Genetic variation and evolutionary characteristics of Echovirus 11: new variant within genotype D5 associated with neonatal death found in China.

Echovirus 11 (E11) has gained attention owing to its association with severe neonatal infections. From 2018 to 2023, a surge in severe neonatal cases and fatalities linked to a novel variant of genotype D5 was documented in China, France, and Italy. However, the prevention and control of E11 variants have been hampered by limited background data on the virus circulation and genetic variance. Therefore, the present study investigated the circulating dynamics of E11 and the genetic variation and molecular evolution of genotype D5 through the collection of strains from the national acute flaccid paralysis (AFP) and hand, foot, and mouth disease (HFMD) surveillance system in China during 2000-2022 and genetic sequences published in the GenBank database. The results of this study revealed a prevalent dynamic of E11 circulation, with D5 being the predominant genotype worldwide. Further phylogenetic analysis of genotype D5 indicated that it could be subdivided into three important geographic clusters (D5-CHN1: 2014-2019, D5-CHN2: 2016-2022, and D5-EUR: 2022-2023). Additionally, variant-specific (144) amino acid mutation sites and positive-selection pressure sites (132, 262) were identified in the VP1 region. Cluster-specific recombination patterns were also identified, with CVB5, E6, and CVB4 as the major recombinant viruses. These findings provide a preliminary landscape of E11 circulation worldwide and basic scientific data for further study of the pathogenicity of E11 variants.

Coxsackievirus A10 impairs nail regeneration and induces onychomadesis by mimicking DKK1 to attenuate Wnt signaling.

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/ß-catenin signaling by restraining LDL receptor-related protein 6 (LRP6) phosphorylation and ß-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 (GSK3ß 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.

Genomic epidemiology of CVA10 in Guangdong, China, 2013-2021.

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

Immunogenicity and immunodominant linear B-cell epitopes of a new DNA-based tetravalent vaccine against four major enteroviruses causing hand, foot, and mouth disease.

Hand, foot, and mouth disease (HFMD) poses a significant public health threat primarily caused by four major enteroviruses: enterovirus 71 (EV71), coxsackieviruses A16, A10, and A6. Broadly protective immune responses are essential for complete protection against these major enteroviruses. In this study, we designed a new tetravalent immunogen for HFMD, validated it in silico, in vivo evaluated the immunogenicity of the DNA-based tetravalent vaccine in mice, and identified immunogenic B-cell and T-cell epitopes. A new tetravalent immunogen, VP1me, was designed based on the chimeric protein and epitope-based vaccine principles. It contains a complete EV71 VP1 protein and six reported neutralizing B-cell epitopes derived from the four major enteroviruses causing HFMD. In silico validation using multiple immunoinformatic tools indicated good attributes of the VP1me immunogen suitable for vaccine development. The VP1me-based DNA vaccine efficiently induced both humoral and cellular immune responses in BALB/cAJcl mice. A combination of in silico prediction and immunoassays enabled the identification of immunogenic linear B-cell and CD8 T-cell epitopes within the VP1me immunogen. Immunodominant linear B-cell epitopes were identified in six regions of VP1me, with one epitope located at the N-terminus of the VP1 protein (aa 9-23) regarded as a novel epitope. Interestingly, some B-cell epitopes could also induce the CD8 T-cell response, suggesting their dual functions in immune stimulation. These results lay the groundwork for further development of VP1me as a new vaccine candidate.

Evolution, recombination and geographic spreading of global Coxsackievirus A6.

BACKGROUND: The 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. METHODS: From 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. RESULTS: The 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. CONCLUSIONS: Based 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.

Effects of extreme meteorological factors and high air pollutant concentrations on the incidence of hand, foot and mouth disease in Jining, China.

