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Introduction: Coxsackievirus A6 (CV-A6) has emerged as the predominant epidemic strain responsible for hand, foot and mouth disease (HFMD). CV-A6 infection can result in severe clinical manifestations, including encephalitis, meningitis, and potentially life-threatening central nervous system disorders. Our previous research findings demonstrated that neonatal mice infected with CV-A6 exhibited limb weakness, paralysis, and ultimately succumbed to death. However, the underlying mechanism of CV-A6-induced nervous system injury remains elusive. Numerous reports have highlighted the pivotal role of miRNAs in various viral infections., Methods: Separately established infection and control groups of mice were used to create miRNA profiles of the brain tissues before and after CV-A6 transfection, followed by experimental verification, prediction, and analysis of the results., Results: At 2 days post-infection (dpi), 4 dpi, and 2dpi vs 4dpi, we identified 175, 198 and 78 significantly differentially expressed miRNAs respectively using qRT-PCR for validation purposes. Subsequently, we predicted target genes of these differentially expressed miRNAs and determined their potential targets through GO (Gene Ontology) enrichment analysis and KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analysis. Finally, we verified the miRNA-mRNA pairing via double luciferase experiments while confirming functional enrichment of target genes through Western Blotting analyses., Discussion: The results from this study suggest that transcriptional regulation, neuronal necrosis, pro-inflammatory cytokine release, and antiviral immunity are all implicated in the pathogenesis of central nervous system injury in mice infected with CV-A6. Brain injury resulting from CV-A6 infection may involve multiple pathways, including glial cell activation, neuronal necrosis, synaptic destruction, degenerative diseases of the nervous system. It can even encompass destruction of the blood-brain barrier, leading to central nervous system injury. The dysregulated miRNAs and signaling pathways discovered in this study provide valuable insights for further investigations into the pathogenesis of CV-A6., Competing Interests: Authors YS, JW, SQ, and SS were employed by the company Wuhan Institute of Biological Products Co. Ltd. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Sun, Hao, Wu, Qian, Shen and Yu.)

Hand, foot, and mouth disease (HFMD) is a common viral infection primarily affecting children. It causes vesicles on the skin and inside the mouth. Although most cases get better on their own, severe cases can lead to complications such as brain stem encephalitis, meningoencephalitis, acute flaccid paralysis, and pulmonary edema. Hand, foot, and mouth disease is caused by various enteroviruses, with enterovirus A71 (EV-A71) and coxsackievirus A16 being the most common. However, recent studies have shown a shift in the molecular epidemiology of HFMD-causing pathogens, with coxsackievirus A6 and coxsackievirus A10 causing more infections. In addition, extensive recombination events have been identified among enterovirus strains, which may have a role in faster evolution and extinction of dominant enterovirus serotypes. Other strains of enterovirus can also cause severe complications, and there has been an increase in mortality associated with brain stem encephalitis in children under 3 years of age and teenagers. Currently, there are no effective antiviral therapies available to treat enterovirus infections. Vaccines against EV-A71 have been approved and are now used in mainland China. Studying the changing epidemiology of HFMD pathogens and the evolution patterns of its causative agents is crucial in developing effective prevention and control strategies. Increased interest in the molecular epidemiology of HFMD causative agents has led to a better understanding of the critical drivers of HFMD outbreaks, which can inform efforts to prevent and control the disease.

