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In this study, we investigated the features of co-infection with SARS-CoV-2 and the enterovirus vaccine strain LEV8 of coxsackievirus A7 or enterovirus A71 for Vero E6 cells and Syrian hamsters. The investigation of co-infection with SARS-CoV-2 and LEV-8 or EV-A71 in the cell model showed that a competitive inhibitory effect for these viruses was especially significant against SARS-CoV-2. Pre-infection with enteroviruses in the animals caused more than a 100-fold decrease in the levels of SARS-CoV-2 virus replication in the respiratory tract and more rapid clearance of infectious SARS-CoV-2 from the lower respiratory tract. Co-infection with SARS-CoV-2 and LEV-8 or EV-A71 also reduced the severity of clinical manifestations of the SARS-CoV-2 infection in the animals. Additionally, the histological data illustrated that co-infection with strain LEV8 of coxsackievirus A7 decreased the level of pathological changes induced by SARS-CoV-2 in the lungs. Research into the chemokine/cytokine profile demonstrated that the studied enteroviruses efficiently triggered this part of the antiviral immune response, which is associated with the significant inhibition of SARS-CoV-2 infection. These results demonstrate that there is significant viral interference between the studied strain LEV-8 of coxsackievirus A7 or enterovirus A71 and SARS-CoV-2 in vitro and in vivo. [ABSTRACT FROM AUTHOR]

The open-source drug library, namely, MMV Pandemic Response Box, contains 153 antiviral agents, a chemically and pharmacologically diverse mixture of early-stage, emerging anti-infective scaffolds, and mature compounds currently undergoing clinical development. Hence, the Pandemic Response Box might contain compounds that bind and interfere with target molecules or cellular pathways that are conserved or shared among the closely related viruses with enterovirus A71 (EV-A71). This study aimed to screen antiviral agents included in the Pandemic Response Box for repurposing to anti-EV-A71 activity and investigate the inhibitory effects of the compounds on viral replication. The compounds' cytotoxicity and ability to rescue infected cells were determined by % cell survival using an SRB assay. The hit compounds were verified for anti-EV-A71 activity by virus reduction assays for viral RNA copy numbers, viral protein synthesis, and mature particle production using qRT-PCR, Western blot analysis, and CCID50 assay, respectively. It was found that some of the hit compounds could reduce EV-A71 genome replication and protein synthesis. D-D7 (2-pyridone-containing human rhinovirus 3C protease inhibitor) exhibited the highest anti-EV-A71 activity. Even though D-D7 has been originally indicated as a polyprotein processing inhibitor of human rhinovirus 3C protease, it could be repurposed as an anti-EV-A71 agent. [ABSTRACT FROM AUTHOR]

Human enterovirus A71 (EV‐A71) is a significant etiological agent responsible for epidemics of hand, foot, and mouth disease (HFMD) in Asia‐Pacific regions. There are presently no licensed antivirals against EV‐A71, and the druggable target for EV‐A71 remains very limited. The phenotypic hit 10,10′‐bis(trifluoromethyl) marinopyrrole A derivative, herein termed MPA‐CF3, is a novel potent small‐molecule inhibitor against EV‐A71, but its pharmacological target(s) and antiviral mechanisms are not defined. Here, quantitative chemoproteomics deciphered the antiviral target of MAP‐CF3 as host factor coatomer subunit zeta‐1 (COPZ1). Mechanistically, MPA‐CF3 disrupts the interaction of COPZ1 with the EV‐A71 nonstructural protein 2C by destabilizing COPZ1 upon binding. The destruction of this interaction blocks the coatomer‐mediated transport of 2C to endoplasmic reticulum, and ultimately inhibits EV‐A71 replication. Taken together, our study disclosed that MPA‐CF3 can be a structurally novel host‐targeting anti‐EV‐A71 agent, providing a structural basis for developing the COPZ1‐targeting broad‐spectrum antivirals against enteroviruses. The mechanistic elucidation of MPA‐CF3 against EV‐A71 may offer an alternative COPZ1‐involved therapeutic pathway for enterovirus infection. [ABSTRACT FROM AUTHOR]

Enterovirus A71 (EV-A71) is among the most neuropathogenic non-polio enterovirus types and, in rare instances, can lead to severe or even fatal outcomes, particularly in children under 5 years of age. This case study presents clinical and microbiological findings from the initial documented severe pediatric EV-A71 case in Finland, identified in May 2019. The nearcomplete genome sequence confirms that the EV-A71 strain belongs to the newly identified recombinant C1-like EV-A71 genetic lineage, which emerged in 2015 and has since been circulating in Europe, causing severe cases among children in various European countries. Enhanced environmental surveillance revealed widespread circulation of EV-A71 in Finland in 2019. However, the overall number of EV clinical cases remained lower than in previous years. [ABSTRACT FROM AUTHOR]

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. 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. 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). These findings reiterate the need to continue the development of a multivalent vaccine or combined vaccines, and have implications for introducing optimized vaccination strategies. [ABSTRACT FROM AUTHOR]

Small animal models play an important role in investigating and revealing the molecular determinants and mechanisms underlying neuro-virulence of enterovirus A71 (EV-A71). In our previous study, we successfully developed two mouse cell-line replication competent EV-A71 strains (EV71:TLLm and EV71:TLLmv) which were capable of inducing neuro-invasion in BALB/c mice. The more virulent EV71:TLLmv exhibited ability to induce acute encephalomyelitis accompanied by neurogenic pulmonary oedema. EV71:TLLcho virus strain was generated from EV71:TLLm by a series of passages in CHO-K1 cells. EV71:TLLcho demonstrated a broader range of infectivity across various mammalian cell lines and exhibited complete cytopathic effects (CPE) within 48 hours post-inoculation in comparison to EV71:TLLm or EV71:TLLmv. EV71:TLLcho consistently yielded higher levels of viral replication at all time points examined. In comparison to EV71:TLLm, EV71:TLLcho consistently induced more severe disease and increased mortality in one-week old BALB/c mice. However, unlike mice challenged with EV71:TLLmv, none of the mice challenged with EV71:TLLcho progressed to severe acute encephalomyelitis and developed neurogenic pulmonary oedema. [ABSTRACT FROM AUTHOR]

Background: Enterovirus A71 (EV-A71) is capable of causing hand, foot and mouth disease (HFMD), which may lead to neurological sequelae and even death. As EV-A71 is resistant to environmental changes and mutates easily, there is still a lack of effective treatments or globally available vaccines. Aim of Review: For more than 50 years since the HFMD epidemic, related drug research has been conducted. Progress in this area can promote the further application of existing potential drugs and develop more efficient and safe antiviral drugs, and provide useful reference for protecting the younger generation and maintaining public health security. Key Scientific Concepts of Review: At present, researchers have identified hundreds of EV-A71 inhibitors based on screening repurposed drugs, targeted structural design, and rational modification of previously effective drugs as the main development strategies. This review systematically introduces the current potential drugs to inhibit EV-A71 infection, including viral inhibitors targeting key sites such as the viral capsid, RNA-dependent RNA polymerase (RdRp), 2C protein, internal ribosome entry site (IRES), 3C proteinase (3Cpro), and 2A proteinase (2Apro), starting from each stage of the viral life cycle. Meanwhile, the progress of host-targeting antiviral drugs and their development are summarized in terms of regulating host immunity, inhibiting autophagy or apoptosis, and regulating the cellular redox environment. In addition, the current clinical methods for the prevention and treatment of HFMD are summarized and discussed with the aim of providing support and recommendations for the treatment of enterovirus infections including EV-A71.

