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Enteroviruses contain multiple serotypes and can cause severe neurological complications. The intricate life cycle of enteroviruses involving dynamic virus-receptor interaction hampers the development of broad therapeutics and vaccines. Here, using function-based screening, we identify a broadly therapeutic antibody h1A6.2 that potently protects mice in lethal models of infection with both enterovirus A71 and coxsackievirus A16 through multiple mechanisms, including inhibition of the virion-SCARB2 interactions and monocyte/macrophage-dependent Fc effector functions. h1A6.2 mitigates inflammation and improves intramuscular mechanics, which are associated with diminished innate immune signalling and preserved tissue repair. Moreover, cryogenic electron microscopy structures delineate an adaptive binding of h1A6.2 to the flexible and dynamic nature of the VP2 EF loop with a binding angle mimicking the SCARB2 receptor. The coordinated binding mode results in efficient binding of h1A6.2 to all viral particle types and facilitates broad neutralization of enterovirus, therefore informing a promising target for the structure-guided design of pan-enterovirus vaccine., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Human Enterovirus 71 (EV71) has emerged as one of the predominant causative agents of hand, foot and mouth disease (HFMD) with global impact. Despite the inactivated vaccine being licensed, other vaccine candidates based on advanced technology platforms are under development. In this report, we rationally designed and constructed two DNA-launched live attenuated vaccine candidates (pDL-EV71) under the control of specific promoters. In vitro and in vivo transfection with pDL-EV71 driven by the CMV promoter successfully yielded fully infectious EV71. More importantly, the administration of pDL-EV71 did not cause clinical symptoms following intracranial or intramuscular inoculation in neonatal and IFNα/βR -/- mice, demonstrating its safety profile. Moreover, a single-dose or two-dose immunization with pDL-EV71 elicited robust neutralizing antibodies against EV71 as well as an antigen-specific cellular response in mice. A single-dose immunization with 10 ​μg of pDL-EV71 conferred complete protection against lethal EV71 infection in neonates born to immunized maternal mice. Overall, our present results demonstrate that pDL-EV71 is a safe and effective vaccine candidate against EV71 for further development., Competing Interests: Conflict of interest Prof. Cheng-Feng Qin is an editorial board member for Virologica Sinica and was not involved in the editorial review or the decision to publish this article. All authors declare that there are no competing interests., (Copyright © 2024 The Authors. Publishing services by Elsevier B.V. All rights reserved.)

Innate immune cells have been acknowledged as trainable in recent years. While intestinal tuft cells are recognized for their crucial roles in the host defense against intestinal pathogens, there remains uncertainty regarding their trainability. Enterovirus 71 (EV71), a prevalent enterovirus that primarily infects children but rarely infects adults. At present, there is a significant expansion of intestinal tuft cells in the EV71-infected mouse model, which is associated with EV71-induced interleukin-25 (IL-25) production. Further, we found that IL-25 pre-treatment at 2 weeks old mouse enabled tuft cells to acquire immune memory. This was evidenced by the rapid expansion and stronger response of IL-25-trained tuft cells in response to EV71 infection at 6 weeks old, surpassing the reactivity of naïve tuft cells in mice without IL-25-trained progress. Interestingly, IL-25-trained intestinal tuft cells exhibit anti-enteroviral effect via producing a higher level of IL-25. Mechanically, IL-25 treatment upregulates spermidine/spermine acetyl-transferase enzyme (SAT1) expression, mediates intracellular polyamine deficiency, further inhibits enterovirus replication. In summary, tuft cells can be trained by IL-25, which supports faster and higher level IL-25 production in response to EV71 infection and further exhibits anti-enteroviral effect via SAT1-mediated intracellular polyamine deficiency. Given that IL-25 can be induced by multiple gut microbes during human growth and development, including shifts in gut flora abundance, which may partially explain the different susceptibility to enteroviral infections between adults and children., (© 2024. The Author(s).)

Vaccine co-administration can efficiently increase vaccination uptake and timely immunization. This study aimed to evaluate the safety of the enterovirus 71 (EV71) vaccine administered alone or concurrently with other vaccines in infants 6-11 months. A total of 3,769 EV71 vaccine doses were administered to children in the active surveillance area, of which 1,909 were administered concurrently with other vaccines and 1,860 doses were administered alone. Active surveillance was conducted to observe adverse events (AEs) within 0-7 and ≥8 days after vaccination and to determine the incidence of reported AEs. The overall AE incidence was 2.12% (95% CI: 1.66%-2.58%), with 1.56% (95% CI:1.00%-2.12%) for the EV71 vaccine alone and 2.67% (95% CI: 1.95%-3.40%) for simultaneous administration of the EV71 vaccine and other vaccines ( x 2  = 5.612, p = .018). The solicited local AE incidence was 1.00% (95% CI: 0.55%-1.44%) in the EV71 vaccine co-administration group and 0.59% (95% CI: 0.24%-0.94%) in the EV71 vaccine alone group ( x 2  = 1.946, p = .018). The solicited systemic AE incidence was 1.68% (95% CI: 1.10%-2.25%) and 0.86% (95% CI: 0.44%-1.28%) in the EV71 vaccine co-administered and EV71 vaccine alone groups, respectively ( x 2  = 4.990, p = .025). No serious vaccine-related AEs were reported. Fever was the most common AE; no difference was observed in the incidence rate of fever between the two groups ( x 2  = 3.467, p = .063). Overall, AE incidence following EV71 vaccination alone or concurrently with other vaccines was acceptable; concurrent vaccination did not increase AE risk or severity.

Trial Registration Number: NCT04993365 (ClinicalTrials.gov).

Coxsackievirus A2 (CVA2) is associated with multiple diseases in children. Currently, there is limited research on immunological detection methods for CVA2. Herein, the VP1 gene of CVA2 strain 201711, belonging to cluster 2 within genotype D, was analyzed. The structures of VP1 from CVA2 strains 201711, 7-1 and 12-1, enterovirus A71 (EV-A71) strain 201713, coxsackievirus A16 (CVA16) strain 201717, and coxsackievirus A6 (CVA6) strain JLS10 were compared. The Escherichia coli BL21(DE3)/pET vector system was employed to express the recombinant protein containing the entire VP1 of CVA2 strain 201711. Mice were immunized with the purified protein, and the sera were collected and used to specifically identify the VP1 in CVA2-infected RD cells by Western blot and immunofluorescence assay. There was no evident cross-reactivity of the sera with the VP1 of EV-A71, CVA16, and CVA6 strains mentioned above. Therefore, this study provided mouse-specific anti-CVA2 VP1 polyclonal antibodies for CVA2 detection., 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.)