Coxsackievirus A16 (CV-A16) and coxsackievirus A10 (CV-A10), more commonly etiological agents of hand, foot and mouth disease (HFMD), are capable of causing severe neurological syndromes with high fatalities, but their neuropathogenesis has rarely been studied. Mounting evidence indicated that pyroptosis is an inflammatory form of cell death that might be widely involved in the pathogenic mechanisms of neurotropic viruses. Our study was designed to examine the effects of NLRP3-mediated pyroptosis in CV-A16- and CV-A10-induced inflammatory neuropathologic formation. In this work, it was showed that SH-SY5Y cells were susceptible to CV-A16 and CV-A10, and meanwhile their infections could result in a decreasing cell viability and an increasing LDH release as well as Caspase1 activation. Moreover, CV-A16 and CV-A10 infections triggered NLRP3-mediated pyroptosis and promoted the release of inflammatory cytokines. Additionally, activated NLRP3 accelerated the pyroptosis formation and aggravated the inflammatory response, but inhibited NLRP3 had a dampening effect on the above situation. Finally, it was further revealed that NLRP3 agonist enhanced the viral replication, but NLRP3 inhibitor suppressed the viral replication, suggesting that NLRP3-driven pyroptosis might support CV-A16 and CV-A10 production in SH-SY5Y cells. Together, our findings demonstrated a mechanism by which CV-A16 and CV-A10 induce inflammatory responses by evoking NLRP3 inflammasome-regulated pyroptosis, which in turn further stimulated the viral replication, providing novel insights into the pathogenesis of CV-A16 and CV-A10 infections., Competing Interests: Declaration of competing interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024. Published by Elsevier B.V.)

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

Background: EV71 is one of the important pathogens of Hand-foot-and-mouth disease (HFMD), which causes serious neurological symptoms. Several studies have speculated that there will be interaction between 5'UTR and 3D protein. However, whether 5'UTR interacts with the 3D protein in regulating virus replication has not been clarified., Methods: Four 5'UTR mutation sites (nt88C/T, nt90-102-3C, nt157G/A and nt574T/A) and two 3D protein mutation sites (S37N and R142K) were mutated or co-mutated using virulent strains as templates. The replication of these mutant viruses and their effect on autophagy were determined., Results: 5'UTR single-point mutant strains, except for EGFP-EV71(nt90-102-3C), triggered replication attenuation. The replication ability of them was weaker than that of the parent strain the virulent strain SDLY107 which is the fatal strain that can cause severe neurological complications. While the replication level of the co-mutant strains showed different characteristics. 5 co-mutant strains with interaction were screened: EGFP-EV71(S37N-nt88C/T), EGFP-EV71(S37N-nt574T/A), EGFP-EV71(R142K-nt574T/A), EGFP-EV71(R142K-nt88C/T), and EGFP-EV71(R142K-nt157G/A). The results showed that the high replicative strains significantly promoted the accumulation of autophagosomes in host cells and hindered the degradation of autolysosomes. The low replicative strains had a low ability to regulate the autophagy of host cells. In addition, the high replicative strains also significantly inhibited the phosphorylation of AKT and mTOR., Conclusions: EV71 5'UTR interacted with the 3D protein during virus replication. The co-mutation of S37N and nt88C/T, S37N and nt574T/ A, R142K and nt574T/A induced incomplete autophagy of host cells and promoted virus replication by inhibiting the autophagy pathway AKT-mTOR. The co-mutation of R142K and nt88C/T, and R142K and nt157G/A significantly reduced the inhibitory effect of EV71 on the AKT-mTOR pathway and reduced the replication ability of the virus., (© 2024. The Author(s).)