We report on a 2023 outbreak of severe hand, foot, and mouth disease in southern Vietnam caused by an emerging lineage of enterovirus A71 subgenogroup B5. Affected children were significantly older than those reported during previous outbreaks. The virus should be closely monitored to assess its potential for global dispersal.

A positive-sense (+) single-stranded RNA (ssRNA) virus (e.g. enterovirus A71, EV-A71) depends on viral polypeptide translation for initiation of virus replication after entry. We reported that EV-A71 hijacks Hsp27 to induce hnRNP A1 cytosol redistribution to initiate viral protein translation, but the underlying mechanism is still elusive. Here, we show that phosphorylation-deficient Hsp27-3A (Hsp27S15/78/82A) and Hsp27S78A fail to translocate into the nucleus and induce hnRNP A1 cytosol redistribution, while Hsp27S15A and Hsp27S82A display similar effects to the wild type Hsp27. Furthermore, we demonstrate that the viral 2A protease (2Apro) activity is a key factor in regulating Hsp27/hnRNP A1 relocalization. Hsp27S78A dramatically decreases the IRES activity and viral replication, which are partially reduced by Hsp27S82A. However, Hsp27S15A displays the same activity as the wild-type Hsp27. Peptide S78 potently suppresses EV-A71 protein translation and reproduction through blockage of EV-A71-induced Hsp27 phosphorylation and Hsp27/hnRNP A1 relocalization. A point mutation (S78A) on S78 impairs its inhibitory functions on Hsp27/hnRNP A1 relocalization and viral replication. Taken together, we demonstrate the importance of Ser78 phosphorylation of Hsp27 regulated by virus infection in nuclear translocation, hnRNP A1 cytosol relocation, and viral replication, suggesting a new path (such as peptide S78) for target-based antiviral strategy.

ABSTRACTThe 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

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. [ABSTRACT FROM AUTHOR]

[Display omitted] • The structural characteristics of EV71 and the mechanism of infection were reviewed. • Anti-EV71 drug candidates for different targets based on in vitro , animal, and clinical data were summarized. • The main therapeutic approaches for EV71 infection were discussed, and suggestions were made for further drug development. Enterovirus A71 (EV-A71) is capable of causing hand, foot and mouth disease (HFMD), which may lead to neurological sequelae and even death. As EV-A71 is resistant to environmental changes and mutates easily, there is still a lack of effective treatments or globally available vaccines. For more than 50 years since the HFMD epidemic, related drug research has been conducted. Progress in this area can promote the further application of existing potential drugs and develop more efficient and safe antiviral drugs, and provide useful reference for protecting the younger generation and maintaining public health security. At present, researchers have identified hundreds of EV-A71 inhibitors based on screening repurposed drugs, targeted structural design, and rational modification of previously effective drugs as the main development strategies. This review systematically introduces the current potential drugs to inhibit EV-A71 infection, including viral inhibitors targeting key sites such as the viral capsid, RNA-dependent RNA polymerase (RdRp), 2C protein, internal ribosome entry site (IRES), 3C proteinase (3Cpro), and 2A proteinase (2Apro), starting from each stage of the viral life cycle. Meanwhile, the progress of host-targeting antiviral drugs and their development are summarized in terms of regulating host immunity, inhibiting autophagy or apoptosis, and regulating the cellular redox environment. In addition, the current clinical methods for the prevention and treatment of HFMD are summarized and discussed with the aim of providing support and recommendations for the treatment of enterovirus infections including EV-A71. [ABSTRACT FROM AUTHOR]

Enterovirus A71 (EV71), coxsackievirus A16 (CVA16), and coxsackievirus B3 (CVB3) are pathogenic members of the Picornaviridae family that cause a range of diseases, including severe central nervous system complications, myocarditis, and pancreatitis. Despite the considerable public health impact of these viruses, no approved antiviral treatments are currently available. In the present study, we confirmed the potential of saucerneol, a compound derived from Saururus chinensis, as an antiviral agent against EV71, CVA16, and CVB3. In the in vivo model, saucerneol effectively suppressed CVB3 replication in the pancreas and alleviated virus-induced pancreatitis. The antiviral activity of saucerneol is associated with increased mitochondrial ROS (mROS) production. In vitro inhibition of mROS generation diminishes the antiviral efficacy of saucerneol. Moreover, saucerneol treatment enhanced the phosphorylation of STING, TBK-1, and IRF3 in EV71- and CVA16-infected cells, indicating that its antiviral effects were mediated through the STING/TBK-1/IRF3 antiviral pathway, which was activated by increased mROS production. Saucerneol is a promising natural antiviral agent against EV71, CVA16, and CVB3 and has potential against virus-induced pancreatitis and myocarditis. Further studies are required to assess its safety and efficacy, which is essential for the development of effective antiviral strategies against these viruses. [ABSTRACT FROM AUTHOR]

Enterovirus A71 (EV-A71) can induce severe neurological complications and even fatal encephalitis in children, and it has caused several large outbreaks in Taiwan since 1998. We previously generated VP1 codon-deoptimized (VP1-CD) reverse genetics (rg) EV-A71 viruses (rgEV-A71s) that harbor a high-fidelity (HF) 3D polymerase. These VP1-CD-HF rgEV-A71s showed lower replication kinetics in vitro and decreased virulence in an Institute of Cancer Research (ICR) mouse model of EV-A71 infection, while still retaining their antigenicity in comparison to the wild-type virus. In this study, we aimed to further investigate the humoral and cellular immune responses elicited by VP1-CD-HF rgEV-A71s to assess the potential efficacy of these EV-A71 vaccine candidates. Following intraperitoneal (i.p.) injection of VP1-CD-HF rgEV-A71s in mice, we observed a robust induction of EV-A71-specific neutralizing IgG antibodies in the antisera after 21 days. Splenocytes isolated from VP1-CD-HF rgEV-A71s-immunized mice exhibited enhanced proliferative activities and cytokine production (IL-2, IFN-, IL-4, IL-6, and TNF-a) upon re-stimulation with VP1-CD-HF rgEV-A71, as compared to control mice treated with adjuvant only. Importantly, administration of antisera from VP1-CD-HF rgEV-A71s-immunized mice protected against lethal EV-A71 challenge in neonatal mice. These findings highlight that our generated VP1-CD-HF rgEV-A71 viruses are capable of inducing both cellular and humoral immune responses, supporting their potential as next-generation EV-A71 vaccines for combating EV-A71 infection. IMPORTANCE EV-A71 can cause severe neurological diseases and cause death in young children. Here, we report the development of synthetic rgEV-A71s with the combination of codon deoptimization and high-fidelity (HF) substitutions that generate genetically stable reverse genetics (rg) viruses as potential attenuated vaccine candidates. Our work provides insight into the development of low-virulence candidate vaccines through a series of viral genetic editing for maintaining antigenicity and genome stability and suggests a strategy for the development of an innovative next-generation vaccine against EV-A71. [ABSTRACT FROM AUTHOR]