Enterovirus 71 (EV-71) has strong neurotropism, and it is the main pathogen causing severe hand, foot, and mouth disease (HFMD). In clinical observations, significant differences were observed in the severity and prognosis of HFMD among children who were also infected with EV-71. Genetic differences among individuals could be one of the important causes of differences in susceptibility to EV-71-induced HFMD. As P-selectin glycoprotein ligand-1 (PSGL-1) is an important receptor of EV-71, the correlation between single-nucleotide polymorphisms (SNPs) in PSGL-1 and the susceptibility to severe HFMD following EV-71 infection is worth studying. Given the role of PSGL-1 in immunity, the correlations between PSGL-1 SNPs and the immune status after EV-71 infection are also worth studying. Meanwhile, PSGL-1 variable number of tandem repeats (VNTR) represents a research hotspot in cardiovascular and cerebrovascular diseases, but PSGL-1 VNTR polymorphism has not been investigated in HFMD caused by EV-71 infection. In this study, specific gene fragments were amplified by polymerase chain reaction, and PSGL-1 VNTR sequences were genotyped using an automatic nucleic acid analyzer. The correlations of PSGL-1 VNTR polymorphism with the susceptibility to EV-71-associated severe HFMD and the post-infection immune status were analyzed. The PSGL-1 VNTR A allele was identified as a susceptible SNP for severe HFMD. The risk of severe HFMD was higher for AA + AB genotype carriers than for BB genotype carriers. The counts of peripheral blood lymphocyte subsets were lower in AA + AB genotype carries than in BB genotype carries. In conclusion, PSGL-1 VNTR polymorphism is associated with the susceptibility to EV-71-induced severe HFMD and the immune status after infection. PSGL-1 VNTR might play a certain role in the pathogenesis of severe cases., (© 2024. The Author(s).)

Enteroviruses are important human pathogens with diverse serotypes, posing a major challenge to develop vaccines for individual serotypes, the success of polio vaccines in controlling and eradicating polio, along with the recent emergence and high prevalence of enterovirus-caused infectious diseases, highlights the importance of enterovirus vaccine development. Given our previous report on enteroviruses weakened by the 2 A S/T125A mutation, we assessed the potential of the EV-A71 2A-125A mutant as a vaccine candidate to address this challenge. We found that the 2A-125A mutant caused transient mild symptoms, low viral loads, and no significant pathological changes mild pathological changes in hSCARB2-KI mice, producing long-lasting cross-neutralizing antibodies against two EV-A71 wild strains. Pre-exposure to the 2A-125A mutant substantially protected against the EV-A71 Isehara wild-type strain, causing minor pathologies, significantly reducing muscle and lung inflammation, and preventing neurological damage, with reduced viral loads in vivo. Pre-exposure also distinctly suppressed the expression of pro-inflammatory cytokines, correlating to the severity of clinical symptoms. Collectively, the EV-A71 2A-125A mutant was attenuated and could generate a robust and protective immune response, suggesting its potential as a vaccine candidate and global solution for specific enterovirus vaccine development., (© 2024 Wiley Periodicals LLC.)

Background: Hand, foot, and mouth disease (HFMD) is a major public health issue in China while temperature and humidity are well-documented predictors. However, evidence on the combined effect of temperature and humidity is still limited. It also remains unclear whether such an effect could be modified by the enterovirus 71 (EV71) vaccination., Methods: Based on 320,042 reported HFMD cases during the summer months between 2012 and 2019, we conducted a study utilizing Distributed Lag Non-Linear Models (DLNM) and time-varying DLNM to examine how China's HFMD EV71 vaccine strategy would affect the correlation between meteorological conditions and HFMD risk., Results: The incidence of HFMD changed with the Discomfort Index in an arm-shaped form. The 14-day cumulative risk of HFMD exhibited a statistically significant increase during the period of 2017-2019 (following the implementation of the EV71 vaccine policy) compared to 2012-2016 (prior to the vaccine implementation). For the total population, the range of relative risk (RR) values for HFMD at the 75th, 90th, and 99th percentiles increased from 1.082-1.303 in 2012-2016 to 1.836-2.022 in 2017-2019. In the stratified analyses, Han Chinese areas show stronger relative growth, with RR values at the 75th, 90th, and 99th percentiles increased by 14.3%, 39.1%, and 134.4% post-vaccination, compared to increases of 22.7%, 41.6%, and 38.8% in minority areas. Similarly, boys showed greater increases (24.4%, 47.7%, 121.5%) compared to girls (8.1%, 28.1%, 58.3%). Additionally, the central Guizhou urban agglomeration displayed a tendency for stronger relative growth compared to other counties., Conclusions: Although the EV71 vaccine policy has been implemented, it hasn't effectively controlled the overall risk of HFMD. There's been a shift in the main viral subtypes, potentially altering population susceptibility and influencing HFMD occurrences. The modulating effects of vaccine intervention may also be influenced by factors such as race, sex, and economic level., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Sun et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Introduction: The Hand, Foot and Mouth Disease (HFMD), caused by enterovirus 71 infection, is a global public health emergency. Severe HFMD poses a significant threat to the life and well-being of children. Numerous studies have indicated that the occurrence of severe HFMD is associated with cytokine storm. However, the precise molecular mechanism underlying cytokine storm development remains elusive, and there are currently no safe and effective treatments available for severe HFMD in children., Methods: In this study, we established a mouse model of severe HFMD to investigate the molecular mechanisms driving cytokine storm. We specifically analyzed metabolic disturbances, focusing on arginine/ornithine metabolism, and assessed the potential therapeutic effects of spermine, an ornithine metabolite., Results: Our results identified disturbances in arginine/ornithine metabolism as a pivotal factor driving cytokine storm onset in severe HFMD cases. Additionally, we discovered that spermine effectively mitigated the inflammatory injury phenotype observed in mice with severe HFMD., Discussion: In conclusion, our findings provide novel insights into the molecular mechanisms underlying severe HFMD from a metabolic perspective while offering a promising new strategy for its safe and effective treatment., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Zhang, Yang, Peng, Tian, Lv, Zeng, Jiang, Cheng, Yang, Zhong, Lu and Zhu.)

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., 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 Ltd. All rights reserved.)

Hand, foot, and mouth disease (HFMD) is a common infectious disease caused by enterovirus 71 (EV71) that frequently affects children, leading to severe infections in some cases. In general, when infection occurs, the body upregulates inflammatory responses to eliminate pathogenic microorganisms to protect the host from infection. However, EV71 may inhibit host's innate immunity to promote virus infection. At present, it is not fully understood how EV71 hijack the host cells for its own replication. Toll-like receptor 4 (TLR4), a natural immune receptor, historically associated with bacterial endotoxin-induced inflammatory responses. However, it is still unclear whether and how TLR4 is altered during EV71 infection. In this study, we observed a reduction in both TLR4 protein and gene transcript levels in RD, GES-1, and Vero cells following EV71 infection, as detected by RT-qPCR, immunofluorescence staining and western blot. Furthermore, we observed that the TLR4 downstream molecules of MYD88, p-NF-κB p65, p-TBK1 and related inflammatory cytokines were also reduced, suggesting that antiviral innate immune and inflammatory response were suppressed. To determine the impact of TLR4 changes on EV71 infection, we interfered EV71-infected RD cells with TLR4 agonist or inhibitor and the results showed that activation of TLR4 inhibited EV71 replication, while inhibition of TLR4 promote EV71 replication. Besides, EV71 replication was also promoted in TLR4 siRNA-transfected and EV71-infected RD cells. This suggests that down-regulation the expression of TLR4 by EV71 can inhibit host immune defense to promote EV71 self-replication. This novel mechanism may be a strategy for EV71 to evade host immunity., Competing Interests: The authors declare the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Hao, Wang, Lu, Li and Zhang.)