Coxsackievirus A10 (CVA10) has recently emerged as one of the major causative agents of hand, foot, and mouth disease. CVA10 may also cause a variety of complications. No approved vaccine or drug is currently available for CVA10. The residues of CVA10 critical for viral attachment, infectivity and in vivo pathogenicity have not been identified by experiment. Here, we report the identification of CVA10 residues important for binding to cellular receptor KREMEN1. We identified VP2 N142 as a key receptor-binding residue by screening of CVA10 mutants resistant to neutralization by soluble KREMEN1 protein. The receptor-binding residue N142 is exposed on the canyon rim but highly conserved in all naturally occurring CVA10 strains, which provides a counterexample to the canyon hypothesis. Residue N142 when mutated drastically reduced receptor-binding activity, resulting in decreased viral attachment and infection in cell culture. More importantly, residue N142 when mutated reduced viral replication in limb muscle and spinal cord of infected mice, leading to lower mortality and less severe clinical symptoms. Additionally, residue N142 when mutated could decrease viral binding affinity to anti-CVA10 polyclonal antibodies and a neutralizing monoclonal antibody and render CVA10 resistant to neutralization by the anti-CVA10 antibodies. Overall, our study highlights the essential role of VP2 residue N142 of CVA10 in the interactions with KREMEN1 receptor and neutralizing antibodies and viral virulence in mice, facilitating the understanding of the molecular mechanisms of CVA10 infection and immunity. Our study also provides important information for rational development of antibody-based treatment and vaccines against CVA10 infection., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Li et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Enterovirus A71 (EV-A71) is a human pathogen that causes hand, foot, and mouth disease, which can progress to severe neurological disease. EV-A71 infects humans via the human scavenger receptor B2 (hSCARB2). It can also infect neonatal mice experimentally. Wild-type (WT) EV-A71 strains replicate primarily in the muscle of neonatal mice; however, susceptibility lasts only for a week after birth. Mouse-adapted (MA) strains, which can be obtained by serial passages in neonatal mice, are capable of infecting both muscle and neurons of the central nervous system. It is not clear how the host range and tropism of EV-A71 are regulated and why neonatal mice lose their susceptibility during development. We hypothesized that EV-A71 infection in neonatal mice is mediated by mouse Scarb2 (mScarb2) protein. Rhabdomyosarcoma (RD) cells expressing mScarb2 were prepared. Both WT and MA strains infected mScarb2-expressing cells, but the infection efficiency of the WT strain was much lower than that of the MA strain. Infection by WT and MA strains in vivo was abolished completely in Scarb2 -/- mice. Scarb2 +/- mice, in which Scarb2 expression was approximately half of that in Scarb2 +/+ mice, showed a milder pathology than Scarb2 +/+ mice after infection with the WT strain. The Scarb2 expression level in muscle decreased with aging, which was consistent with the reduced susceptibility of aged mice to infection. These results indicated that EV-A71 infection is mediated by mScarb2 and that the severity of the disease, the spread of virus, and the susceptibility period are modulated by mScarb2 expression. IMPORTANCE EV-A71 infects humans naturally but can also infect neonatal mice. The tissue tropism and severity of EV-A71 disease are determined by several factors, among which the virus receptor is thought to be important. We show that EV-A71 can infect neonatal mice using mScarb2. However, the infection efficiency of WT strains via mScarb2 is so low that an elevated virus-receptor interaction associated with mouse adaptation mutation and decrease in mScarb2 expression level during development modulate the severity of the disease, the spread of virus, and the susceptibility period in the artificial neonatal mice model.

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

Enterovirus 71 can cause severe hand, foot, and mouth disease (HFMD) in children. However, little is known about the mechanism of inflammatory disorders caused by EV71 infection and why severe cases are mainly children aged under-three. In current study, using mRNA microarray assay, the differential expression of Placenta-specific 8 (PLAC8) was identified in mice brain. In addition, we found that PLAC8 expression was down-regulated with age in mice lung tissues and human peripheral blood. Then, we further proved that PLAC8 could promote inflammation progress and disturb Th1/Th2/Th17/Treg related cytokines release after EV71 infection using PLAC8 plasmid over-expressed neonatal mouse model. Our data suggest that PLAC8 might play a crucial role in Th cell differentiation and inflammatory damage caused by EV71 infection in infants. Thus, our findings would help understand the causes of severe inflammatory injury in infants during EV71 infection, and provide new insights into the prevention and control of severe HFMD., (Copyright © 2021. Published by Elsevier Inc.)