Abstract Human enterovirus A71 (EV‐A71) is a significant etiological agent responsible for epidemics of hand, foot, and mouth disease (HFMD) in Asia‐Pacific regions. There are presently no licensed antivirals against EV‐A71, and the druggable target for EV‐A71 remains very limited. The phenotypic hit 10,10′‐bis(trifluoromethyl) marinopyrrole A derivative, herein termed MPA‐CF3, is a novel potent small‐molecule inhibitor against EV‐A71, but its pharmacological target(s) and antiviral mechanisms are not defined. Here, quantitative chemoproteomics deciphered the antiviral target of MAP‐CF3 as host factor coatomer subunit zeta‐1 (COPZ1). Mechanistically, MPA‐CF3 disrupts the interaction of COPZ1 with the EV‐A71 nonstructural protein 2C by destabilizing COPZ1 upon binding. The destruction of this interaction blocks the coatomer‐mediated transport of 2C to endoplasmic reticulum, and ultimately inhibits EV‐A71 replication. Taken together, our study disclosed that MPA‐CF3 can be a structurally novel host‐targeting anti‐EV‐A71 agent, providing a structural basis for developing the COPZ1‐targeting broad‐spectrum antivirals against enteroviruses. The mechanistic elucidation of MPA‐CF3 against EV‐A71 may offer an alternative COPZ1‐involved therapeutic pathway for enterovirus infection.

Enterovirus A71 (EV-A71) is among the most neuropathogenic non-polio enterovirus types and, in rare instances, can lead to severe or even fatal outcomes, particularly in children under 5 years of age. This case study presents clinical and microbiological findings from the initial documented severe pediatric EV-A71 case in Finland, identified in May 2019. The near-complete genome sequence confirms that the EV-A71 strain belongs to the newly identified recombinant C1-like EV-A71 genetic lineage, which emerged in 2015 and has since been circulating in Europe, causing severe cases among children in various European countries. Enhanced environmental surveillance revealed widespread circulation of EV-A71 in Finland in 2019. However, the overall number of EV clinical cases remained lower than in previous years.

Abstract Background Human enteroviruses A71 (EV-A71) and D68 (EV-D68) are the suspected causative agents of hand-foot-and-mouth disease, aseptic meningitis, encephalitis, acute flaccid myelitis, and acute flaccid paralysis in children. Until now, no cure nor mucosal vaccine existed for EV-A71 and EV-D68. Novel mucosal bivalent vaccines are highly important for preventing EV-A71 and EV-D68 infections. Methods In this study, formalin-inactivated EV-A71 and EV-D68 were used as antigens, while PS-G, a polysaccharide from Ganoderma lucidum, was used as an adjuvant. Natural polysaccharides have the characteristics of intrinsic immunomodulation, biocompatibility, low toxicity, and safety. Mice were immunized intranasally with PBS, EV-A71, EV-D68, or EV-A71 + EV-D68, with or without PS-G as an adjuvant. Results The EV-A71 + EV-D68 bivalent vaccine generated considerable EV-A71- and EV-D68-specific IgG and IgA titres in the sera, nasal washes, saliva, bronchoalveolar lavage fluid, and feces. These antibodies neutralized EV-D68 and EV-A71 infectivity. They also cross-neutralized infections by different EV-D68 and EV-A71 sub-genotypes. Furthermore, compared with the PBS group, EV-A71 + EV-D68 + PS-G-vaccinated mice exhibited an increased number of EV-D68- and EV-A71-specific IgA- and IgG-producing cells. In addition, T-cell proliferative responses, and IFN-γ and IL-17 secretion in the spleen were substantially induced when PS-G was used as an adjuvant with EV-A71 + EV-D68. Finally, in vivo challenge experiments demonstrated that the immune sera induced by EV-A71 + EV-D68 + PS-G conferred protection in neonate mice against lethal EV-A71 and EV-D68 challenges as indicated by the increased survival rate and decreased clinical score and viral RNA tissue expression. Taken together, all EV-A71/EV-D68 + PS-G-immunized mice developed potent specific humoral, mucosal, and cellular immune responses to EV-D68 and EV-A71 and were protected against them. Conclusions These findings demonstrated that PS-G can be used as a potential adjuvant for EV-A71 and EV-D68 bivalent mucosal vaccines. Our results provide useful information for the further preclinical and clinical development of a mucosal bivalent enterovirus vaccine against both EV-A71 and EV-D68 infections. Graphical Abstract

Background: Vaccine stability is an important issue for vaccine development, which affects whether the vaccine product is effective within a certain period of time in each progress. Hand, foot, and mouth diseases (HFMD) is an epidemic disease in young children usually caused by Enterovirus A group viruses, and the Enterovirus A71 (EV-A71) had caused several pandemics and public health issues around the world. After two decades of research and development, formalin-inactivated EV-A71 (FI-EV-A71) vaccines are the first to complete the phase III clinical trials for protection against EV-A71 infection. Currently, the shelf life of FI-EV-A71 vaccine product is set to be within 18 months, but the stability and the effectiveness of the FI-EV-A71 whole virion when stored long-term at low temperature remains undetermined. Methods: Assessing the long-term storage properties of viral particles facilitates flexibility in manufacturing of vaccine products. In this study, the stability profiles of FI-EV-A71 vaccine lots and bulks after long-term of low temperature storage were analyzed by protein tests, particle measurement and animal immunization study. Results: After over ten years of storage, the reduction of protein concentration in the FI-EV-A71 bulk samples is less than 30 % and the antigenic content remained in a suspended, particulate state. Both the packed FI-EV-A71 final vaccine products and the FI-EV-A71 antigens adjuvant premix bulk could elicit strong neutralizing responses in mice. Conclusion: After ten years of low temperature storage, the FI-EV-A71 vaccine still presents decent stability and good immunogenicity.

Background: Human enteroviruses A71 (EV-A71) and D68 (EV-D68) are the suspected causative agents of hand-foot-and-mouth disease, aseptic meningitis, encephalitis, acute flaccid myelitis, and acute flaccid paralysis in children. Until now, no cure nor mucosal vaccine existed for EV-A71 and EV-D68. Novel mucosal bivalent vaccines are highly important for preventing EV-A71 and EV-D68 infections. Methods: In this study, formalin-inactivated EV-A71 and EV-D68 were used as antigens, while PS-G, a polysaccharide from Ganoderma lucidum, was used as an adjuvant. Natural polysaccharides have the characteristics of intrinsic immunomodulation, biocompatibility, low toxicity, and safety. Mice were immunized intranasally with PBS, EV-A71, EV-D68, or EV-A71 + EV-D68, with or without PS-G as an adjuvant. Results: The EV-A71 + EV-D68 bivalent vaccine generated considerable EV-A71- and EV-D68-specific IgG and IgA titres in the sera, nasal washes, saliva, bronchoalveolar lavage fluid, and feces. These antibodies neutralized EV-D68 and EV-A71 infectivity. They also cross-neutralized infections by different EV-D68 and EV-A71 sub-genotypes. Furthermore, compared with the PBS group, EV-A71 + EV-D68 + PS-G-vaccinated mice exhibited an increased number of EV-D68- and EV-A71-specific IgA- and IgG-producing cells. In addition, T-cell proliferative responses, and IFN-γ and IL-17 secretion in the spleen were substantially induced when PS-G was used as an adjuvant with EV-A71 + EV-D68. Finally, in vivo challenge experiments demonstrated that the immune sera induced by EV-A71 + EV-D68 + PS-G conferred protection in neonate mice against lethal EV-A71 and EV-D68 challenges as indicated by the increased survival rate and decreased clinical score and viral RNA tissue expression. Taken together, all EV-A71/EV-D68 + PS-G-immunized mice developed potent specific humoral, mucosal, and cellular immune responses to EV-D68 and EV-A71 and were protected against them. Conclusions: These findings demonstrated that PS-G can be used as a potential adjuvant for EV-A71 and EV-D68 bivalent mucosal vaccines. Our results provide useful information for the further preclinical and clinical development of a mucosal bivalent enterovirus vaccine against both EV-A71 and EV-D68 infections. [ABSTRACT FROM AUTHOR]