Background: The evidence on the effects of extreme meteorological conditions and high air pollution levels on incidence of hand, foot and mouth disease (HFMD) is limited. Moreover, results of the available studies are inconsistent. Further investigations are imperative to elucidate the specific issue. Methods: Data on the daily cases of HFMD, meteorological factors and air pollution were obtained from 2017 to 2022 in Jining City. We employed distributed lag nonlinear model (DLNM) incorporated with Poisson regression to explore the impacts of extreme meteorological conditions and air pollution on HFMD incidence. Results: We found that there were nonlinear relationships between temperature, wind speed, PM2.5, SO2, O3 and HFMD. The cumulative risk of extreme high temperature was higher at the 95th percentile (P95th) than at the 90th percentile(P90th), and the RR values for both reached their maximum at 10-day lag (P95th RR = 1.880 (1.261-2.804), P90th RR = 1.787 (1.244-2.569)), the hazardous effect of extreme low temperatures on HFMD is faster than that of extreme high temperatures. The cumulative effect of extreme low wind speeds reached its maximum at 14-day lag (P95th RR = 1.702 (1.389-2.085), P90th RR = 1.498(1.283-1.750)). The cumulative effect of PM2.5 concentration at the P90th was largest at 14-day lag (RR = 1.637 (1.069-2.506)), and the cumulative effect at the P95th was largest at 10-day lag (RR = 1.569 (1.021-2.411)). High SO2 concentration at the P95th at 14-day lag was associated with higher risk for HFMD (RR: 1.425 (1.001-2.030)). Conclusion: Our findings suggest that high temperature, low wind speed, and high concentrations of PM2.5 and SO2 are associated with an increased risk of HFMD. This study not only adds insights to the understanding of the impact of extreme meteorological conditions and high levels of air pollutants on HFMD incidence but also holds practical significance for the development and enhancement of an early warning system for HFMD.

Epidemiological and etiological investigations of hand, foot, and mouth disease in Jiashan, northeastern Zhejiang Province, China, during 2016 to 2022.

Background: Hand, foot, and mouth disease (HFMD) is a common infectious disease in children. Enterovirus A71 (EV71) and coxsackievirus A16 (CA16) have been identified as the predominant pathogens for several decades. In recent years, coxsackievirus A6 (CA6) and coxsackievirus A10 (CA10) have played increasingly important roles in a series of HFMD outbreaks. We performed a retrospective analysis of the epidemiology of HFMD and the spectrum of different viral serotypes, to elucidate the genetic and phylogenetic characteristics of the main serotypes in the Jiashan area during 2016 to 2022. Methods: Descriptive epidemiological methods were used to analyze the time and population distribution of HFMD in Jiashan during 2016 to 2022 based on surveillance data. Molecular diagnostic methods were performed to identify the viral serotypes and etiological characteristics of HFMD. Phylogenetic analyses was based on VP1 region of CA16 and CA6. Results: The average annual incidence rate of HFMD fluctuated from 2016 to 2022. Children aged 1-5 years accounted for 81.65% of cases and boys were more frequently affected than girls. Except when HFMD was affected by the COVID-19 epidemic in 2020 and 2022, epidemics usually peak in June to July, followed by a small secondary peak from October to December and a decline in February. Urban areas had a high average incidence and rural areas had the lowest. Among 560 sample collected in Jiashan, 472 (84.29%) were positive for enterovirus. The most frequently identified serotypes were CA6 (296, 52.86%), CA16 (102, 18.21%), EV71 (16, 2.86%), CA10 (14, 2.50%) and other enteroviruses (44, 7.86%). There were 71 and 142 VP1 sequences from CA16 and CA6, respectively. Substitution of N218D, A220L and V251I was detected in CA16 and may have been related to viral infectivity. Phylogenetic analysis showed that CA16 could be assigned to two genogroups, B1a and B1b, while all the CA6 sequences belonged to the D3a genogroup. Conclusion: CA6 and CA16 were the two major serotypes of enteroviruses circulating in the Jiashan area during 2016 to 2022. Continuous and comprehensive surveillance for HFMD is needed to better understand and evaluate the prevalence and evolution of the associated pathogens.

Quantitation of Enterovirus A71 Empty and Full Particles by Sedimentation Velocity Analytical Ultracentrifugation.