Coxsackievirus A16 (CV-A16) has caused worldwide epidemics of hand, foot, and mouth disease (HFMD) in infants and preschool children. Circular RNAs (circRNAs), a class of noncoding RNA molecules, participate in the progression of viral infectious diseases. Although the function of circRNAs has been a heavily researched topic, their role in CV-A16 infection is still unclear. In this study, the viral effects of CV-A16 on the cellular circRNA transcriptome were investigated using next-generation sequencing technology. The results showed that a total of 8726, 8611, and 6826 circRNAs were identified at 0, 12, and 24 h postinfection, respectively. Moreover, it was found that 1769 and 1192 circRNAs were differentially expressed in at 12 and 24 h postinfection, respectively. The common differentially expressed circRNAs were used for functional annotation analysis, and it was found that the parent genes of differentially expressed circRNAs might be associated with the viral infection process, especially the "Immune system process" in GO analysis and the "Inflammation mediated by chemokine and cytokine signaling pathway" in KEGG analysis. Subsequently, circRNA-miRNA-mRNA regulatory networks were constructed, and the hsa_circ_0004447/hsa-miR-942-5p/MMP2, hsa_circ_0078617/hsa-miR-6780b-5p/MMP2 and hsa_circ_0078617/hsa-miR-5196-5p/MMP2 regulatory axes were identified by enrichment analysis as important networks during the progression of CV-A16 infection. Finally, six dysregulated circRNAs were selected for validation and were verified to be consistent with the sequencing results. Considering all of these results, to the best of our knowledge, this study is the first to present a comprehensive overview of circRNAs induced by CV-A16 infection, and this research demonstrated that a network of enriched circRNAs and circRNA-associated competitive endogenous RNAs (ceRNAs) is involved in the regulation of CV-A16 infection, thereby helping to elucidate the mechanisms underlying CV-A16-host interactions., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Enterovirus A71 (EV-A71) is one of the major etiological agents of hand, foot, and mouth disease (HFMD), and infection occasionally leads to fatal neurological complications in children. However, only inactivated whole-virus vaccines against EV-A71 are commercially available in Mainland China. Furthermore, the mechanisms underlying the infectivity and pathogenesis of EV-A71 remain to be better understood. By adaptation of an EV-A71 B5 strain in monkey Vero cells in the presence of brilliant black BN (E151), an anti-EV-A71 agent, a double mutant with VP1-V238A,K244R emerged whose infection was enhanced by E151. The growth of the reverse genetics (RG) mutant RG/B5-VP1-V238A,K244R (RG/B5-AR) was promoted by E151 in Vero cells but inhibited in other human and murine cells, while its parental wild type, RG/B5-wt, was strongly prevented by E151 from infection in all tested cells. In the absence of E151, RG/B5-AR exhibited defective cell entry/exit, resulting in reduced viral transmission and growth in vitro . It had augmented binding affinity to sulfated glycans, cells, and tissue/organs, which probably functioned as decoys to restrict viral dissemination and infection. RG/B5-AR was also attenuated, with a 355 times higher 50% lethal dose (LD 50 ) and a shorter timing of virus clearance than those of RG/B5-wt in suckling AG129 mice. However, it remained highly immunogenic in adult AG129 mice and protected their suckling mice from lethal EV-A71 challenges through maternal neutralizing antibodies. Overall, discovery of the attenuated mutant RG/B5-AR contributes to better understanding of virulence determinants of EV-A71 and to further development of novel vaccines against EV-A71. IMPORTANCE Enterovirus A71 (EV-A71) is highly contagious in children and has been responsible for thousands of deaths in Asia-Pacific region since the 1990s. Unfortunately, the virulence determinants and pathogenesis of EV-A71 are not fully clear. We discovered that a novel EV-A71 mutant, VP1-V238A,K244R, showed growth attenuation with reduced efficiency of cell entry/exit. In the Vero cell line, which has been approved for manufacturing EV-A71 vaccines, the growth defects of the mutant were compensated by a food dye, brilliant black BN. The mutant also showed augmented binding affinity to sulfated glycans and other cellular components, which probably restricted viral infection and dissemination. Therefore, it was virulence attenuated in a mouse model but still retained its immunogenicity. Our findings suggest the mutant as a promising vaccine candidate against EV-A71 infection.