Enterovirus 71 (EV71) is a significant causative agent of hand, foot, and mouth disease, with potential serious neurologic complications or fatal outcomes. The lack of effective treatments for EV71 infection is attributed to its elusive pathogenicity. Our study reveals that human plasmacytoid dendritic cells (pDCs), the main type I IFN-producing cells, selectively express scavenger receptor class B, member 2 (SCARB2) and P-selectin glycoprotein ligand 1 (PSGL-1), crucial cellular receptors for EV71. Some strains of EV71 can replicate within pDCs and stimulate IFN-α production. The activation of pDCs by EV71 is hindered by Abs to PSGL-1 and soluble PSGL-1, whereas Abs to SCARB2 and soluble SCARB2 have a less pronounced effect. Our data suggest that only strains binding to PSGL-1, more commonly found in severe cases, can replicate in pDCs and induce IFN-α secretion, highlighting the importance of PSGL-1 in these processes. Furthermore, IFN-α secretion by pDCs can be triggered by EV71 or UV-inactivated EV71 virions, indicating that productive infection is not necessary for pDC activation. These findings provide new insights into the interaction between EV71 and pDCs, suggesting that pDC activation could potentially mitigate the severity of EV71-related diseases., (Copyright © 2024 by The American Association of Immunologists, Inc.)

Enterovirus A71 (EV-A71) is a major pathogen causing hand, foot, and mouth disease (HFMD) in children worldwide. It can lead to severe gastrointestinal, pulmonary, and neurological complications. The innate immune system, which rapidly detects pathogens via pathogen-associated molecular patterns or pathogen-encoded effectors, serves as the first defensive line against EV-A71 infection. Concurrently, the virus has developed various sophisticated strategies to evade host antiviral responses and establish productive infection. Thus, the virus-host interactions and conflicts, as well as the ability to govern biological events at this first line of defense, contribute significantly to the pathogenesis and outcomes of EV-A71 infection. In this review, we update recent progress on host innate immune responses to EV-A71 infection. In addition, we discuss the underlying strategies employed by EV-A71 to escape host innate immune responses. A better understanding of the interplay between EV-A71 and host innate immunity may unravel potential antiviral targets, as well as strategies that can improve patient outcomes.

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 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.

Aim: To develop a trivalent DNA vaccine candidate encapsulated in Chitosan-TPP nanoparticles against hand foot and mouth disease (HFMD) and assess its immunogenicity in mice. Materials & methods: Trivalent plasmid carrying the VP1 and VP2 genes of EV-A71, VP1 gene of CV-A16 was encapsulated in Chitosan-TPP nanoparticles through ionic gelation. In vitro characterization and in vivo immunization studies of the CS-TPP-NPs (pIRES-VP121) were performed. Results: Mice administered with CS-TPP NPs (pIRES-VP121) intramuscularly were observed to have the highest IFN-γ response. Sera from mice immunized with the naked pDNA and CS-TPP-NPs (pIRES-VP121) demonstrated good viral clearance against wild-type EV-A71 and CV-A16 in RD cells. Conclusion: CS-TPP-NPs (pIRES-VP121) could serve as a prototype for future development of multivalent HFMD DNA vaccine candidates.

Enterovirus A71 (EV-A71) is one of the major pathogens responsible for hand, foot, and mouth disease (HFMD). Many HFMD outbreaks have been reported throughout the world in the past decades. Compared with other viruses, EV-A71 infection is more frequently associated with severe neurological complications and even death in children. EV-A71 can also infect adults and cause severe complications and death, although such cases are very uncommon. Although fatal cases of EV-A71 infection have been reported, the underlying mechanisms of EV-A71 infection, especially the mode of viral spread into the central nervous system (CNS) and mechanisms of pulmonary edema, which is considered to be the direct cause of death, have not yet been fully clarified, and more studies are needed. Here, we first summarize the pathological findings in various systems of patients with fatal EV-A71 infections, focussing in detail on gross changes, histopathological examination, tissue distribution of viral antigens and nucleic acids, systemic inflammatory cell infiltration, and tissue distribution of viral receptors and their co-localization with viral antigens. We then present our conclusions about viral dissemination, neuropathogenesis, and the mechanism of pulmonary edema in EV-A71 infection, based on pathological findings., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

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

Hand-foot-and-mouth disease is a contagious disease common among children under 5 years old worldwide. It is caused by strains of enterovirus, especially EV-A71, which can lead to severe disease. Vaccines are the only way to fight this disease. Accordingly, it is necessary to establish an efficient and accurate methodology to evaluate vaccine efficacy in vivo . Here, we established a practical method using a hSCARB2 knock-in mouse model, which was susceptible to EV-A71 infection at 5-6 weeks of age, to directly determine the efficacy of vaccines. Unlike traditional approaches, one-week-old hSCARB2 mice were immunized twice with a licensed vaccine, with an interval of a week. The titre of antibodies was measured after 1 week. Mice at 4 weeks of age were challenged with EV-A71 intraperitoneally and intracranially, respectively. The unimmunized hSCARB2 mice displayed systemic clinical symptoms and succumbed to the disease at a rate of approximately 50%. High viral loads were detected in the lungs, brain, and muscles, accompanied by clear pathological changes. The expression of IL-1β, IL-13, IL-17, and TNF-α was significantly upregulated. By contrast, the immunized group was practically normal and indistinguishable from the control mice. These results indicate that the hSCARB2 knock-in mouse is susceptible to infection in adulthood, and the in vivo efficacy of EV-A71 vaccine could be directly evaluated in this mouse model. The method developed here may be used in the development of new vaccines against HFMD or quality control of licensed vaccines.

Coxsackievirus A6 (CV-A6) has been emerging as a major pathogen of hand, foot and mouth disease (HFMD). Study on the pathogenesis of CV-A6 infection and development of vaccines is hindered by a lack of appropriate animal models. Here, we report an actively immunized-challenged mouse model to evaluate the efficacy of a Vero-cell-based, inactivated CV-A6 vaccine candidate. The neonatal Kunming mice were inoculated with a purified, formaldehyde-inactivated CV-A6 vaccine on days 3 and 9, followed by challenging on day 14 with a naturally selected virulent strain at a lethal dose. Within 14 days postchallenge, all mice in the immunized groups survived, while 100% of the Alum-only inoculated mice died. Neutralizing antibodies (NtAbs) were detected in the serum of immunized suckling mice, and the NtAb levels correlated with the survival rate of the challenged mice. The virus loads in organs were reduced, and pathological changes and viral protein expression were weak in the immunized mice compared with those in Alum-only inoculated control mice. Elevated levels of interleukin-4, 6, interferon γ and tumour necrosis factor α were also observed in Alum-only control mice compared with immunized mice. Importantly, the virulent CV-A6 challenge strain was selected quickly and conveniently from a RD cell virus stock characterized with the natural multi-genotypes. The virulent determinants were mapped to V124M and I242 V at VP1. Together, our results indicated that this actively immunized mouse model is invaluable for future studies to develop multivalent vaccines containing the major component of CV-A6 against HFMD.