Enteroviruses encode two protease active sites, in the 2A and 3C coding regions. While they target many host proteins, they first need to be excised from the viral polyprotein in which they are embedded. Polyprotein cleavage can occur either intra-molecularly (in cis) or inter-molecularly (in trans). Previous work suggested that antivirals targeting intra-molecular cleavages could generate inhibitory precursors that can suppress the outgrowth of drug-resistant variants. Therefore, we wanted to evaluate enteroviral cleavage patterns to identify such obligate intra-molecular cleavages as a method of drug target selection. Using translation extracts, we show that cis cleavage of the 2A protease N-terminal junction is conserved across three enterovirus clades, while the mechanism for the N-terminal junction of 3C varies, with enterovirus D68 3C cleavage occurring in cis and poliovirus 3C cleavage occurring in trans. Antiviral agents targeting proteases are generally identified via their ability to block the cleavage of artificial peptide substrates. Here, we show that antivirals identified for their ability to block inter-molecular cleavage can sometimes block intra-molecular cleavage of the protease from its polyprotein but with widely varying efficacy. Additionally, we demonstrate that for three enteroviral species, genomes defective in 2A protease activity suppress the growth of wild-type virus in mixed populations, supporting the hypothesis that preventing intra-molecular cleavage at the VP1·2A junction can create dominantly inhibitory precursors. These data argue that, to reduce the likelihood of drug resistance, protease-targeted antivirals should be evaluated for their ability to block intra-molecular polyprotein cleavages in addition to inter-molecular cleavage of other substrates. [ABSTRACT FROM AUTHOR]

Hand, foot, and mouth disease (HFMD) caused by enterovirus A71 (EV-A71) infection, currently lacks specific preventive and therapeutic interventions. Here, we demonstrated that Pien Tze Huang (PZH) could dose-dependently inhibit EV-A71 replication at the cellular level, resulting in significant reductions in EV-A71 virus protein 1 (VP1) expression and viral yields in Vero and human rhabdomyosarcoma cells. More importantly, we confirmed that PZH could protect mice from EV-A71 infection for the first time, with Ribavirin serving as a positive control. PZH treatment reduced EV-A71 VP1 protein expression, viral yields in infected muscles, and improved muscle pathology. Additionally, we conducted a preliminary mechanism study using quantitative proteomics. The results suggested that the suppression of the PI3K/AKT/mTOR and NF-κB signaling pathways may contribute to the anti-EV-A71 activity of PZH. These findings provide strong evidence supporting the potential therapeutic application of PZH for EV-A71 infection management. [ABSTRACT FROM AUTHOR]

Abstract Background Enterovirus A71 is one of the causative agents of hand, foot, and mouth disease, which is usually a self-limiting disease. Complications of enterovirus infection are also very rare. However, when such complications occur, they can lead to serious neurological diseases or even death. Case presentation In this report, we describe a case of enterovirus A71-associated acute flaccid paralysis in a 13-month-old Caucasian girl that was managed in our hospital. The patient presented with sudden onset of left arm paresis that could not be attributed to any other cause. Establishing a diagnosis was furthermore complicated by negative virological investigations of cerebrospinal fluid and non-pathological radiological findings. A polymerase chain reaction test of the child’s stool sample however tested positive for enterovirus and sequencing results revealed the presence of enterovirus A71. A previous history of febrile gastroenteritis just before the paresis started also supported the suspected diagnosis of enterovirus-associated acute flaccid paralysis. Following this, the child was treated with intravenous immunoglobulin over 5 days and a remarkable improvement was observed in the child’s paresis. Conclusion This case report describes a possible complication of enterovirus A71 infection in a child. It also highlights the prolonged detection of enterovirus in the child’s stool sample as compared with cerebrospinal fluid weeks after the primary infection occurred. Finally, it shows the need for increased clinical and diagnostic awareness especially in the management of sudden and unknown causes of paresis or paralysis in children.

The open-source drug library, namely, MMV Pandemic Response Box, contains 153 antiviral agents, a chemically and pharmacologically diverse mixture of early-stage, emerging anti-infective scaffolds, and mature compounds currently undergoing clinical development. Hence, the Pandemic Response Box might contain compounds that bind and interfere with target molecules or cellular pathways that are conserved or shared among the closely related viruses with enterovirus A71 (EV-A71). This study aimed to screen antiviral agents included in the Pandemic Response Box for repurposing to anti-EV-A71 activity and investigate the inhibitory effects of the compounds on viral replication. The compounds’ cytotoxicity and ability to rescue infected cells were determined by % cell survival using an SRB assay. The hit compounds were verified for anti-EV-A71 activity by virus reduction assays for viral RNA copy numbers, viral protein synthesis, and mature particle production using qRT-PCR, Western blot analysis, and CCID50 assay, respectively. It was found that some of the hit compounds could reduce EV-A71 genome replication and protein synthesis. D-D7 (2-pyridone-containing human rhinovirus 3C protease inhibitor) exhibited the highest anti-EV-A71 activity. Even though D-D7 has been originally indicated as a polyprotein processing inhibitor of human rhinovirus 3C protease, it could be repurposed as an anti-EV-A71 agent.

In this study, we investigated the features of co-infection with SARS-CoV-2 and the enterovirus vaccine strain LEV8 of coxsackievirus A7 or enterovirus A71 for Vero E6 cells and Syrian hamsters. The investigation of co-infection with SARS-CoV-2 and LEV-8 or EV-A71 in the cell model showed that a competitive inhibitory effect for these viruses was especially significant against SARS-CoV-2. Pre-infection with enteroviruses in the animals caused more than a 100-fold decrease in the levels of SARS-CoV-2 virus replication in the respiratory tract and more rapid clearance of infectious SARS-CoV-2 from the lower respiratory tract. Co-infection with SARS-CoV-2 and LEV-8 or EV-A71 also reduced the severity of clinical manifestations of the SARS-CoV-2 infection in the animals. Additionally, the histological data illustrated that co-infection with strain LEV8 of coxsackievirus A7 decreased the level of pathological changes induced by SARS-CoV-2 in the lungs. Research into the chemokine/cytokine profile demonstrated that the studied enteroviruses efficiently triggered this part of the antiviral immune response, which is associated with the significant inhibition of SARS-CoV-2 infection. These results demonstrate that there is significant viral interference between the studied strain LEV-8 of coxsackievirus A7 or enterovirus A71 and SARS-CoV-2 in vitro and in vivo.