Enterovirus A71 (EV-A71) and many members of the Picornaviridae family are neurotropic pathogens of global concern. These viruses are primarily transmitted through the fecal-oral route, and thus suitable animal models of oral infection are needed to investigate viral pathogenesis. An animal model of oral infection was developed using transgenic mice expressing human SCARB2 (hSCARB2 Tg), murine-adapted EV-A71/MP4 virus, and EV-A71/MP4 virus with an engineered nanoluciferase gene that allows imaging of viral replication and spread in infected mice. Next-generation sequencing of EV-A71 genomes in the tissues and organs of infected mice was also performed. Oral inoculation of EV-A71/MP4 or nanoluciferase-carrying MP4 virus stably induced neurological symptoms and death in infected 21-day-old weaned mice. In vivo bioluminescence imaging of infected mice and tissue immunostaining of viral antigens indicated that orally inoculated virus can spread to the central nervous system (CNS) and other tissues. Next-generating sequencing further identified diverse mutations in viral genomes that can potentially contribute to viral pathogenesis. This study presents an EV-A71 oral infection murine model that efficiently infects weaned mice and allows tracking of viral spread, features that can facilitate research into viral pathogenesis and neuroinvasion via the natural route of infection. IMPORTANCE Enterovirus A71 (EV-A71), a positive-strand RNA virus of the Picornaviridae , poses a persistent global public health problem. EV-A71 is primarily transmitted through the fecal-oral route, and thus suitable animal models of oral infection are needed to investigate viral pathogenesis. We present an animal model of EV-A71 infection that enables the natural route of oral infection in weaned and nonimmunocompromised 21-day-old hSCARB2 transgenic mice. Our results demonstrate that severe disease and death could be stably induced, and viral invasion of the CNS could be replicated in this model, similar to severe real-world EV-A71 infections. We also developed a nanoluciferase-containing EV-A71 virus that can be used with this animal model to track viral spread after oral infection in real time. Such a model offers several advantages over existing animal models and can facilitate future research into viral spread, tissue tropism, and viral pathogenesis, all pressing issues that remain unaddressed for EV-A71 infections.

Enterovirus-A71 (EV-A71) associated Hand, foot and mouth disease (HFMD) is a highly contagious viral infection affecting children in Asia-Pacific region and has become a major threat to public health. Although several EV-A71 genotypes (C, D, and G) were isolated in India in recent years, no recognizable outbreak of EV-A71 caused HFMD, Acute Flaccid paralysis (AFP) or encephalitis have been reported so far. It is essential to study the pathogenicity or cell tropism of these Indian isolates in order to understand their tendency to cause disease. We investigated the susceptibility and cytokine responses of indigenous EV-A71 genotypes (D and G) isolated from cases of AFP and genotype C viruses isolated from cases of HFMD and encephalitis, in human cells in-vitro. Although all three EV-A71 genotypes could infect and replicate in human muscle and neuronal cells, the genotype D virus showed a delayed response in human neuronal cells. Quantification of cytokine secretion in response to these isolates followed by confirmation with gene expression assays in human neuronal cells revealed significantly higher secretion of pro-inflammatory cytokines TNF-α IL-8, IL-6, IP-10 (p < 0.001) in G genotype infected cells as compared to pathogenic C genotypes whereas the genotype D virus could not induce any of the inflammatory cytokines. These findings will help to better understand the host response to indigenous EV-A71 genotypes for management of future EV-A71 outbreaks in India, if any., (© 2021. The Author(s).)

Outbreaks of hand, foot, and mouth disease caused by enterovirus-A71 (EV-A71) can result in many deaths, due to central nervous system complications. Outbreaks with many fatalities have occurred sporadically in the Asia-Pacific region and have become a serious public health concern. It is hypothesized that virulent mutations in the EV-A71 genome cause these occasional outbreaks. Analysis of EV-A71 neurovirulence determinants is important, but there are no virulence determinants that are widely accepted among researchers. This is because most studies have been done in artificially infected mouse models and because EV-A71 mutates very quickly to adapt to the artificial host environment. Although EV-A71 uses multiple receptors for infection, it is clear that adaptation-related mutations alter the binding specificity of the receptors and allow the virus to adopt the best entry route for each environment. Such mutations have confused interpretations of virulence in animal models. This article will discuss how environment-adapted mutations in EV-A71 occur, how they affect virulence, and how such mutations can be avoided. We also discuss future perspectives for EV-A71 virulence research.