Toll-like receptors (TLRs) are essential for the protection of the host from pathogen infections by initiating the integration of contextual cues to regulate inflammation and immunity. However, without tightly controlled immune responses, the host will be subjected to detrimental outcomes. Therefore, it is important to balance the positive and negative regulations of TLRs to eliminate pathogen infection, yet avert harmful immunological consequences. This study revealed a distinct mechanism underlying the regulation of the TLR network. The expression of sex-determining region Y-box 4 (Sox4) is induced by virus infection in viral infected patients and cultured cells, which subsequently represses the TLR signaling network to facilitate viral replication at multiple levels by a distinct mechanism. Briefly, Sox4 inhibits the production of myeloid differentiation primary response gene 88 (MyD88) and most of the TLRs by binding to their promoters to attenuate gene transcription. In addition, Sox4 blocks the activities of the TLR/MyD88/IRAK4/TAK1 and TLR/TRIF/TRAF3/TBK1 pathways by repressing their key components. Moreover, Sox4 represses the activation of the nuclear factor kappa-B (NF-κB) through interacting with IKKα/α, and attenuates NF-kB and IFN regulatory factors 3/7 (IRF3/7) abundances by promoting protein degradation. All these contributed to the down-regulation of interferons (IFNs) and IFN-stimulated gene (ISG) expression, leading to facilitate the viral replications. Therefore, we reveal a distinct mechanism by which viral pathogens evade host innate immunity and discover a key regulator in host defense.

Abstract: A 33-year-old man presented with acute painless loss of vision in his right eye after hand-foot-mouth disease (HFMD). Examination confirmed a right optic neuropathy. Neuroimaging and routine evaluations for alternative causes for an optic neuropathy were negative. He was treated with high dose corticosteroids and made an almost complete visual recovery. Postinfectious optic neuritis has been reported after a vast array of infections including: varicella zoster virus, influenza virus, herpes simplex virus, Epstein-Barr Virus, Lyme disease, and many others. Although Coxsackie virus infections are a known cause of HFMD and have been reported to cause maculopathy, to the best of our knowledge, this is the first reported case of optic neuritis after HFMD in the English language ophthalmic literature., Competing Interests: The authors report no conflicts of interest., (Copyright © 2020 by North American Neuro-Ophthalmology Society.)

The reciprocal interactions between pathogens and hosts are complicated and profound. A comprehensive understanding of these interactions is essential for developing effective therapies against infectious diseases. Interferon responses induced upon virus infection are critical for establishing host antiviral innate immunity. Here, we provide a molecular mechanism wherein isoform switching of the host IKKε gene, an interferon-associated molecule, leads to alterations in IFN production during EV71 infection. We found that IKKε isoform 2 (IKKε v2) is upregulated while IKKε v1 is downregulated in EV71 infection. IKKε v2 interacts with IRF7 and promotes IRF7 activation through phosphorylation and translocation of IRF7 in the presence of ubiquitin, by which the expression of IFNβ and ISGs is elicited and virus propagation is attenuated. We also identified that IKKε v2 is activated via K63-linked ubiquitination. Our results suggest that host cells induce IKKε isoform switching and result in IFN production against EV71 infection. This finding highlights a gene regulatory mechanism in pathogen-host interactions and provides a potential strategy for establishing host first-line defense against pathogens.

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

Coxsackievirus A6 (CVA6) is recognized as a major enterovirus type that can cause severe hand, foot, and mouth disease and spread widely among children. Vaccines and antiviral drugs may be developed more effectively based on a stable and easy-to-operate CVA6 mouse infection model. In this study, a wild CVA6-W strain was sub-cultured in newborn mice of different ages (in days), for adaptation. Therefore, a CVA6-A mouse-adapted strain capable of stably infecting the mice was generated, and a fatal model was built. As the result indicated, CVA6-A could infect the 10-day-old mice to generate higher levels of IFN-γ, IL-6, and IL-10. The mice infected with CVA6-A were treated with IFN-α1b at a higher dose, with complete protection. Based on this strain, an animal model with active immunization was built to evaluate antiviral protection by active immunization. The three-day-old mice were pre-immunized with inactivated CVA6 thereby generating IgM and IgG antibodies within 7 days that enabled complete protection of the pre-immunized mice following the CVA6 virus challenge. There were eight mutations in the genome of CVA6-A than in that of CVA6-W, possibly attributed to the virulence of CVA6 in mice. Briefly, the CVA6 infection model of the 10-day-old mice built herein, may serve as an applicable preclinical evaluation model for CVA6 antiviral drugs and vaccine study., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Jiang, Zhang, Lin, Cheng, Lu, Liu, Zhou, Zhong and Tian.)

The pathogenic human enterovirus EV-A71 has raised serious public health concerns. A hallmark of EV-A71 infection is the distortion of host transcriptomes in favour of viral replication. While high-throughput approaches have been exploited to dissect these gene dysregulations, they do not fully capture molecular perturbations at the single-cell level and in a physiologically relevant context. In this study, we applied a single-cell RNA sequencing approach on infected differentiated enterocyte cells (C2BBe1), which model the gastrointestinal epithelium targeted initially by EV-A71. Our single-cell analysis of EV-A71-infected culture provided several lines of illuminating observations: 1) This systems approach demonstrated extensive cell-to-cell variation in a single culture upon viral infection and delineated transcriptomic differences between the EV-A71-infected and bystander cells. 2) By analysing expression profiles of known EV-A71 receptors and entry facilitation factors, we found that ANXA2 was closely correlated in expression with the viral RNA in the infected population, supporting its role in EV-A71 entry in the enteric cells. 3) We further catalogued dysregulated lncRNAs elicited by EV-A71 infection and demonstrated the functional implication of lncRNA CYTOR in promoting EV-A71 replication. Viewed together, our single-cell transcriptomic analysis illustrated at the single-cell resolution the heterogeneity of host susceptibility to EV-A71 and revealed the involvement of lncRNAs in host antiviral response.

