Induced intra- and intermolecular template switching as a therapeutic mechanism against RNA viruses.

Viral RNA-dependent RNA polymerases (RdRps) are a target for broad-spectrum antiviral therapeutic agents. Recently, we demonstrated that incorporation of the T-1106 triphosphate, a pyrazine-carboxamide ribonucleotide, into nascent RNA increases pausing and backtracking by the poliovirus RdRp. Here, by monitoring enterovirus A-71 RdRp dynamics during RNA synthesis using magnetic tweezers, we identify the "backtracked" state as an intermediate used by the RdRp for copy-back RNA synthesis and homologous recombination. Cell-based assays and RNA sequencing (RNA-seq) experiments further demonstrate that the pyrazine-carboxamide ribonucleotide stimulates these processes during infection. These results suggest that pyrazine-carboxamide ribonucleotides do not induce lethal mutagenesis or chain termination but function by promoting template switching and formation of defective viral genomes. We conclude that RdRp-catalyzed intra- and intermolecular template switching can be induced by pyrazine-carboxamide ribonucleotides, defining an additional mechanistic class of antiviral ribonucleotides with potential for broad-spectrum activity.

VP1 codon deoptimization and high-fidelity substitutions in 3D polymerase as potential vaccine strategies for eliciting immune responses against enterovirus A71.

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-γ, IL-4, IL-6, and TNF-α) 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.IMPORTANCEEV-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.

Heterogeneous nuclear ribonucleoprotein A3 binds to the internal ribosomal entry site of enterovirus A71 and affects virus replication in neural cells.

Enterovirus A71 (EV-A71) belongs to the genus Enterovirus of the Picornaviridae family and often causes outbreaks in Asia. EV-A71 infection usually causes hand, foot, and mouth disease and can even affect the central nervous system, causing neurological complications or death. The 5'-untranslated region (5'-UTR) of EV-A71 contains an internal ribosome entry site (IRES) that is responsible for the translation of viral proteins. IRES-transacting factors can interact with the EV-A71 5'-UTR to regulate IRES activity. Heterogeneous nuclear ribonucleoprotein (hnRNP) A3 is a member of the hnRNP A/B protein family of RNA-binding proteins and is involved in RNA transport and modification. We found that hnRNP A3 knockdown promoted the replication of EV-A71 in neural calls. Conversely, increasing the expression of hnRNP A3 within cells inhibits the growth of EV-A71. HnRNP A3 can bind to the EV-A71 5'-UTR, and knockdown of hnRNP A3 enhances the luciferase activity of the EV-A71 5'-UTR IRES. The localization of hnRNP A3 shifts from the nucleus to the cytoplasm of infected cells during viral infection. Additionally, EV-A71 infection can increase the protein expression of hnRNP A3, and the protein level is correlated with efficient viral growth. Based on these findings, we concluded that hnRNP A3 plays a negative regulatory role in EV-A71 replication within neural cells.

Recent Advances in Enterovirus A71 Infection and Antiviral Agents.

Enterovirus A71 (EV-A71) is one of the major causative agents of hand, foot, and mouth disease (HFMD) that majorly affects children. Most of the time, HFMD is a mild disease but can progress to severe complications, such as meningitis, brain stem encephalitis, acute flaccid paralysis, and even death. HFMD caused by EV-A71 has emerged as an acutely infectious disease of highly pathogenic potential in the Asia-Pacific region. In this review, we introduced the properties and life cycle of EV-A71, and the pathogenesis and the pathophysiology of EV-A71 infection, including tissue tropism and host range of virus infection, the diseases caused by the virus, as well as the genes and host cell immune mechanisms of major diseases caused by enterovirus 71 (EV-A71) infection, such as encephalitis and neurologic pulmonary edema. At the same time, clinicopathologic characteristics of EV-A71 infection were introduced. There is currently no specific medication for EV-A71 infection, highlighting the urgency and significance of developing suitable anti-EV-A71 agents. This overview also summarizes the targets of existing anti-EV-A71 agents, including virus entry, translation, polyprotein processing, replication, assembly and release; interferons; interleukins; the mitogen-activated protein kinase, phosphatidylinositol 3-kinase, and protein kinase B signaling pathways; the oxidative stress pathway; the ubiquitin-proteasome system; and so on. Furthermore, it overviews the effects of natural products, monoclonal antibodies, and RNA interference against EV-A71. It also discusses issues limiting the research of antiviral drugs. This review is a systematic and comprehensive summary of the mechanism and pathological characteristics of EV-A71 infection, the latest progress of existing anti-EV-A71 agents. It would provide better understanding and guidance for the research and application of EV-A71 infection and antiviral inhibitors.