MicroRNAs (miRNAs) are crucial in the process of host-pathogen interaction. In this study, we established a screening system for miRNAs of target genes to detect the effect of miRNAs on Enterovirus 71 (EV71) replication in rhabdomyosarcoma (RD) cells. A 3'-untranslated region (UTR) dual-luciferase assay was performed to confirm putative miRNA targets in EV71 genome. Firstly, 13 fragments of EV71 genome were inserted into the vector pMIR, and luciferase activities were analyzed to identify the putative miRNAs of target genes. The expression of the reporter protein was significantly downregulated in cells transfected with the vector containing gene VP3. Then we screened for miRNAs that might target to VP3 through online analysis software. In addition, Western blot, real-time PCR, virus titration, and morphological changes were considered to examine the effects of miRNAs on virus replication. The results suggested that miR-18a and miR-452 repress the reproduction of EV71 virus by binding to VP3. Moreover, EV71 infection also affected the expression of endogenous miR-18a and miR-452. In addition, no significant cytotoxic effects were observed. The results from this study suggest that the intracellular miRNAs may play vital roles in the host-virus interaction.

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

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

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

Enterovirus 71 (EV71) can cause a severe hand-foot-mouth disease in children. However, the precise mechanism of EV71-associated disease, particularly the neuropathogenesis and pulmonary disorder, is still not fully understood because no suitable animal models are available. The human scavenger receptor class B, member 2 (hSCARB2), is a cellular receptor for EV71. Here, we generated a novel knock-in (KI) mouse model using the CRISPR/Cas9 system to insert the hSCARB2 gene into the mouse Rosa26 locus to study the pathogenesis of EV71. The hSCARB2 KI mice infected with clinical isolates of EV71 showed neurological symptoms, such as ataxia, paralysis, and death. Viral replication was detected in mainly astrocytes and a limited number of neurons and microglia, accompanied by gliosis. Vascular leakage and alveoli filled with erythrocytes were detected, suggesting that edema and hemorrhage, which are observed in human patients, also occurred in EV71-infected KI mice. In addition, proinflammatory cytokines and chemokines were significantly increased in the serum of infected KI mice. These pathological features of the KI mice after infection resembled those of EV71 encephalomyelitis in humans. Therefore, our KI mouse model is suitable to study the pathogenesis of EV71 and is of great significance for development of antiviral drugs and vaccines to treat or prevent EV71 infection. IMPORTANCE Enterovirus 71 (EV71) is associated with severe hand-foot-mouth disease. Recently, outbreaks of EV71 infection with high mortality have been reported in the Asia-Pacific region, posing a great challenge for global public health. To date, the precise mechanism of EV71-induced disease, particularly the neuropathogenesis and respiratory disorders, is still not fully understood because no suitable animal models are available. Human scavenger receptor class B, member 2 (hSCARB2), has been identified as a cellular receptor for EV71. Here, we introduce a novel CRISPR/Cas9-mediated hSCARB2 knock-in (KI) mouse model for the study of EV71 pathogenesis, which is of great significance for the development of antiviral drugs and vaccines., (Copyright © 2021 Jin et al.)