Enterovirus A71 (EV-A71) is a significant hand-foot-mouth disease (HFMD) etiology. The inactivated EV-A71 vaccines were approved in China in 2016. However, the seroprevalence of EV-A71 after the vaccine application and its potential association with the EV-A71 epidemic in the population are rarely studied. In this study, we analyzed the incidence of EV-A71 infection and seroepidemiology in Guangzhou City, China. From 2019 to 2021, 167,920 clinically confirmed HFMD cases were reported in Guangzhou. In 6,868 enterovirus-positive samples, Coxsackievirus A6 and Coxsackievirus A16 were dominant genotypes, and only 3 EV-A71-positive samples were detected, highlighting the deficient epidemic activity of EV-A71. Microneutralization assay was performed on 1,000 representative serum samples. Notably, the seroprevalence and geometric mean titer (GMT) decreased significantly in 2020, and that in the < 3-year age group were increased and even higher than that in 3–5-year age group in 2019 and 2021, which was contrary to our previous surveillance result and other studies in Guangzhou. Furthermore, a moderate decline of GMT level was observed following the vaccination, but the seropositive serums were still detected for 49 months after second immunization, suggesting the long-term persistence of the immunity. Our seroepidemiology study revealed relatively higher neutralizing antibody activity in the susceptible population after the EV-A71 vaccine was adopted in 2016 in Guangzhou. It may be one of the reasons for the lower epidemic activity of EV-A71 in Guangzhou from 2019 to 2021.

Enterovirus A71 (EV‐A71) is a highly contagious virus that poses a major threat to global health, representing the primary etiological agent for hand–foot and mouth disease (HFMD) and neurological complications. It has been established that interferon signaling is critical to establishing a robust antiviral state in host cells, mainly mediated through the antiviral effects of numerous interferon‐stimulated genes (ISGs). The host restriction factor SHFL is a novel ISG with broad antiviral activity against various viruses through diverse underlying molecular mechanisms. Although SHFL is widely acknowledged for its broad‐spectrum antiviral activity, it remains elusive whether SHFL inhibits EV‐A71. In this work, we validated that EV‐A71 triggers the upregulation of SHFL both in cell lines and in a mouse model. Knockdown and overexpression of SHFL in EVA71‐infected cells suggested that this factor could markedly suppress EV‐A71 replication. Our findings further revealed an intriguing mechanism of SHFL that it could interact with the nonstructural proteins 3Dpol of EV‐A71 and promoted the degradation of 3Dpol through the ubiquitin–proteasome pathway. Furthermore, the zinc‐finger domain and the 36 amino acids (164–199) of SHFL were crucial to the interaction between SHFL and EV‐A71 3Dpol. Overall, these findings broadened our understanding of the pivotal roles of SHFL in the interaction between the host and EV‐A71. [ABSTRACT FROM AUTHOR]

Hand, foot and mouth disease (HFMD) is a childhood disease caused by enterovirus A71 (EV-A71) and coxsackievirus A16 (CV-A16). Capsid loops are important epitopes for EV-A71 and CV-A16. Seven chimeric EV-A71 (ChiE71) involving VP1 BC (45.5% similarity), DE, EF, GH and HI loops, VP2 EF loop and VP3 GH loop (91.3% similarity) were substituted with corresponding CV-A16 loops. Only ChiE71-1-BC, ChiE71-1-EF, ChiE71-1-GH and ChiE71-3-GH were viable. EV-A71 and CV-A16 antiserum neutralized ChiE71-1-BC and ChiE71-1-EF. Mice immunized with inactivated ChiE71 elicited high IgG, IFN-γ, IL-2, IL-4 and IL-10. Neonatal mice receiving passive transfer of WT EV-A71, ChiE71-1-EF and ChiE71-1-BC immune sera had 100%, 80.0% and no survival, respectively, against lethal challenges with EV-A71, suggesting that the substituted CV-A16 loops disrupted EV-A71 immunogenicity. Passive transfer of CV-A16, ChiE71-1-EF and ChiE71-1-BC immune sera provided 40.0%, 20.0% and 42.9% survival, respectively, against CV-A16. One-day-old neonatal mice immunized with WT EV-A71, ChiE71-1-BC, ChiE71-1-EF and CV-A16 achieved 62.5%, 60.0%, 57.1%, and no survival, respectively, after the EV-A71 challenge. Active immunization using CV-A16 provided full protection while WT EV-A71, ChiE71-1-BC and ChiE71-1-EF immunization showed partial cross-protection in CV-A16 lethal challenge with survival rates of 50.0%, 20.0% and 40%, respectively. Disruption of a capsid loop could affect virus immunogenicity, and future vaccine design should include conservation of the enterovirus capsid loops. [ABSTRACT FROM AUTHOR]

Background: Enterovirus A71 is one of the causative agents of hand, foot, and mouth disease, which is usually a self-limiting disease. Complications of enterovirus infection are also very rare. However, when such complications occur, they can lead to serious neurological diseases or even death. Case presentation: In this report, we describe a case of enterovirus A71-associated acute flaccid paralysis in a 13-month-old Caucasian girl that was managed in our hospital. The patient presented with sudden onset of left arm paresis that could not be attributed to any other cause. Establishing a diagnosis was furthermore complicated by negative virological investigations of cerebrospinal fluid and non-pathological radiological findings. A polymerase chain reaction test of the child's stool sample however tested positive for enterovirus and sequencing results revealed the presence of enterovirus A71. A previous history of febrile gastroenteritis just before the paresis started also supported the suspected diagnosis of enterovirus-associated acute flaccid paralysis. Following this, the child was treated with intravenous immunoglobulin over 5 days and a remarkable improvement was observed in the child's paresis. Conclusion: This case report describes a possible complication of enterovirus A71 infection in a child. It also highlights the prolonged detection of enterovirus in the child's stool sample as compared with cerebrospinal fluid weeks after the primary infection occurred. Finally, it shows the need for increased clinical and diagnostic awareness especially in the management of sudden and unknown causes of paresis or paralysis in children. [ABSTRACT FROM AUTHOR]

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.

Enterovirus A71 (EV-A71) is one of the causative agents of hand-foot-mouth disease, which can be associated with neurocomplications of the central nervous system. A limited understanding of the virus's biology and pathogenesis has led to the unavailability of effective anti-viral treatments. The EV-A71 RNA genome carries type I internal ribosomal entry site (IRES) at 5′ UTR that plays an essential role in the viral genomic translation. However, the detailed mechanism of IRES-mediated translation has not been elucidated. In this study, sequence analysis revealed that the domains IV, V, and VI of EV-A71 IRES contained the structurally conserved regions. The selected region was transcribed in vitro and labeled with biotin to use as an antigen for selecting the single-chain variable fragment (scFv) antibody from the naïve phage display library. The so-obtained scFv, namely, scFv #16-3, binds specifically to EV-A71 IRES. The molecular docking showed that the interaction between scFv #16-3 and EV-A71 IRES was mediated by the preferences of amino acid residues, including serine, tyrosine, glycine, lysine, and arginine on the antigen-binding sites contacted the nucleotides on the IRES domains IV and V. The so-produced scFv has the potential to develop as a structural biology tool to study the biology of the EV-A71 RNA genome. [ABSTRACT FROM AUTHOR]