The enterovirus A71 (EV71) inactivated vaccine is an effective intervention to control the spread of the virus and prevent EV71-associated hand, foot, and mouth disease (HFMD). It is widely administered to infants and children in China. The empty particles (EPs) and full particles (FPs) generated during production have different antigenic and immunogenic properties. However, the antigen detection methods currently used were established without considering the differences in antigenicity between EPs and FPs. There is also a lack of other effective analytical methods for detecting the different particle forms, which hinders the consistency between batches of products. In this study, we analyzed the application of sedimentation velocity analytical ultracentrifugation (SV-AUC) in characterizing the EPs and FPs of EV71. Our results showed that the proportions of the two forms could be quantified simultaneously by SV-AUC. We also determined the repeatability and accuracy of this method and found that both parameters were satisfactory. We assessed SV-AUC for bulk vaccine quality control, and our findings indicated that SV-AUC can be used effectively to analyze the percentage of EPs and FPs and monitor the consistency of the process to ensure the quality of the vaccine.

Impact of the COVID-19 outbreak and interventions on hand, foot and mouth disease in Zhengzhou, China, 2014-2022: a retrospective study.

BACKGROUND: Since December 2019, COVID-19 has spread rapidly around the world, and studies have shown that measures to prevent COVID-19 can largely reduce the spread of other infectious diseases. This study explored the impact of the COVID-19 outbreak and interventions on the incidence of HFMD. METHODS: We gathered data on the prevalence of HFMD from the Children's Hospital Affiliated to Zhengzhou University. An autoregressive integrated moving average model was constructed using HFMD incidence data from 2014 to 2019, the number of cases predicted from 2020 to 2022 was predicted, and the predicted values were compared with the actual measurements. RESULTS: From January 2014 to October 2022, the Children's Hospital of Zhengzhou University admitted 103,995 children with HFMD. The average number of cases of HFMD from 2020 to 2022 was 4,946, a significant decrease from 14,859 cases from 2014 to 2019. We confirmed the best ARIMA (2,0,0) (1,1,0)12 model. From 2020 to 2022, the yearly number of cases decreased by 46.58%, 75.54%, and 66.16%, respectively, compared with the forecasted incidence. Trends in incidence across sexes and ages displayed patterns similar to those overall. CONCLUSIONS: The COVID-19 outbreak and interventions reduced the incidence of HFMD compared to that before the outbreak. Strengthening public health interventions remains a priority in the prevention of HFMD.

Hand, foot, and mouth disease presenting with a testicular mass in an adult.

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.

Chinese parents' intention to vaccinate their 0-5-year-old children with the EV-71 vaccine against hand, foot, and mouth disease and willingness-to-pay.

Background: This study aimed to determine the intention and willingness-to-pay (WTP) of Chinese parents/guardians to vaccinate their children with the EV-71 vaccine. Knowledge levels about hand, foot, and mouth disease (HFMD) and the EV-71 vaccine were also investigated. Methods: A cross-sectional, self-administered online survey was conducted between November 2022 and March 2023. A stratified multi-stage random sampling method was used to recruit parents/guardians of children aged 0-5 years in southeastern China. Results: A total of 3,626 complete responses were received. The mean knowledge score of HFMD was 9.99 (±4.23) out of a total of 14 points. The majority of the participants reported a somewhat willing intent (58.8%), followed by an extremely willing intent (28.9%). Participants who did not consider the EV-71 vaccine expensive (OR = 2.94, 95%CI 2.45-3.53) perceived that the EV-71 vaccine is effective (OR = 2.73, 95%CI 1.52-4.90), and a high knowledge level of HFMD (OR = 1.90, 95%CI 1.57-2.29) had the highest significant odds of having an extremely willing intent to vaccinate their children with the EV-71 vaccine. The median (interquartile range [IQR]) of WTP for the EV-71 vaccine was CNY¥200/USD$28 (IQR CNY¥100-400/USD$14-56). The highest marginal WTP for the vaccine was mainly influenced by the perceived high cost of the vaccine. Those participants who did not consider the EV-71 vaccine expensive had more than 10 times higher odds of vaccinating their children (OR = 10.86, 95%CI 8.49-13.88). Perceived susceptibility, perceived benefits, and perceived barriers were also significant influencing factors in the highest marginal WTP. Conclusion: The findings demonstrate the importance of improving health promotion and reducing the barriers to EV-71 vaccination. Therefore, it is important to improve health promotion and reduce the barriers to EV-71 vaccination.