Enterovirus A-71 (EV71) is a global, highly contagkkious pathogen responsible for severe cases of hand-food-mouth-disease (HFMD). The use of vaccines eliciting cross neutralizing antibodies (NTAbs) against the different circulating EV71 sub-genotypes is important for preventing HFMD outbreaks. Here, we tested the cross-neutralizing activities induced by EV71 genotype/sub-genotype A, B0-B4, C1, C2, C4, and C5 viruses using rats. Differences were noted in the cross-neutralization of the 10 sub-genotypes tested but there were generally good levels of cross-neutralization except against genotype A virus, against which neutralization antibody titres (NTAb) where the lowest with NTAbs being the highest against sub-genotype B4. Moreover, NTAb responses induced by C4, B4, C1, and C2 viruses were homogenous, with values of maximum/minimum NTAb ratios (MAX/MIN) against all B and C viruses ranging between 4.0 and 6.0, whereas MAX/MIN values against B3 and A viruses were highly variable, 48.0 and 256.0, respectively. We then dissected the cross-neutralizing ability of sera from infants and children and rats immunized with C4 EV71 vaccines. Cross-neutralizing titers against the 10 sub-genotypes were good in both vaccinated infants and children and rats with the MAX/MIN ranging from 1.8-3.4 and 5.1-7.1, respectively, which were similar to those found in naturally infected patients (2.8). Therefore, we conclude that C4 EV71 vaccines can provide global protection to infants and children against HFMD caused by different sub-genotypes.

Hand, foot, and mouth disease (HFMD) recently emerged as a global public threat. The licensure of inactivated enterovirus A71 (EV-A71) vaccine was the first step in using a vaccine to control HFMD. New challenges arise from changes in the pathogen spectrum while vaccines directed against other common serotypes are in the preclinical stage. The mission of a broad-spectrum prevention strategy clearly favors multivalent vaccines. The development of multivalent vaccines was attempted via the simple combination of potent monovalent vaccines or the construction of chimeric vaccines comprised of epitopes derived from different virus serotypes. The present review summarizes recent advances in HFMD vaccine development and discusses the next steps toward a safe and effective HFMD vaccine that is capable of establishing a cross-protective antibody response.

Enterovirus A71 (EV-A71) and coxsackievirus A16 (CA16) are major etiological agents of hand foot and mouth disease (HFMD) in children, which may result in fatal neurological complications. The development of safe, cost effective vaccines against HFMD, especially for use in developing countries, is still a top public health priority. We have successfully generated a stable, cold-adapted, temperature sensitive/conditional lethal EV-A71 through adaptive culturing in Vero cells at incrementally lower cultivation temperatures. An additional 40 passages at an incubation temperature of 28 °C, and a temperature reversion study at an incubation temperature of 37 °C and 39.5 °C, reveals the virus's phenotypic and genetic stability at the predefined culture conditions. Six unique mutations (two in noncoding regions and four in nonstructural protein-coding genes) in combination may have contributed to its stable phenotype and inability to fully revert to its original wild phenotype. The safety and immunogenicity of this stable, cold-adapted, temperature sensitive/conditional lethal EV-A71 was performed in six monkeys. None of the inoculated monkeys developed any obvious clinical illness except one which developed a transient spike of fever. No gross postmortem lesion or abnormal histological finding was noted for all monkeys at autopsy. No virus was reisolated although EV-A71 specific RNA was detected in serum samples collected on both day 4 and day 8 postinoculation. Only EV-A71 RNA and viral antigen were detected in the spleen homogenate and peripheral blood mononuclear cells, respectively, collected on day 4. The two remaining monkeys developed good humoral immune response on day 14 and day 30 post-inoculation.

Background: Enterovirus 71 (EV71) infection contributes to hand, foot, and mouth disease (HFMD) with severe neurogenic complications, leading to higher morbidity. In addition to their typical roles in coagulation, platelets could serve as essential immune regulatory cells to play a key role in the pathogenesis of this viral infection., Methods: Platelet parameters were measured using an automatic hematology analyzer. T-helper type 1 (Th1) and Th2 cells were analyzed by flow cytometry. The levels of cytokines and key transcription factors were determined., Results: The levels of platelet count and plateletcrit were positively associated with the severity of HFMD. Th1 and Th2 cells as well as their corresponding cytokines were increased in the severe group compared to the healthy volunteers. Moreover, the levels of platelets were negatively correlated with the level of interferon-γ (IFN-γ), but positively correlated with the frequency of Th1 cells. Coculture of platelets and naive CD4 + T cells showed that platelets from mild patients promote Th1 cell differentiation and IFN-γ secretion., Conclusions: Our study has shown for the first time that the distinct roles of platelets are responsible for the regulation of pathogenic CD4 + T cell differentiation and function in the pathogenesis of HFMD caused by EV71., Impact: Our study has shown for the first time that the distinct roles of platelets are responsible for the regulation of pathogenic CD4 + T cell differentiation and function in the pathogenesis of HFMD caused by EV71. For the first time, we have discovered the role of platelets in children's HFMD caused by EV71 infection, which may provide a better treatment for HFMD in the future. This article describes new discoveries in platelet immunity.

In this study, we analyzed ten CVA10 strains were genotyped and cultured for 10 generations to detect plaque morphology, pathogenicity, growth and other characteristics. Mice were injected with live and inactivated virus to detect neutralizing antibody titers. The results suggested that all CVA10 strains fell into Genotype C. Each strain cultured on KMB 17 and Vero cells, increased from 1st generation onwards to peak in the 3rd and 4th, and the titer at which each became infectious ranged from 5.0 to 6.5 and 6.0 to 7.0 lgCCID 50 /ml, respectively. Two-day-old BALB/c mice were selected and inoculated intracerebral with the CVA10 strains, Limb paralysis was significant as early as 3 d; paralysis of all limbs for 50% of affected mice. LT 50 was approximately 6 d, all died within 8 d. Ten strains induced good immune response, the GMT value of booster immunizations was higher than that of initial immunization. This provide reference points for selecting CVA10 vaccine candidates., (Copyright © 2020. Published by Elsevier Inc.)