Human enterovirus causes various clinical manifestations in the form of rashes, febrile illness, flu-like illness, uveitis, hand–foot–mouth disease (HFMD), herpangina, meningitis, and encephalitis. Enterovirus A71 and coxsackievirus are significant causes of epidemic HFMD worldwide, especially in children aged from birth to five years old. The enterovirus genotype variants causing HFMD epidemics have been reported increasingly worldwide in the last decade. We aim to use simple and robust molecular tools to investigate human enteroviruses circulating among kindergarten students at genotype and subgenotype levels. With the partial 5′-UTR sequencing analysis as a low-resolution preliminary grouping tool, ten enterovirus A71 (EV-A71) and coxsackievirus clusters were identified among 18 symptomatic cases and 14 asymptomatic cases in five kindergartens in Bangkok, Thailand, between July 2019 and January 2020. Two occurrences of a single clone causing an infection cluster were identified (EV-A71 C1-like subgenotype and coxsackievirus A6). Random amplification-based sequencing using MinION (Oxford Nanopore Technology) helped identify viral transmission between two closely related clones. Diverse genotypes co-circulating among children in kindergartens are reservoirs for new genotype variants emerging, which might be more virulent or better at immune escape. Surveillance of highly contagious enterovirus in communities is essential for disease notifications and controls. [ABSTRACT FROM AUTHOR]

Enteroviruses (EVs) are positive-sense RNA viruses, with over 50,000 nucleotide sequences publicly available. While most human infections are typically associated with mild respiratory symptoms, several different EV types have also been associated with severe human disease, especially acute flaccid paralysis (AFP), particularly with endemic members of the EV-B species and two pandemic types-EV-A71 and EV-D68-that appear to be responsible for recent widespread outbreaks. Here we review the recent literature on the prevalence, characteristics, and circulation dynamics of different enterovirus types and combine this with an analysis of the sequence coverage of different EV types in public databases (e.g., the Virus Pathogen Resource). This evaluation reveals temporal and geographic differences in EV circulation and sequence distribution, highlighting recent EV outbreaks and revealing gaps in sequence coverage. Phylogenetic analysis of the EV genus shows the relatedness of different EV types. Recombination analysis of the EV-A species provides evidence for recombination as a mechanism of genomic diversification. The absence of broadly protective vaccines and effective antivirals makes human enteroviruses important pathogens of public health concern.

Abstract Background Enterovirus A71 (EV A71) is one of the most important enteroviruses related to morbidity and mortality in children worldwide. This study aimed to analyse the secular trend of EV A71 in Taiwan from 1998 to 2020 and to evaluate the effectiveness of infection control measures. Methods We collected the epidemiological data of EV A71 from disease surveillance systems in Taiwan. We analysed the association between the secular trend of EV A71 and preventive measures such as hand washing, case isolation, and suspension of classes. Results The incidence of enterovirus infections with severe complications (EVSC) decreased from 16.25 per 100,000 children under six in 1998 to less than 9.73 per 100,000 children under six after 2012 (P = 0.0022). The mortality rate also decreased significantly, from 3.52 per 100,000 children under six in 1998 to 0 per 100,000 children under six in 2020 (P

Enterovirus A71 can cause serious neurological disease in young children. Animal models for EV-A71 are needed to evaluate potential antiviral therapies. Existing models have limitations, including lack of lethality or crucial disease signs. Here we report the development of an EV-A71 model in 28-day-old mice. Virus was serially passaged until it produced consistent lethality and rear-limb paralysis. Onset of disease occurred between days 6–9 post-infection, with mortality following weight loss and neurological signs on days 9–14. In addition, a single administration of human intravenous immunoglobulin at doses of 200, 400 and 800 mg/kg at 4h post-infection was evaluated in the model. Protection from weight loss, neurological signs, and mortality (between 50 and 89%) were observed at doses of 400 mg/kg or greater. Based on these results, IVIG was selected for use as a positive control in this acute model, and suggest that IVIG is a potential therapeutic for EV-A71 infections. • This report describes a new model for EV-A71 infection in 28-day-old mice for evaluation of antiviral therapeutics. • This report also describes the process of mouse-adaptation of EV-A71. • The mouse-adapted virus shows consistent lethality and relevant disease signs, including rear-limb paralysis in mice. • Seventeen antiviral compounds shown to have in vitro activity against EV-A71 were evaluated in this model. • Only human intravenous immunoglobulin showed protection, and was selected for use as a positive control in this model. [ABSTRACT FROM AUTHOR]

Enterovirus A71 (EV71), coxsackievirus A16 (CVA16), and coxsackievirus B3 (CVB3) are pathogenic members of the Picornaviridae family that cause a range of diseases, including severe central nervous system complications, myocarditis, and pancreatitis. Despite the considerable public health impact of these viruses, no approved antiviral treatments are currently available. In the present study, we confirmed the potential of saucerneol, a compound derived from Saururus chinensis, as an antiviral agent against EV71, CVA16, and CVB3. In the in vivo model, saucerneol effectively suppressed CVB3 replication in the pancreas and alleviated virus-induced pancreatitis. The antiviral activity of saucerneol is associated with increased mitochondrial ROS (mROS) production. In vitro inhibition of mROS generation diminishes the antiviral efficacy of saucerneol. Moreover, saucerneol treatment enhanced the phosphorylation of STING, TBK-1, and IRF3 in EV71- and CVA16-infected cells, indicating that its antiviral effects were mediated through the STING/TBK-1/IRF3 antiviral pathway, which was activated by increased mROS production. Saucerneol is a promising natural antiviral agent against EV71, CVA16, and CVB3 and has potential against virus-induced pancreatitis and myocarditis. Further studies are required to assess its safety and efficacy, which is essential for the development of effective antiviral strategies against these viruses.

Enterovirus A71 (EV-A71) is a significant human pathogen, especially in children. EV-A71 infection is one of the leading causes of hand, foot, and mouth diseases (HFMD), and can lead to neurological complications such as acute flaccid myelitis (AFM) in severe cases. Although three EV-A71 vaccines are available in China, they are not broadly protective and have reduced efficacy against emerging strains. There is currently no approved antiviral for EV-A71. Significant progress has been made in developing antivirals against EV-A71 by targeting both viral proteins and host factors. However, viral capsid inhibitors and protease inhibitors failed in clinical trials of human rhinovirus infection due to limited efficacy or side effects. This review discusses major discoveries in EV-A71 antiviral development, analyzes the advantages and limitations of each drug target, and highlights the knowledge gaps that need to be addressed to advance the field forward.

Enterovirus A71 (EV-A71) causes major outbreaks of hand, foot, and mouth disease (HFMD) in many countries, most frequently affecting children, and a small proportion of cases may lead to death. Currently, no vaccine is available in most endemic regions, and no licenced treatments for EV-A71 infection are available. Here, we characterize a human monoclonal antibody (HuMAb), E1, by screening a Fab antibody phage library derived from patients who recovered from EV-A71 infection. E1 exhibits strong neutralizing activity against EV-A71 virus in cells. The cryo-electron microscopy (cryo-EM) structures of the EV-A71 virion in complex with E1 Fab fragments demonstrated that E1 recognized an epitope formed by residues in the BC and HI loops of VP1. In a mouse model, E1 effectively protected against lethal EV-A71 challenge in both prophylactic and therapeutic treatment. In particular, E1 significantly reduces virus titers and muscle damage. E1 might represent a potential adjunct to EV-A71 treatment. [ABSTRACT FROM AUTHOR]