Emerging Enterovirus A71 Subgenogroup B5 Causing Severe Hand, Foot, and Mouth Disease, Vietnam, 2023.

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.

Differential inhibition of intra- and inter-molecular protease cleavages by antiviral compounds.

IMPORTANCE: Most protease-targeted antiviral development evaluates the ability of small molecules to inhibit the cleavage of artificial substrates. However, before they can cleave any other substrates, viral proteases need to cleave themselves out of the viral polyprotein in which they have been translated. This can occur either intra- or inter-molecularly. Whether this process occurs intra- or inter-molecularly has implications for the potential for precursors to accumulate and for the effectiveness of antiviral drugs. We argue that evaluating candidate antivirals for their ability to block these cleavages is vital to drug development because the buildup of uncleaved precursors can be inhibitory to the virus and potentially suppress the selection of drug-resistant variants.

Enterovirus-A71 exploits RAB11 to recruit chaperones for virus morphogenesis.

BACKGROUND: Enterovirus 71 (EV-A71) causes Hand, Foot and Mouth Disease (HFMD) in children and has been associated with neurological complications. The molecular mechanisms involved in EV-A71 pathogenesis have remained elusive. METHODS: A siRNA screen in EV-A71 infected-motor neurons was performed targeting 112 genes involved in intracellular membrane trafficking, followed by validation of the top four hits using deconvoluted siRNA. Downstream approaches including viral entry by-pass, intracellular viral genome quantification by qPCR, Western blot analyses, and Luciferase reporter assays allowed determine the stage of the infection cycle the top candidate, RAB11A was involved in. Proximity ligation assay, co-immunoprecipitation and multiplex confocal imaging were employed to study interactions between viral components and RAB11A. Dominant negative and constitutively active RAB11A constructs were used to determine the importance of the protein's GTPase activity during EV-A71 infection. Mass spectrometry and protein interaction analyses were employed for the identification of RAB11A's host interacting partners during infection. RESULTS: Small GTPase RAB11A was identified as a novel pro-viral host factor during EV-A71 infection. RAB11A and RAB11B isoforms were interchangeably exploited by strains from major EV-A71 genogroups and by Coxsackievirus A16, another major causative agent of HFMD. We showed that RAB11A was not involved in viral entry, IRES-mediated protein translation, viral genome replication, and virus exit. RAB11A co-localized with replication organelles where it interacted with structural and non-structural viral components. Over-expression of dominant negative (S25N; GDP-bound) and constitutively active (Q70L; GTP-bound) RAB11A mutants had no effect on EV-A71 infection outcome, ruling out RAB11A's involvement in intracellular trafficking of viral or host components. Instead, decreased ratio of intracellular mature viral particles to viral RNA copies and increased VP0:VP2 ratio in siRAB11-treated cells supported a role in provirion maturation hallmarked by VP0 cleavage into VP2 and VP4. Finally, chaperones, not trafficking and transporter proteins, were found to be RAB11A's top interacting partners during EV-A71 infection. Among which, CCT8 subunit from the chaperone complex TRiC/CCT was further validated and shown to interact with viral structural proteins specifically, representing yet another novel pro-viral host factor during EV-A71 infection. CONCLUSIONS: This study describes a novel, unconventional role for RAB11A during viral infection where it participates in the complex process of virus morphogenesis by recruiting essential chaperone proteins.

Proteomic and metabonomic analysis uncovering Enterovirus A71 reprogramming host cell metabolic pathway.

Enterovirus A71 (EV71) infection can cause hand, foot, and mouth disease (HFMD) and severe neurological complications in children. However, the biological processes regulated by EV71 remain poorly understood. Herein, proteomics and metabonomics studies were conducted to uncover the mechanism of EV71 infection in rhabdomyosarcoma (RD) cells and identify potential drug targets. Differential expressed proteins from enriched membrane were analyzed by isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomics technology. Twenty-six differential proteins with 1.5-fold (p < 0.05) change were detected, including 14 upregulated proteins and 12 downregulated proteins. The upregulated proteins are mainly involved in metabolic process, especially in the glycolysis pathway. Alpha-enolase (ENO1) protein was found to increase with temporal dependence following EV71 infection. The targeted metabolomics analysis revealed that glucose absorption and glycolysis metabolites were increased after EV71 infection. The glycolysis pathway was inhibited by knocking down ENO1 or the use of a glycolysis inhibitor (dichloroacetic acid [DCA]); and we found that EV71 infection was inhibited by depleting ENO1 or using DCA. Our study indicates that EV71 may reprogram glucose metabolism by activating glycolysis, and EV71 infection can be inhibited by interrupting the glycolysis pathway. ENO1 may be a potential target against EV71, and DCA could act as an inhibitor of EV71.