Background: Since 1997, epidemics of hand, foot, and mouth disease associated with enterovirus A71 (EV-A71) have affected children younger than 5 years in the Asia-Pacific region, including mainland China. EV-A71 vaccines have been licensed for use in children aged 6-71 months in China, but not for infants younger than 6 months. We aimed to assess the dynamics of maternal EV-A71 antibodies to inform choice of potential vaccination strategies to protect infants younger than 6 months, because they have a substantial burden of disease., Methods: We did a longitudinal cohort study with mother-neonate pairs in local hospitals in southern China during 2013-18. We collected cord blood from neonates and venous blood from mothers at delivery. We followed up and collected blood samples from the children at ages 2, 4, 6, 12, 24, and 36 months and tested for the presence of neutralising antibodies against EV-A71 with virus neutralisation assays. Seropositivity, or protective titre, was defined as a neutralisation antibody titre of 16 or higher. We estimated the seroprevalence, geometric mean titre (GMT), and transfer ratio of maternal antibodies. We used a binomial distribution to derive the 95% CIs of seroprevalence. Seropositivity between mothers and neonates was compared by use of an agreement (κ), while GMTs were compared by use of paired Student's t tests., Findings: Between Sept 20, 2013, and Oct 14, 2015, 1054 mothers with 1066 neonates were enrolled. The EV-A71 GMT was similar among pairs of neonates (22·7, 95% CI 20·8-24·9) and mothers (22·1, 95% CI 20·2-24·1; p=0·20). The mean transfer ratio of maternal antibodies was 1·03 (95% CI 0·98-1·08). Although 705 (66%) of 1066 neonates acquired protective concentrations of EV-A71 antibodies from mothers, these declined rapidly, with a half-life of 42 days (95% CI 40-44). The time to loss of protective immunity was extended to 5 months in neonates with mothers who had titres of 128 or higher. By age 30 months, 28% of children had become seropositive because of natural infection., Interpretation: EV-A71 maternal antibodies were efficiently transferred to neonates, but declined quickly to below the protective threshold, particularly among those whose mothers had low antibody titres. Our findings suggest that maternal vaccination could be explored to provide neonatal protection against EV-A71 through maternal antibodies. Catch-up vaccination between ages 6 months to 5 years could provide protection to the approximately 30-90% of children that have not had natural EV-A71 infection by that age., Funding: National Science Fund for Distinguished Young Scholars, National Natural Science Foundation of China., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Coxsackievirus A5 (CV-A5) has recently emerged as a main hand, foot, and mouth disease (HFMD) pathogen. Following a large-scale vaccination campaign against enterovirus 71 (EV-71) in China, the number of HFMD-associated cases with EV-71 was reduced, especially severe and fatal cases. However, the total number of HFMD cases remains high, as HFMD is also caused by other enterovirus serotypes. A multivalent HFMD vaccine containing 4 or 6 antigens of enterovirus serotypes is urgently needed. A formaldehyde-inactivated CV-A5 vaccine derived from Vero cells was used to inoculate newborn Kunming mice on days 3 and 10. The mice were challenged on day 14 with a mouse-adapted CV-A5 strain at a dose that was lethal for 14-day-old suckling mice. Within 14 days postchallenge, groups of mice immunized with three formulations, empty particles (EPs), full particles (FPs), and a mixture of the EP and FP vaccine candidates, all survived, while 100% of the mock-immunized mice died. Neutralizing antibodies (NtAbs) were detected in the sera of immunized mice, and the NtAb levels were correlated with the survival rate of the challenged mice. The virus loads in organs were reduced, and pathological changes and viral protein expression were weak or not observed in the immunized mice compared with those in alum-inoculated control mice. Another interesting finding was the identification of CV-A5 dense particles (DPs), facilitating morphogenesis study. These results demonstrated that the Vero cell-adapted CV-A5 strain is a promising vaccine candidate and could be used as a multivalent HFMD vaccine component in the future. IMPORTANCE The vaccine candidate strain CV-A5 was produced with a high infectivity titer and a high viral particle yield. Three particle forms, empty particles (EPs), full particles (FPs), and dense particles (DPs), were obtained and characterized after purification. The immunogenicities of EP, FP, and the EP and FP mixture were evaluated in mice. Mouse-adapted CV-A5 was generated as a challenge strain to infect 14-day-old mice. An active immunization challenge mouse model was established to evaluate the efficacy of the inactivated vaccine candidate. This animal model mimics vaccination, similar to immune responses of the vaccinated. The animal model also tests protective efficacy in response to the vaccine against the disease. This work is important for the preparation of multivalent vaccines against HFMD caused by different emerging strains., (Copyright © 2021 American Society for Microbiology.)

Enterovirus 71 (EV71) is a positive single-stranded RNA (ssRNA) virus from the enterovirus genus of Picornaviridae family and causes diseases ranged from the mild disease of hand, foot and mouth disease (HFMD) to the severe disease of neurological involvement in young children. TLR7 is an intracellular pattern recognition receptor (PRR) recognizing viral ssRNA. In this study, we investigated the role of TLR7 in EV71 infection in mouse pups (10-12 days old) and found that wild-type (WT) and TLR7 knock-out (TLR7KO) mice infected with EV71 showed similar limb paralysis at the onset and peak of the disease, comparable loss of motor neurons, and similar levels of antiviral molecules in the spinal cord. These results suggest that TLR7 is not the absolute PRR for EV71 in the spinal cord. Interestingly, TLR7KO mice infected with EV71 exhibited significantly delayed recovery from limb paralysis compared with WT mice. TLR7KO mice infected with EV71 showed significantly decreased levels of IgM and IgG2, important antibodies for antiviral humoral immunity. Furthermore, TLR7KO mice infected with EV71 showed a decrease of germinal center B cells in the spleen compared with WT mice. Altogether, our study suggests that TLR7 plays a critical role in anti-viral humoral immunity rather than in being a PRR in the spinal cord during EV71 infection in young mice., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Lin, Lu, Chuang, Kuo, Lin, Li, Wang, Hsueh and Liao.)

BACKGROUND Enterovirus 71 (EV71) is the pathogen most likely to cause HFMD in young children (1-5 years old). A small number of virion protein (VP) vaccine candidates are considered as the protective molecules in EV71 models. This study aimed to observe comprehensive immunogenicity for a promising EV71 vaccine depending on VP1 in neonatal mouse EV71 models. MATERIAL AND METHODS VP1 was isolated from patients and associated peptides were synthesized. EV71 particles were inactivated and mixed with Freund's complete adjuvant to prepare peptide vaccines. An EV71 vaccine was administered to establish the mouse model and the mice were infected with EV71. Hematoxylin and eosin staining was used to examine inflammatory response in EV71-infected neonatal mice. A semi-quantitative reverse transcription-polymerase chain reaction assay was performed to evaluate the levels of EV71 virus in skeletal muscle, small intestines, and brain tissues. RESULTS Three peptides were selected from 20 VP1 peptides due to their exhibition of the highest immunogenicity. The peptide injection improved inflammation and decreased EV71 particle levels in muscle, small intestines, and brain tissues. The injection also decreased lesions in the small intestines of EV71-infected mice and protected brain tissues from the EV71 infection. CONCLUSIONS The present study confirmed the immuno-protective effects of VP1 vaccine transplantation in mice infected with EV71 virus. Our results provide valuable information that can be used in further studies investigating the specific mechanism of the anti-EV71 vaccine.

Coxsackievirus A6 (CV-A6) and Coxsackievirus A10 (CV-A10) have been emerging as the prevailing serotypes and overtaking Enterovirus A71 (EV-A71) and Coxsackievirus A16 (CV-A16) in most areas as main pathogens of hand, foot and mouth disease (HFMD) in China since 2013. To investigate whole etiological spectrum following EV-A71 vaccination of approximate 40,000 infants and young children in Xiangyang, enteroviruses were serotyped in 4415 HFMD cases from October 2016 to December 2017 using Real Time and conventional PCR and cell cultures. Of the typeable 3201 specimen, CV-A6 was the predominant serotype followed by CV-A16, CV-A10, CV-A5, CV-A2 and EV-A71 with proportions of 59.54%, 15.31%, 11.56%, 4.56%, 3.78% and 3.03%, respectively. Other 12 minor serotypes were also detected. The results demonstrated that six major serotypes of enteroviruses were co-circulating, including newly emerged CV-A2 and CV-A5. A dramatic decrease of EV-A71 cases was observed, whereas the total cases remained high. Multivalent vaccines against major serotypes are urgently needed for control of HFMD.