Enteroviruses, such as EV-A71 and CVA16, mainly infect the human gastrointestinal tract. Human coronaviruses, including SARS-CoV and SARS-CoV-2, have been variably associated with gastrointestinal symptoms. We aimed to optimize the human intestinal organoids and hypothesize that these optimized intestinal organoids can recapitulate enteric infections of enterovirus and coronavirus. We demonstrate that the optimized human intestinal organoids enable better simulation of the native human intestinal epithelium, and that they are significantly more susceptible to EV-A71 than CVA16. Higher replication of EV-A71 than CVA16 in the intestinal organoids triggers a more vigorous cellular response. However, SARS-CoV and SARS-CoV-2 exhibit distinct dynamics of virus-host interaction; more robust propagation of SARS-CoV triggers minimal cellular response, whereas, SARS-CoV-2 exhibits lower replication capacity but elicits a moderate cellular response. Taken together, the disparate profile of the virus-host interaction of enteroviruses and coronaviruses in human intestinal organoids may unravel the cellular basis of the distinct pathogenicity of these viral pathogens., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Enterovirus A71 (EV-A71) is a causative agent of hand, foot and mouth disease and occasionally causes death in children. Its infectivity and pathogenesis, however, remain to be better understood. Three sulfonated azo dyes, including acid red 88 (Ar88), were identified to enhance the infectivity of EV-A71, especially isolates with VP1-98K, 145E (-KE), by mainly promoting viral genome release in vitro. Enzymatic removal of sulfated glycosaminoglycans (GAGs) or knockout of xylosyltransferase II (XT2) responsible for biosynthesis of sulfated GAGs weakened the Ar88 enhanced EV-A71 infection. Ar88 is proposed to prevent the -KE variants from being trapped by sulfated GAGs at acidic pH and to facilitate the viral interaction with uncoating factors for genome release in endosomes. The results suggest dual roles of sulfated GAGs as attachment factors and as decoys during host interaction of EV-A71 and caution that these artificial dyes in our environment can enhance viral infection., (Copyright © 2020. Published by Elsevier Inc.)

Background: To analyze the prevalence of latent infection of pathogens of hand, foot, and mouth disease (HFMD) in Chinese healthy population and its influencing factors, so as to provide reference for the prevention and control of HFMD., Methods: A systematic literature searching about the incidence of latent infection of HFMD was conducted in Chinese and English databases. The inclusion and exclusion criteria of the retrieved literature were established. The qualified literatures were screened and the data were extracted. The pooled rate and its 95% confidence interval was used to assess the latent infection rate of HFMD pathogens in healthy Chinese population, and subgroup analysis was conducted based on gender and age. All statistical analyses were performed using the STATA version 12.0 software., Results: A total of 31 literatures were included in this meta-analysis. The recessive infection rate of HFMD pathogens reported in the literature of Chinese healthy people ranged from 4.59% to 44.12%. The results of meta-analysis showed that the latent infection rate of human enteroviruses (HEVs) in healthy Chinese population was 17.5% (14.9-20.1%), among which, the latent infection rates of EV-A71, CV-A16, and other HEVs were 3.3% (2.2-4.4%), 1.7% (1.0-2.5%), and 15.1% (11.1-17.1%), respectively. The latent infection rates of HEVs in healthy men and women in China were 16.7% (12.9-20.4%) and 14.4% (10.8-18.0%), respectively. The latent infection rates of HEVs in the healthy population aged 0 to 5 years and over 5 years were 24.4% (20.4-28.5%) and 9.4% (6.5-12.2%), respectively. Meta regression showed that the factors affecting the latent infection rate of HEVs in Chinese healthy population included sampling period, sampling area, and study population., Conclusion: The latent infection rate of HEVs is high in healthy people in China, but it is mainly caused by other enteroviruses. The latent infection rate of HEVs in male was higher than that of female and was greater in people aged 0 to 5 than that of aged over 5 years. Limited by the quantity and quality of the included studies, more high-quality studies are needed for further verification in the future., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2021 the Author(s). Published by Wolters Kluwer Health, Inc.)