Antiviral Activity of trans-Hexenoic Acid against Coxsackievirus B and Enterovirus A71.

Enterovirus infections are life-threatening viral infections which occur mainly among children and are possible causes of viral outbreak. Until now, treatment and management of infections caused by members of the genus Enterovirus largely depended on supportive care, and no antiviral medications are currently approved for the treatment of most of these infections. The urgency of discovering new therapeutic options for the treatment of enterovirus infection is increasing. In the present study, we identified that trans-2-hexenoic acid (THA), a natural product from a dietary source, possesses antiviral activity against coxsackievirus B (CVB) and enterovirus A71 (EV-A71) in a dose-dependent manner. We found that THA possesses antiviral activity at 50% effective concentrations (EC50) of 2.9 µM and 3.21 µM against CVB3 and EV-A71 infections, respectively. The time of addition assay revealed that THA inhibits both CVB3 and EV-A71 replication at the entry stage of infection. Additional results from this study further suggest that THA inhibits viral replication by blocking viral entry. Given that THA has received approval as a food additive, treatment of enterovirus infections with THA might be a safe therapeutic option or could pave the way for semisynthetic manufacturing of more antiviral drugs in the future.

Neutralization of African enterovirus A71 genogroups by antibodies to canonical genogroups.

Enterovirus 71 (EV-A71) is a major public health problem, causing a range of illnesses from hand-foot-and-mouth disease to severe neurological manifestations. EV-A71 strains have been phylogenetically classified into eight genogroups (A to H), based on their capsid-coding genomic region. Genogroups B and C have caused large outbreaks worldwide and represent the two canonical circulating EV-A71 subtypes. Little is known about the antigenic diversity of new genogroups as compared to the canonical ones. Here, we compared the antigenic features of EV-A71 strains that belong to the canonical B and C genogroups and to genogroups E and F, which circulate in Africa. Analysis of the peptide sequences of EV-A71 strains belonging to different genogroups revealed a high level of conservation of the capsid residues involved in known linear and conformational neutralization antigenic sites. Using a published crystal structure of the EV-A71 capsid as a model, we found that most of the residues that are seemingly specific to some genogroups were mapped outside known antigenic sites or external loops. These observations suggest a cross-neutralization activity of anti-genogroup B or C antibodies against strains of genogroups E and F. Neutralization assays were performed with diverse rabbit and mouse anti-EV-A71 sera, anti-EV-A71 human standards and a monoclonal neutralizing antibody. All the batches of antibodies that were tested successfully neutralized all available isolates, indicating an overall broad cross-neutralization between the canonical genogroups B and C and genogroups E and F. A panel constituted of more than 80 individual human serum samples from Cambodia with neutralizing antibodies against EV-A71 subgenogroup C4 showed quite similar cross-neutralization activities between isolates of genogroups C4, E and F. Our results thus indicate that the genetic drift underlying the separation of EV-A71 strains into genogroups A, B, C, E and F does not correlate with the emergence of antigenically distinct variants.

3,4-Dicaffeoylquinic Acid from the Medicinal Plant Ilex kaushue Disrupts the Interaction Between the Five-Fold Axis of Enterovirus A-71 and the Heparan Sulfate Receptor.