EV-A120 is a recently identified serotype of the enterovirus A species. Only one full-length genomic sequence is currently available in GenBank, and very few studies have been conducted on EV-A120 globally. Thus, additional information and research on EV-A120 are needed to explore its genetic characteristics, phylogeny, and relationship with enteroviral disease. In this study, we report the phylogenetic characteristics of a EV-A120 strain ( Q0082 /XZ/CHN/2000) from Tibet, China. The amino acid sequence similarity and nucleotide sequence similarity of the full-length genomic sequence of this EV-A120 strain and the EV-A120 prototype strain were 96.3% and 79.9%, respectively, showing an evolutionary trend. Recombination analysis found intraspecies recombination in the 5' -UTR , 2B , 2C , and 3D regions. Serum neutralization testing of the EV-A120 ( Q0082 ) strain was also carried out. Low serum-positive rates and geometric mean titres (GMTs) indicated that the extent of EV-A120 transmission and exposure in the population was very limited compared with that in the outbreaks of EV-A71 and CV-A16 in China since 2008. The EV-A120 strain ( Q0082 ) is non-temperature sensitive, indicating its potential to spread in the population. In summary, this study reports the full-length genomic sequence of EV-A120 and provides important information for its global molecular epidemiology.

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.)

Background: Scarce information exists about immunity to hand, foot, and mouth disease (HFMD) among household contacts of index cases in Vietnam and what that means for reducing ongoing HFMD transmission in the community., Methods: We analyzed neutralizing antibodies (NT) and the incidence of enterovirus (EVs) infection among household contacts of index cases in a province where HFMD remains endemic. Throat swab and 2 mL blood samples from household contacts were collected at enrollment, during and after 2 weeks follow-up., Results: The incidence of EV-A71 infection among household contacts was 40/84 (47.6%, 95% Cl: 36.9-58.3%), compared with 106/336 (31.5%, 95% Cl: 26.6-36.5%) for CV-A6 and 36/107 (33.6%, 95% Cl: 24.7-42.6%) for CV-A16. The incidence of CV-A6 infection was fairly constant across ages; in contrast, CV-A71 and CV-A16 had some variation across ages. At baseline, higher geometric mean titer (GMT) of EV-A71, CV-A6, and CV-A16 antibody titers was found for 25-34-year groups (range 216.3 to 305.0) compared to the other age groups. There was a statistically significant difference in GMT values of CV-A6 and CV-A16 between those who had an infection or did not have infection among households with an index case of these serotypes., Conclusions: Our results indicated that adults were becoming infected with HFMD and could be contributing to the transmission. There is, therefore, a need for considering the household setting as an additional target for intervention programs for HFMD., Competing Interests: The author declares that there are no conflicts of interest., (Copyright © 2020 C. Q. Hoang et al.)

Thailand is one of the countries in the Asia-pacific region that has been most affected by the Enterovirus-A71 (EV-A71) epidemic. An individual who is susceptible to EV-A71 may also be infected asymptomatically, thus, a serological assay is a useful tool to estimate the cumulative incidence of infection in the community and to provide guidance for vaccination scheduling. There have been several candidate EV-A71 vaccines, of which three have been approved and licensed in China. The population target for EV-A71 vaccine is children younger than three years of age. In Thailand, there are limited data available on the seroprevalence of EV-A71 neutralizing (NT) antibodies and the timing of seroconversion in children. This study aims to investigate the seroprevalence and seroconversion rate of EV-A71 NT antibody in a cohort of Thai children. Sera were collected at the King Chulalongkorn Memorial Hospital in Bangkok, Thailand from 100 children between 2015 and 2020. Maternal sera were collected on the day of delivery. Serum samples from children were collected at birth (month 0) and at 2, 7, 18, 24, 36, and 48 months of age to test for EV-A71 NT antibody titers using an enzyme-linked immunosorbent assay (ELISA)-based microneutralization test. The seroprotection rate (NT antibody ≥1:16) in children at months 0, 2, 7, 18, 24, 36, and 48 was 81.0%, 60.0%, 9.0%, 10.0%, 13.0%, 17.0%, and 37.1%, respectively. The seroprotection rate was lowest at month 7 due to waning of the maternal antibody and the immunity of children increased with increasing age. At 48 months of age, less than 40% of children were seroprotected. Children at the age of 6 months should be considered a primary target for vaccination., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Enterovirus 71 (EV71)-neutralizing antibodies correlate with protection and have potential as therapeutic agents. We isolate and characterize a panel of plasmablast-derived monoclonal antibodies from an infected child whose antibody response focuses on the plateau epitope near the icosahedral 3-fold axes. Eight of a total of 19 antibodies target this epitope and three of these potently neutralize the virus. Representative neutralizing antibodies 38-1-10A and 38-3-11A both confer effective protection against lethal EV71 challenge in hSCARB2-transgenic mice. The cryo-electron microscopy structures of the EV71 virion in complex with Fab fragments of these potent and protective antibodies reveal the details of a conserved epitope formed by residues in the BC and HI loops of VP2 and the BC and HI loops of VP3 spanning the region around the 3-fold axis. Remarkably, the two antibodies interact with the epitope in quite distinct ways. These plateau-binding antibodies provide templates for promising candidate therapeutics.

Background: Hand, foot, and mouth disease (HFMD) has posed a serious threat to children's health. Three inactivated monovalent enterovirus 71 (EV71) vaccines are proved to be highly efficacious in phase III clinical trials and are now available in China., Methods: We analyzed the citywide surveillance data on HFMD cases in Chengdu during 2009-2018, and estimated cumulative first-dose EV71 vaccination coverage among children eligible to EV71 vaccination after August 2016 in Chengdu. Time series susceptible-infected-recovered model was developed to analyze basic reproduction number and herd immunity threshold of HFMD. Overall and serotype-specific HFMD incidences and severity risks were compared before and after the EV71 vaccination., Results: Among 3 laboratory-identified serotype categories, i.e. EV71, coxsackievirus A16 (CV-A16), and other enteroviruses, the major serotype attributed to HFMD has been changing across years. The cumulative first-dose EV71 vaccination coverage rate was estimated as 60.8% during the study period in Chengdu. By contrast, herd immunity threshold for EV71-related HFMD was 94.0%. After introduction of EV71 vaccines, the overall incidence of HFMD increased 60.8%, mainly driven by 173.7% and 11.8% increased in HFMD caused by other enteroviruses and CV-A16, respectively, which offset a significant reduction in the incidence of HFMD caused by EV71. The overall case-severity risk decreased from 1.4% to 0.3%, with significantly declined presented in all serotype categories., Conclusions: The incidence and severity of EV71-related HFMD decreased following implementation of EV71 vaccination. Developing multivalent vaccines and strengthening laboratory-based surveillance could further decline burden of HFMD.