Cordycepin Inhibits Enterovirus A71 Replication and Protects Host Cell from Virus-Induced Cytotoxicity through Adenosine Action Pathway.

Enterovirus A71 (EV-A71) infection typically causes mild illnesses, such as hand-foot-and-mouth disease (HFMD), but occasionally leads to severe or fatal neurological complications in infants and young children. Currently, there is no specific antiviral treatment available for EV-A71 infection. Thus, the development of an effective anti-EV-A71 drug is required urgently. Cordycepin, a major bioactive compound found in Cordyceps fungus, has been reported to possess antiviral activity. However, its specific activity against EV-A71 is unknown. In this study, the potency and role of cordycepin treatment on EV-A71 infection were investigated. Results demonstrated that cordycepin treatment significantly reduced the viral load and viral ribonucleic acid (RNA) level in EV-A71-infected Vero cells. In addition, EV-A71-mediated cytotoxicity was significantly inhibited in the presence of cordycepin in a dose-dependent manner. The protective effect can also be extended to Caco-2 intestinal cells, as evidenced by the higher median tissue culture infectious dose (TCID50) values in the cordycepin-treated groups. Furthermore, cordycepin inhibited EV-A71 replication by acting on the adenosine pathway at the post-infection stage. Taken together, our findings reveal that cordycepin could be a potential antiviral candidate for the treatment of EV-A71 infection.

Epidemiological and Genetic Characterization of Coxsackievirus A6-Associated Hand, Foot, and Mouth Disease in Gwangju, South Korea, in 2022.

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

Age-time-specific transmission of hand-foot-and-mouth disease enterovirus serotypes in Vietnam: A catalytic model with maternal immunity.

Hand, foot and mouth disease (HFMD) is highly prevalent in the Asia Pacific region, particularly in Vietnam. To develop effective interventions and efficient vaccination programs, we inferred the age-time-specific transmission patterns of HFMD serotypes enterovirus A71 (EV-A71), coxsackievirus A6 (CV-A6), coxsackievirus A10 (CV-A10), coxsackievirus A16 (CV-A16) in Ho Chi Minh City, Vietnam from a case data collected during 2013-2018 and a serological survey data collected in 2015 and 2017. We proposed a catalytic model framework with good adaptability to incorporate maternal immunity using various mathematical functions. Our results indicate the high-level transmission of CV-A6 and CV-A10 which is not obvious in the case data, due to the variation of disease severity across serotypes. Our results provide statistical evidence supporting the strong association between severe illness and CV-A6 and EV-A71 infections. The HFMD dynamic pattern presents a cyclical pattern with large outbreaks followed by a decline in subsequent years. Additionally, we identify the age group with highest risk of infection as 1-2 years and emphasise the risk of future outbreaks as over 50% of children aged 6-7 years were estimated to be susceptible to CV-A16 and EV-A71. Our study highlights the importance of multivalent vaccines and active surveillance for different serotypes, supports early vaccination prior to 1 year old, and points out the potential utility for vaccinating children older than 5 years old in Vietnam.

Effectiveness of Enterovirus 71 inactivated vaccines against hand, foot, and mouth disease: A test-negative case-control study.