While infections by enterovirus A71 (EV-A71) are generally self-limiting, they can occasionally lead to serious neurological complications and death. No licensed therapies against EV-A71 currently exist. Using anti-virus-induced cytopathic effect assays, 3,4-dicaffeoylquinic acid (3,4-DCQA) from Ilex kaushue extracts was found to exert significant anti-EV-A71 activity, with a broad inhibitory spectrum against different EV-A71 genotypes. Time-of-drug-addition assays revealed that 3,4-DCQA affects the initial phase (entry step) of EV-A71 infection by directly targeting viral particles and disrupting viral attachment to host cells. Using resistant virus selection experiments, we found that 3,4-DCQA targets the glutamic acid residue at position 98 (E98) and the proline residue at position 246 (P246) in the 5-fold axis located within the VP1 structural protein. Recombinant viruses harboring the two mutations were resistant to 3,4-DCQA-elicited inhibition of virus attachment and penetration into human rhabdomyosarcoma (RD) cells. Finally, we showed that 3,4-DCQA specifically inhibited the attachment of EV-A71 to the host receptor heparan sulfate (HS), but not to the scavenger receptor class B member 2 (SCARB2) and P-selectin glycoprotein ligand-1 (PSGL1). Molecular docking analysis confirmed that 3,4-DCQA targets the 5-fold axis to form a stable structure with the E98 and P246 residues through noncovalent and van der Waals interactions. The targeting of E98 and P246 by 3,4-DCQA was found to be specific; accordingly, HS binding of viruses carrying the K242A or K244A mutations in the 5-fold axis was successfully inhibited by 3,4-DCQA.The clinical utility of 3,4-DCQA in the prevention or treatment of EV-A71 infections warrants further scrutiny. IMPORTANCE The canyon region and the 5-fold axis of the EV-A71 viral particle located within the VP1 protein mediate the interaction of the virus with host surface receptors. The three most extensively investigated cellular receptors for EV-A71 include SCARB2, PSGL1, and cell surface heparan sulfate. In the current study, a RD cell-based anti-cytopathic effect assay was used to investigate the potential broad spectrum inhibitory activity of 3,4-DCQA against different EV-A71 strains. Mechanistically, we demonstrate that 3,4-DCQA disrupts the interaction between the 5-fold axis of EV-A71 and its heparan sulfate receptor; however, no effect was seen on the SCARB2 or PSGL1 receptors. Taken together, our findings show that this natural product may pave the way to novel anti-EV-A71 therapeutic strategies.

SHFL inhibits enterovirus A71 infection by triggering degradation of viral 3Dpol protein via the ubiquitin-proteasome pathway.

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.

A novel mucosal bivalent vaccine of EV-A71/EV-D68 adjuvanted with polysaccharides from Ganoderma lucidum protects mice against EV-A71 and EV-D68 lethal challenge.

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.

Insights into enterovirus a-71 antiviral development: from natural sources to synthetic nanoparticles.

Enteroviruses are pathogens responsible for several diseases, being enterovirus A71 (EVA71) the second leading cause of hand, foot, and mouth disease (HFMD), especially in Asia-Pacific countries. HFMD is mostly common in infants and children, with mild symptoms. However, the disease can result in severe nervous system disorders in children as well as in immunosuppressed adults. The virus is highly contagious, and its transmission occurs via fecal-oral, oropharyngeal secretions, and fomites. The EVA71 burdens the healthy systems and economies around the world, however, up to date, there is no antiviral approved to treat infected individuals and the existent vaccines are not available or approved to be used worldwide. In this context, an extensive literature research was conducted to describe and summarize the recent advances in natural and/or synthetic compounds with antiviral activity against EVA71. The summarized data presented here might simply encourage the future studies in EVA71 antiviral development, by encouraging further research encompassing these compounds or even the application of the techniques and technologies to improve or produce new antiviral molecules.

Enterovirus-A71 exploits peripherin and Rac1 to invade the central nervous system.

Enterovirus-A71 (EV-A71) has been associated with severe neurological forms of hand, foot, and mouth disease (HFMD). EV-A71 infects motor neurons at neuromuscular junctions (NMJs) to invade the central nervous system (CNS). Here, we investigate the role of peripherin (PRPH) during EV-A71 infection, a type III intermediate neurofilament involved in neurodegenerative conditions. In mice infected with EV-A71, PRPH co-localizes with viral particles in the muscles at NMJs and in the spinal cord. In motor neuron-like and neuroblastoma cell lines, surface-expressed PRPH facilitates viral entry, while intracellular PRPH influences viral genome replication through interactions with structural and non-structural viral components. Importantly, PRPH does not play a role during infection with coxsackievirus A16, another causative agent of HFMD rarely associated with neurological complications, suggesting that EV-A71 ability to exploit PRPH represents a unique attribute for successful CNS invasion. Finally, we show that EV-A71 also exploits some of the many PRPH-interacting partners. Of these, small GTP-binding protein Rac1 represents a potential druggable host target to limit neuroinvasion of EV-A71.

Insight into the Enterovirus A71: A review.

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.

Analysis of the epidemiological trends of enterovirus A in Asia and Europe.