Enterovirus A71 (EV-A71), the pathogen responsible for the seasonal hand-foot-and-mouth epidemics, can cause significant mortality in infants and young children. The vaccine against EV-A71 could potentially prevent virus-induced neurological complications and mortalities occurring due to the high risk of poliomyelitis-like paralysis and fatal encephalitis. It is known that polysaccharide purified from Ganoderma lucidum (PS-G) can effectively modulate immune function. Here, we used PS-G as an adjuvant with the EV-A71 mucosal vaccine and studied its effects. Our data showed that PS-G-adjuvanted EV-A71 generated significantly better IgA and IgG in the serum, saliva, nasal wash, bronchoalveolar lavage fluid (BALF), and feces. More importantly, these antibodies could neutralize the infectivity of EV-A71 (C2 genotype) and cross-neutralize the B4, B5, and C4 genotypes of EV-A71. In addition, more EV-A71-specific IgA- and IgG- secreting cells were observed with the used of a combination of EV-A71 and PS-G. Furthermore, T-cell proliferative responses and IFN-γ and IL-17 secretions levels were notably increased in splenocytes when the EV-A71 vaccine contained PS-G. We also found that levels of IFN-γ and IL-17 released in Peyer's patch cells were significantly increased in EV-A71, after it was combined with PS-G. We further demonstrated that both CD4 + and CD8 + T cells were able to generate IFN-γ and IL-17 in the spleen. An easy-accessed model of hybrid hSCARB2 + / + /stat-1 - / - mice was used for EV-A71 infection and pathogenesis. We infected the mouse model with EV-A71, which was premixed with mouse sera immunized with the EV-A71 vaccine with or without PS-G. Indeed, in the EV-A71 + PS-G group, the levels of VP1-specific RNA sequences in the brain, spinal cord, and muscle decreased significantly. Finally, hSCARB2-Tg mice immunized via the intranasal route with the PS-G-adjuvanted EV-A71 vaccine resisted a subsequent lethal oral EV-A71 challenge. Taken together, these results demonstrated that PS-G could potentially be used as an adjuvant for the EV-A71 mucosal vaccine., (Copyright © 2020 Lin, Shih, Cheng, Chin, Liou, Lee and Chiang.)

An outbreak of the hand-foot-mouth disease with severe neurological cases, mainly caused by the genotype C1 enterovirus A71 (EV-A71), occurred in Taiwan between 2018 and early 2019. In the recent decade, the most dominant EV-A71 genotypes in Taiwan were B5 and C4 but changed to C1 in 2018. Antibody-mediated immunity plays a key role in limiting the EV-A71 illness in humans. However, the level of neutralizing activities against genotype C1 virus by human polyclonal and monoclonal antibodies (MAbs) remains largely unclear. In the study, we demonstrated that that 39% (9 in 23) of post-infection sera from the genotype B5- or C4-infected patients in 2014-2017 exhibit reduced titers with the 2018-2019 genotype C1 viruses than with the earlier B5 and C4 viruses tested. This finding with polyclonal sera is confirmed with human MAbs derived from genotype B5 virus-infected individuals. The 2018-2019 genotype C1 virus is resistant to the majority of canyon-targeting human MAbs, which may be associated with the residue change near or at the bottom of the canyon region on the viral capsid. The remaining three antibodies (16-2-11B, 16-3-4D, and 17-1-12A), which target VP1 S241 on the 5-fold vertex, VP3 E81 on the 3-fold plateau and VP2 D84 on the 2-fold plateau of genotype C1 viral capsid, respectively, retained neutralizing activities with variable potencies. These neutralizing antibodies were also found to be protective against a lethal challenge of the 2018-2019 genotype C1 virus in an hSCARB2-transgenic mice model. These results indicate that the EV-A71-specific antibody response may consist of a fraction of poorly neutralizing antibodies against 2018-2019 genotype C1 viruses among a subset of previously infected individuals. Epitope mapping of protective antibodies that recognize the emerging genotype C1 virus has implications for anti-EV-A71 MAbs and the vaccine field., Competing Interests: The authors have declared that no competing interests exist.

Enterovirus 71 (EV71) is one of the main pathogens of hand, foot and mouth disease (HFMD) and the main pathogen of severe HFMD. In 2015, three EV71 vaccines were successively marketed in China as powerful prevention and control tools for HFMD caused by EV71. To understand the efficacy, immunogenicity, safety and quality stability of the domestic EV71 vaccine after entering into the market and analyze potential problems in its application, this article incorporates research regarding the immune effect, population effect, safety, quality testing and evaluation results, vaccination willingness and vaccination behavior survey to explore the vaccination strategies for the donll stic EV71 vaccine. EV71 vaccine has good immunogenicity, safety, protective efficacy, and good quality stability after entering into the market, however, only a few study focused on its safety when inoculating with other immunization planning vaccines simultaneously. Strengthen safety monitoring and discuss the safety of the EV71 vaccine especially when simultaneously inoculate with other immunization program vaccines are still necessary. Enterovirus evolution and recombination, whilst the probable impact of the EV71 vaccine can be the reason for future changes of HFMD epidemic strains, hence continuous monitoring of antigenic mutations and genetic evolution of enterovirus should be responded to. Encouraging the R&D of polyvalent vaccines against HFMD is also necessary. Parents' lack of HFMD and EV71 vaccine knowledge was common, therefore HFMD knowledge should be strengthened at the same time when introducing the EV71 vaccine to the public. Also, it should be emphasized that the EV71 vaccine can only prevent HFMD caused by EV71.

Purpose: To explore the molecular genetic mechanisms underlying different responsiveness to Enterovirus 71 (EV71) vaccine., Methods: We recruited 10,245 healthy children into a phase 3 clinical trial to evaluate the efficacy of EV71 vaccine in 2012. Fifty subjects from the trial were divided into the potent immune response group (20 subjects) and the ineffective immune response group (30 subjects). Whole-exome sequencing was performed for these 50 samples and we conducted bioinformatics analyses based on online public database., Results: A total of 222,180 germline variants were detected across 50 subjects. Single nucleotide variant (SNV)-based screening of the subjects with potent or ineffective immune response allowed the identification of a potentially detrimental heterozygous missense variant (c.3784C>T) in EEA1. We also retained TRIM59 and ABCA7 genes that contain different loss of function (LoF) variants shared in two cases and involved in the immune response process. Then, we conducted high-resolution typing of 9 classical HLA genes, HLA-DRB1*03:01, HLA-DQA1*05:01 and HLA-DQB1*02:01 alleles were frequently (recurrence ≥5) observed only in ineffective immune responders., Conclusions: Our study is a meaningful attempt on the comparison of genomic profiles between potent and ineffective immune responders induced by EV71 vaccine, and several candidate potentially detrimental genes were identified., (Copyright © 2020 Jiangsu Provincial Center for Disease Control and Prevention. Published by Elsevier Ltd.. All rights reserved.)