Enterovirus A71 is a major causative pathogen of hand, foot and mouth disease. It has become a global public health threat, and is especially important for infants and young children in the Asian‐Pacific countries. The enterovirus A71 is a non‐enveloped virus of the Picornaviridae family having a single‐stranded positive‐sense RNA genome of about 7.4 kb which encodes the structural and nonstructural proteins. Currently there are no US FDA‐approved vaccines or antiviral therapy available against enterovirus A71 infection. Although enterovirus A71 vaccines have been licenced in China, clinically approved vaccines for widespread vaccination programs are lacking. Substantial progress has recently been achieved on understanding the structure and function of enterovirus A71 proteins together with information on the viral genetic diversity and geographic distribution. The present review is intended to provide an overview on our current understanding of the molecular biology and epidemiology of enterovirus A71 which will aid the development of vaccines, therapeutics and other control strategies so as to bolster the preparedness for future enterovirus A71 outbreaks. [ABSTRACT FROM AUTHOR]

Abstract Background Acute flaccid myelitis is a serious condition of the spinal cord. More than 80% of patients experience a mild respiratory illness or fever consistent with a viral infection prior to acute flaccid myelitis development. Enterovirus A71 is known to circulate in Denmark, and has previously been associated with severe neurological symptoms. In this case report we describe acute flaccid rhombencephalomyelitis with radiculitis in an infant with an enterovirus infection. Case presentation The 8-month-old male of Asian origin presented with fever and gastrointestinal symptoms, followed by severe neurological deficits such as flaccid paralysis of the neck and upper extremities. An initial magnetic resonance imaging scan of the brain was normal, and the boy was treated for encephalitis. A follow-up magnetic resonance imaging scan of the brain and spinal cord 1 week later showed the development of pathological symmetrical gray matter hyperintensity lesions on T2-weighted images in the brainstem and upper medulla spinalis, and nerve enhancement in the terminal thread of the spinal cord and the cervical roots; findings consistent with rhombencephalomyelitis with radiculitis causing flaccid paralysis. Enterovirus A71 was detected in both nasopharyngeal and fecal specimens. Other differential diagnostic etiologies of viral and bacterial encephalitis, including poliovirus, were excluded. Conclusions This is the first case in Denmark of a patient diagnosed with acute flaccid rhombencephalomyelitis strongly linked to an enterovirus A71 infection. This case emphasizes the diagnostic importance of combining a history of respiratory and/or gastrointestinal illness, fever, and delayed onset of varying degrees of paralysis with progressive characteristic spinal and brain lesions. Analysis of respiratory, fecal, and cerebrospinal samples for the presence of enterovirus, and eliminating other differential pathogens, is essential to confirm the diagnosis.

Hand, foot and mouth disease (HFMD) is a childhood disease caused by enterovirus A71 (EV-A71) and coxsackievirus A16 (CV-A16). Capsid loops are important epitopes for EV-A71 and CV-A16. Seven chimeric EV-A71 (ChiE71) involving VP1 BC (45.5% similarity), DE, EF, GH and HI loops, VP2 EF loop and VP3 GH loop (91.3% similarity) were substituted with corresponding CV-A16 loops. Only ChiE71-1-BC, ChiE71-1-EF, ChiE71-1-GH and ChiE71-3-GH were viable. EV-A71 and CV-A16 antiserum neutralized ChiE71-1-BC and ChiE71-1-EF. Mice immunized with inactivated ChiE71 elicited high IgG, IFN-γ, IL-2, IL-4 and IL-10. Neonatal mice receiving passive transfer of WT EV-A71, ChiE71-1-EF and ChiE71-1-BC immune sera had 100%, 80.0% and no survival, respectively, against lethal challenges with EV-A71, suggesting that the substituted CV-A16 loops disrupted EV-A71 immunogenicity. Passive transfer of CV-A16, ChiE71-1-EF and ChiE71-1-BC immune sera provided 40.0%, 20.0% and 42.9% survival, respectively, against CV-A16. One-day-old neonatal mice immunized with WT EV-A71, ChiE71-1-BC, ChiE71-1-EF and CV-A16 achieved 62.5%, 60.0%, 57.1%, and no survival, respectively, after the EV-A71 challenge. Active immunization using CV-A16 provided full protection while WT EV-A71, ChiE71-1-BC and ChiE71-1-EF immunization showed partial cross-protection in CV-A16 lethal challenge with survival rates of 50.0%, 20.0% and 40%, respectively. Disruption of a capsid loop could affect virus immunogenicity, and future vaccine design should include conservation of the enterovirus capsid loops.

Abstract Background The increase of inflammation-inducing enterobacteria was recently observed in severe hand, foot, and mouth disease (HFMD) caused by Enterovirus A71 (EV-A71). This study aimed to verify the occurrence of bacterial translocation (BT) and further explore the contributory role of BT to severity of EV-A71-mediated HFMD cases. Methods Serum specimens from 65 mild and 65 severe EV-A71-associated HFMD cases and 65 healthy children were collected. EV-A71 VP1 in serum, inflammatory mediators including C-reactive protein, IL-1β, IL-6, interferon-γ and tumor necrosis factor-α, BT related biomarkers including Claudin-3, intestinal fatty acid binding protein, lipopolysaccharide (LPS), soluble CD14 (sCD14) and endotoxin core antibody were measured by ELISA. Bacterial DNA (BactDNA) fragments were quantified by quantified PCR (qPCR). Rhabdomyosarcoma (RD) or SH-SY5Y cells, infected with LPS-pre-incubated EV-A71 or transfected with plasmid containing viral 2Apro or mRNA containing viral internal ribosomal entry site (IRES), were post-treated with or without LPS in vitro. EV-A71 RNA and viral or cellular proteins were determined by qPCR and western blot, respectively. Results Compared to mild HFMD patients, remarkably higher inflammatory mediators as well as BT-related biomarkers except BactDNA were observed in severe HFMD cases (all P

Enterovirus A71 (EV-A71) is a major pathogen that causes severe and fatal cases of hand-foot-and-mouth disease (HFMD), which is an infectious disease that endangers children's health. However, the pathogenic mechanisms underlying these severe clinical and pathological features remain incompletely understood. Metabolism and stress are known to play critical roles in multiple stages of the replication of viruses. Lipid metabolism and ER stress is an important characterization post viral infection. EV-A71 infection alters the perturbations of intracellular lipid homeostasis and induces ER stress. The characterizations induced by viral infections are essential for optimal virus replication and may be potential antiviral targets. In this study, we found that the addition of the chemical drug of ER stress, PKR IN, an inhibitor, or Tunicamycin, an activator, could significantly reduce viral replication with the decrease of lipid. The replication of viruses was reduced by Chemical reagent TOFA, an inhibitor of acetyl-CoA carboxylase (ACC) or C75, an inhibitor of fatty acid synthase (FASN), while enhanced by oleic acid (OA), which is a kind of exogenous supplement of triacylglycerol. The pharmacochemical reagent of carnitine palmitoyltransferase 1 (CPT1) called Etomoxir could knock down CPT1 to induce EV-A71 replication to decrease. This suggests that lipid, rather than ER stress, is the main factor affecting EV-A71 replication. In conclusion, this study revealed that it is the ß-oxidation of lipid that plays a core role, not ER stress, which is only a concomitant change without restrictive effect, on virus replication. [ABSTRACT FROM AUTHOR]