The Enterovirus A71 (EV-A71) vaccine was introduced in China in December 2015 as a preventive measure against hand, foot, and mouth disease (HFMD) caused by EV-A71. However, the effectiveness of the vaccine (VE) in real-world settings needs to be evaluated. We conducted a test-negative case-control study to assess the effectiveness of EV-A71 vaccines in preventing EV-A71-associated HFMD. Children aged 6-71 months with HFMD were enrolled as participants. The case group comprised those who tested positive for EV-A71, while the control group comprised those who tested negative for EV-A71. To estimate VE, a logistic regression model was employed, adjusting for potential confounders including age, gender, and clinical severity. In total, 3223 children aged 6 to 71 months were included in the study, with 162 in the case group and 3061 in the control group. The proportion of children who received EV-A71 vaccination was significantly lower in the case group compared to the control group (p < .001). The overall VEadj was estimated to be 90.8%. The VEadj estimates for partially and fully vaccinated children were 90.1% and 90.9%, respectively. Stratified by age group, the VEadj estimates were 88.7% for 6 to 35-month-olds and 95.5% for 36 to 71-month-olds. Regarding disease severity, the VEadj estimates were 86.3% for mild cases and 100% for severe cases. Sensitivity analysis showed minimal changes in the VE point estimates, with most changing by no more than 1% point. Our study demonstrates a high level of vaccine effectiveness against EV-A71-HFMD, especially in severe cases. Active promotion of EV-A71 vaccination is an effective strategy in preventing EV-A71 infections.

Modeling and analysis of the effect of optimal virus control on the spread of HFMD.

A within-host and between-host hand, foot and mouth disease (HFMD) mathematical model is established and the affect of optimal control in its within-host part on HFMD transmission is studied. Through define two basic reproduction numbers, by using the fast-slow system analysis method of time scale, the global stabilities of the between-host (slow) system and within-host (fast) system are researched, respectively. An optimal control problem with drug-treatment control on coupled within-host and between-host HFMD model is formulated and analysed theoretically. Finally, the purposed optimal control measures are applied to the actual HFMD epidemic analysis in Zhejiang Province, China from April 1, 2021 to June 30, 2021. The numerical results show that the drug control strategies can reduce the virus load per capita and can effectively prevent large-scale outbreaks of HFMD.

Effectiveness of enterovirus A71 vaccine against pediatric HFMD and disease profile of post-vaccination infection.

BACKGROUND: Vaccination has been proven effective against infection with enterovirus A71 (EV-A71) in clinical trials, but vaccine effectiveness in real-world situations remains incompletely understood. Furthermore, it is not clear whether previous vaccination will result in symptom attenuation among post-vaccinated cases. METHODS: Based on long-term data extracted from the only designed referral hospital for infectious diseases, we used a test-negative case-control design and multivariate logistic regression models to analyze the effectiveness of EV-A71 vaccine against hand, foot and mouth disease (HFMD). And then, generalized linear regression models were used to evaluate the associations between prior vaccination and disease profiles. RESULTS: We selected 4883 inpatients for vaccine efficacy estimations and 2188 inpatients for disease profile comparisons. Vaccine effectiveness against EV-A71-induced HFMD for complete vaccination was 63.4 % and 51.7 % for partial vaccination. The vaccine effectiveness was higher among cases received the first dose within 12 months. No protection was observed against coxsackievirus (CV) A6-, CV-A10- or CV-A16-associated HFMD among children regardless of vaccination status. Completely vaccinated cases had shorter hospital stay and disease course compared to unvaccinated cases (P < 0.05). CONCLUSIONS: These findings reiterate the need to continue the development of a multivalent vaccine or combined vaccines, and have implications for introducing optimized vaccination strategies.

Inhibition of EV71 replication by an interferon-stimulated gene product L3HYPDH.

Enterovirus 71 (EV71) is the common causative agent of hand-foot-mouth disease (HFMD). Despite evidence in mice model suggested that the interferon (IFN) signaling pathways play a role in defending against this virus, knowledge on the IFN-mediated antiviral response is still limited. Here we identified an IFN-stimulated gene (ISG) called L3HYPDH, whose expression inhibits EV71 replication. Mapping assay indicated that amino acids 61-120 and 295-354 are critical for its optimal antiviral activity. Mechanismly, L3HYPDH specifically inhibits protein translation mediated by EV71 internal ribosome entry site (IRES). Our data thus uncovered a new mechanism utilized by the host cell to restrict EV71 replication.