BACKGROUND: Enteroviruses have been in massive, cyclical epidemics worldwide. An in-depth understanding of the international epidemiological characteristics of Enterovirus A (EVA) is critical to determining its clinical significance and total disease burden. Although much research has been conducted on EVA epidemiology, there is still a lack of a comprehensive overview of EVA epidemiological characteristics and trends. OBJECTIVE: EVA nucleic acid sequences from the NCBI virus database were used to summarize the epidemic time (based on the time of specimen collection), spatial and serotype distribution of EVA, and to analyze EVA isolated from cerebrospinal fluid specimens. METHODS: EVA sequences were searched in NCBI Virus by keyword ("Enterovirus A″ or "EVA") to screen sequences released before December 2021 and sort them to analyze EVA by year, geographic region and serotype prevalence. RESULTS: The results found 23,041 retrieved nucleic acid sequences with precise collection dates and geographical regions as of December 2021, with Asia accounting for 87%, Europe for 11% and Africa and the Americas for only 2%. Overall, EV-A71, CVA6 and CVA16 are a few of the main prevalent serotypes; and the prevalence characteristics of the different serotypes change over time from place to place. CONCLUSION: The prevalence of different serotypes of EVA varies considerably over time and space, and we focused on analysing the epidemiological characteristics of EVAs in Asia and Europe and EVAs that invade the nervous system. This study will likely provide important clues for prevention, control and future research in virological surveillance, disease management and vaccine development.

Isolation and Characterization of scFv Antibody against Internal Ribosomal Entry Site of Enterovirus A71.

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.

Temporal Proteomic and Phosphoproteomic Analysis of EV-A71-Infected Human Cells.

Human enterovirus A71 (EV-A71), a member of the Picornaviridae family, is one of the main etiological viruses that lead to hand, foot, and mouth disease (HFMD). We utilized a multiplex tandem mass tag-based quantitative proteomic technique to monitor the alternation of the whole cell proteome and phosphoproteome of human rhabdomyosarcoma cells over the course of EV-A71 infection. We successfully quantified more than 7000 host proteins and 17,000 phosphosites, of which 80 proteins and nearly 1700 phosphosites were significantly regulated upon viral infection. We found that Myc proto-oncogene protein level decreased significantly, benefiting EV-A71 replication. Multiple signaling pathways were regulated in phosphorylation events that converge for protein translation, cell cycle control, and cell survival. Numerous host factors targeted by virus proteins are phosphoproteins. These factors are involved in host translational initiation, unfolded protein response, endoplasmic reticulum stress, and stress granule formation, and their phosphorylation may play key roles in the virus life cycle. Notably, we identified three conserved phosphorylation sites on viral polyproteins that have not been previously reported. Our study provides valuable resources for a systematic understanding of the interaction between the host cells and the EV-A71 at the protein and the post-translational level.

Virulence of Enterovirus A71 Fluctuates Depending on the Phylogenetic Clade Formed in the Epidemic Year and Epidemic Region.

Although epidemics of hand, foot, and mouth disease (HFMD) caused by enterovirus A71 (EV-A71) have occurred worldwide, the Asia-Pacific region has seen large sporadic outbreaks with many severe neurological cases. This suggests that the virulence of the circulating viruses fluctuates in each epidemic and that HFMD outbreaks with many severe cases occur when highly virulent viruses are circulating predominantly, which has not been experimentally verified. Here, we analyzed 32 clinically isolated strains obtained in Japan from 2002 to 2013, along with 27 Vietnamese strains obtained from 2015 to 2016 that we characterized previously using human SCARB2 transgenic mice. Phylogenetic analysis of the P1 region classified them into five clades belonging to subgenogroup B5 (B5-I to B5-V) and five clades belonging to subgenogroup C4 (C4-I to C4-V) according to the epidemic year and region. Interestingly, clades B5-I and B5-II were very virulent, while clades B5-III, B5-IV, and B5-V were less virulent. Clades C4-II, C4-III, C4-IV, and C4-V were virulent, while clade C4-I was not. The result experimentally showed for the first time that several clades with different virulence levels emerged one after another. The experimental virulence evaluation of circulating viruses using SCARB2 transgenic mice is helpful to assess potential risks of circulating viruses. These results also suggest that a minor nucleotide or amino acid substitution in the EV-A71 genome during circulation causes fluctuations in virulence. The data presented here may increase our understanding of the dynamics of viral virulence during epidemics. IMPORTANCE Outbreaks of hand, foot, and mouth disease (HFMD) with severe enterovirus A71 (EV-A71) cases have occurred repeatedly, mainly in Asia. In severe cases, central nervous system complications can lead to death, making it an infectious disease of importance to public health. An unanswered question about this disease is why outbreaks of HFMD with many severe cases sometimes occur. Here, we collected EV-A71 strains that were prevalent in Japan and Vietnam over the past 20 years and evaluated their virulence in a mouse model of EV-A71 infection. This method clearly revealed that viruses belonging to different clades have different virulence, indicating that the method is powerful to assess the potential risks of the circulating viruses. The results also suggested that factors in the virus genome cause an outbreak with many severe cases and that further studies facilitate the prediction of large epidemics of EV-A71 in the future.