory factors from activated immune-related cells and caused lung barrier damage and pulmonary edema.

Evolutionary and Genetic Recombination Analyses of Coxsackievirus A6 Variants Associated with Hand, Foot, and Mouth Disease Outbreaks in Thailand between 2019 and 2022.

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

Genetic characteristics of Coxsackievirus A6 from children with hand, foot and mouth disease in Beijing, China, 2017-2019.

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. 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. 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. 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.

Diagnostic performance of different specimens in detecting enterovirus A71 in children with hand, foot and mouth disease

Hand, foot and mouth disease (HFMD) is a major public health problem among children in the Asia-Pacific region. The optimal specimen for HFMD virological diagnosis remains unclear. Enterovirus A71 (EV-A71) neutralizing antibody titres detected in paired sera were considered the reference standard for calculating the sensitivity, specificity, positive and negative predictive value of throat swabs, rectal swabs, stool, blood samples and cerebrospinal fluid (CSF) by RT-PCR or ELISA assay. In this study, clinical samples from 276 HFMD patients were collected for analysing the sensitivity of different kind of specimens. Our results showed that stool had the highest sensitivity (88%, 95% CI: 74%–96%) and agreement with the reference standard (91%). The order of diagnostic yield for EV-A71 infection was stool sample ​≥ ​rectal swab ​> ​throat swab ​> ​blood sample ​> ​CSF sample, and using a combination of clinical samples improved sensitivity for enterovirus detection. The sensitivity of ELISA for IgM antibody detection in sterile-site specimens was significantly higher than that of RT-PCR (serum/plasma: 62% vs. 2%, CSF: 47% vs. 0%) (P ​< ​0.002). In conclusion, our results suggest that stool has the highest diagnostic yield for EV-A71-infected HFMD. If stool is unavailable, rectal swabs can be collected to achieve a similar diagnostic yield. Otherwise, throat swabs may be useful in detecting positive samples. Although IgM in blood or CSF is diagnostically accurate, it lacks sensitivity, missing 40%–50% of cases. The higher proportion of severe cases and shorter interval between onset and sampling contributed to the increase in congruency between clinical testing and the serological reference standard.