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

Background: Enterovirus A71 (EV A71) is one of the most important enteroviruses related to morbidity and mortality in children worldwide. This study aimed to analyse the secular trend of EV A71 in Taiwan from 1998 to 2020 and to evaluate the effectiveness of infection control measures.Methods: We collected the epidemiological data of EV A71 from disease surveillance systems in Taiwan. We analysed the association between the secular trend of EV A71 and preventive measures such as hand washing, case isolation, and suspension of classes.Results: The incidence of enterovirus infections with severe complications (EVSC) decreased from 16.25 per 100,000 children under six in 1998 to less than 9.73 per 100,000 children under six after 2012 (P = 0.0022). The mortality rate also decreased significantly, from 3.52 per 100,000 children under six in 1998 to 0 per 100,000 children under six in 2020 (P < 0.0001). The numbers of EVSC and fatalities were significantly higher in the years when EV A71 accounted for more than 10% of the annual predominant serotypes (p < 0.05). After the implementation of many non-pharmaceutical interventions in 2012, the incidence of EVSC and mortality rate decreased significantly (p < 0.001).Conclusions: After implementing active enterovirus surveillance and preventive measures, we found that the incidence of EVSC and fatalities due to EV A71 in Taiwan decreased significantly from 1998 to 2020. Continuous surveillance and strengthened infection control policies are still needed in the future. [ABSTRACT FROM AUTHOR]

Aim: Molecular characterization of enterovirus A71 (EV-A71) and coxsackievirus A24 (CVA-24) strains isolated during neurological diseases surveillance activities in Tunisian patients. Materials & methods: Specimens were obtained from two Tunisian children and analyzed for enterovirus with quantitative reverse transcription PCR (RT-qPCR). We sequenced the whole genome of strains detected. Results: The EV-A71 sequence reported was assigned to subgenogroup C2 with a high nucleotide sequence identity to other EV-A71 C2 detected in other countries, which suggests virus migration. Interestingly, in the VP1 coding region, the Tunisian CV-A24 strain displayed high sequence divergence from other CV-A24, which confirms that it represents a new genogroup. Conclusion: This is the first report of EV-A71 in Tunisia and the first report of a CV-A24 strain causing aseptic meningitis. [ABSTRACT FROM AUTHOR]

Background: Enterovirus A71 (EVA71) is one of the main pathogens causing hand, foot and mouth disease (HFMD). In China, the proportion of cases of HFMD caused by EVA71 is known to be significantly lower following EVA71 vaccination; however, infection with EVA71 can still occur after vaccination. Methods: The complete genomic sequences of EVA71-KM18A and KM18B (from two rare cases of EVA71 infection following vaccination) were obtained. Phylogenetic analysis, nucleotide mutation analysis, recombinant analysis and comparative analysis of amino acid mutations were performed. Results: Phylogenetic analysis determined that the EVA71 strains belonged to the C4a subgenotype. The KM18A and KM18B strains were highly similar to the vaccine strains. For the KM18B strain, there were some obvious homologous recombination signals in the 5’non-coding region, region 2A, region 2C and region 3D. Amino acid mutations were observed in the SP55 (position 729) and 71-6 (position 500) conformational neutralizing epitopes of the KM18A and KM18B strains. Conclusions: These amino acid mutations may affect the SP55 and 71-6 conformational neutralizing epitopes and change their spatial conformation, thereby weakening vaccine effectiveness.

Enterovirus A71 (EV-A71) là tác nhân chính gây nên biến chứng nguy hiểm của bệnh Tay Chân Miệng, có thể dẫn đến thương tật, và tử vong ở trẻ nhỏ. Hiện nay, vaccine và hợp chất điều trị EV-A71 còn nhiều hạn chế, chưa được áp dụng rộng rãi. Vì thế, nhiều nghiên cứu tìm giải pháp thay thế hoặc bổ trợ đang được tiến hành, trong đó có bẫy virus. Với chức năng chính là bắt, cố định virus, ngăn sự xâm nhiễm, thể bám của bẫy cấu trúc từ thụ thể liên kết EV-A71, cụ thể là SCARB2. Trong nghiên cứu này, tiểu phần thụ thể tái tổ hợp SCARB2 dung hợp foldon peptide được dòng hóa, biểu hiện, và tinh sạch nhằm tạo thể bám ở dạng trimer hóa có ái lực cao với virus. Vector pET22b-scrb2-IIIx3 được cấu trúc trong E. coli DH5α, protein được biểu hiện trong E. coli BL21(DE3), và chỉ thu được cấu hình monomer, phần lớn ở dạng thể vùi. Việc tái gấp cuộn và tinh sạch protein được tiến hành nhằm đưa protein trở về dạng tan có hoạt tính sinh học. Những kết quả trên cung cấp thêm nhiều thông...

Long-term neurological and neurodevelopmental sequelae are a concerning issue for people with Enterovirus A71 (EV-A71) central nervous system (CNS) infection. Unfortunately, no longitudinal prospective clinical study has systematically investigated the consequences of EV-A71 CNS infection during early life on the later development of other psychiatric disorders. In this naturalistic longitudinal follow-up design, we followed forty-three youth, who got EV-A71 CNS involvement 6–18 years ago and were enrolled in other EV-A71 clinical studies then. Their psychiatric presentation, emotional/behavioral problems, and cognitive issues were examined using a psychiatrist-conducted diagnostic interview, parent- and self-rated questionnaires, and neuropsychological tests, respectively. We compared the prevalence of psychiatric disorders in youth with EV-A71 CNS involvement to a nationally representative cohort. Emotion/behavior and cognition in EV-A71-CNS-infected youth were compared to those in a matched community-based sample of healthy controls and youth with attention-deficit/hyperactivity disorder (ADHD). Compared to a national sample (absolute ADHD prevalence 10.1%), youth with EV-A71 CNS involvement had three times the odds of receiving an ADHD diagnosis (standardized prevalence ratio, 95% CI = 1.8, 4.2; absolute ADHD prevalence 34.9%). No other psychiatric diagnoses were more common in EV-A71-CNS-infected youth. Compared to community-based ADHD youth, EV-A71-CNS-infected youth with psychiatric disorders showed comparable core ADHD symptoms, opposition/defiance, autistic features, and suboptimal sustained attention performance (based on the Conners’ Continuous Performance Test), all of which were more severe than healthy controls. EV-A71-CNS-infected youth without psychiatric disorders showed comparable autistic features to EV-A71-CNS-infected youth with psychiatric disorders and ADHD youth. EV-A71 CNS involvement may cause long-term, adverse psychiatric outcomes that develop into an ADHD diagnosis alongside social/communication/emotion problems and autistic features. We recommend earlier identification and intervention of these problems among these children.

Enterovirus A71 (EV-A71) is one of the causative agents of hand-foot-mouth disease, which can be associated with neurocomplications of the central nervous system. A limited understanding of the virus’s biology and pathogenesis has led to the unavailability of effective anti-viral treatments. The EV-A71 RNA genome carries type I internal ribosomal entry site (IRES) at 5′ UTR that plays an essential role in the viral genomic translation. However, the detailed mechanism of IRES-mediated translation has not been elucidated. In this study, sequence analysis revealed that the domains IV, V, and VI of EV-A71 IRES contained the structurally conserved regions. The selected region was transcribed in vitro and labeled with biotin to use as an antigen for selecting the single-chain variable fragment (scFv) antibody from the naïve phage display library. The so-obtained scFv, namely, scFv #16-3, binds specifically to EV-A71 IRES. The molecular docking showed that the interaction between scFv #16-3 and EV-A71 IRES was mediated by the preferences of amino acid residues, including serine, tyrosine, glycine, lysine, and arginine on the antigen-binding sites contacted the nucleotides on the IRES domains IV and V. The so-produced scFv has the potential to develop as a structural biology tool to study the biology of the EV-A71 RNA genome.