Enterovirus A71 (EV-A71), a positive-stranded RNA virus of the Picornaviridae family, may cause neurological complications or fatality in children. We examined specific factors responsible for this virulence using a chemical genetics approach. Known compounds from an anti-EV-A71 herbal medicine, Salvia miltiorrhiza (Danshen), were screened for anti-EV-A71. We identified a natural product, rosmarinic acid (RA), as a potential inhibitor of EV-A71 by cell-based antiviral assay and in vivo mouse model. Results also show that RA may affect the early stage of viral infection and may target viral particles directly, thereby interfering with virus-P-selectin glycoprotein ligand-1 (PSGL1) and virus-heparan sulfate interactions without abolishing the interaction between the virus and scavenger receptor B2 (SCARB2). Sequencing of the plaque-purified RA-resistant viruses revealed a N104K mutation in the five-fold axis of the structural protein VP1, which contains positively charged amino acids reportedly associated with virus-PSGL1 and virus-heparan sulfate interactions via electrostatic attraction. The plasmid-derived recombinant virus harbouring this mutation was confirmed to be refractory to RA inhibition. Receptor pull-down showed that this non-positively charged VP1-N104 is critical for virus binding to heparan sulfate. As the VP1-N104 residue is conserved among different EV-A71 strains, RA may be useful for inhibiting EV-A71 infection, even for emergent virus variants. Our study provides insight into the molecular mechanism of virus-host interactions and identifies a promising new class of inhibitors based on its antiviral activity and broad spectrum effects against a range of EV-A71.

Enterovirus 71 (EV71) infection causes hand, foot, and mouth disease (HFMD), and even fatal neurological complications. However, the mechanisms underlying EV71 neurological pathogeneses are largely unknown. This study reveals a distinct mechanism by which EV71 induces apoptosis and autophagy in neural cells. EV71 non-structure protein 3D (also known as RNA-dependent RNA polymerase, RdRp) interacts with the peroxisomal protein acyl-CoA oxidase 1 (ACOX1), and contributes to ACOX1 downregulation. Further studies demonstrate that EV71 reduces peroxisome numbers. Additionally, knockdown of ACOX1 or peroxin 19 (PEX19) induces apoptosis and autophagy in neural cells including human neuroblastoma (SK-N-SH) cells and human astrocytoma (U251) cells, and EV71 infection induces neural cell death through attenuating ACOX1 production. Moreover, EV71 infection and ACOX1 knockdown facilitate reactive oxygen species (ROS) production and attenuate the cytoprotective protein deglycase (DJ-1)/Nuclear factor erythroid 2-related factor 2 (NRF2)/Heme oxygenase 1 (HO-1) pathway (DJ-1/NRF2/HO-1) , which collectively result in ROS accumulation in neural cells. In conclusion, EV71 downregulates ACOX1 protein expression, reduces peroxisome numbers, enhances ROS generation, and attenuates the DJ-1 /NRF2/HO-1 pathway, thereby inducing apoptosis and autophagy in neural cells. These findings provide new insights into the mechanism underlying EV71-induced neural pathogenesis, and suggest potential treatments for EV71-associated diseases.

Enterovirus 71 (EV-A71) and coxsackievirus A16 (CV-A16) are the major pathogens responsible for hand, foot and mouth disease (HFMD), but the mechanism by which these viruses cause disease remains unclear. In this study, we used transcriptome sequencing technology to investigate changes in the transcriptome profiles after infection with EV-A71 and CV-A16 in human bronchial epithelial (16HBE) cells. Using systematic bioinformatics analysis, we then searched for useful clues regarding the pathogenesis of HFMD. As a result, a total of 111 common differentially expressed genes were present in both EV-A71- and CV-A16-infected cells. A trend analysis of these 111 genes showed that 91 of them displayed the same trend in EV-A71 and CV-A16 infection, including 49 upregulated genes and 42 downregulated genes. These 91 genes were further used to conduct GO, pathway, and coexpression network analysis. It was discovered that enriched GO terms (such as histone acetylation and positive regulation of phosphorylation) and pathways (such as glycosylphosphatidylinositol (GPI)-anchor biosynthesis and DNA replication) might be closely associated with the pathogenic mechanism of these two viruses, and key genes (such as TBCK and GPC) might be involved in the progression of HFMD. Finally, we randomly selected 10 differentially expressed genes for qRT-PCR to validate the transcriptome sequencing data. The experimental qRT-PCR results were roughly in agreement with the results of transcriptome sequencing. Collectively, our results provide clues to the mechanism of pathogenesis of HFMD induced by EV-A71 and CV-A16.

Enteroviruses are one of the most important causes of viral encephalitis in the neonatal period. However, the non-specificity of the symptoms presented renders its diagnosis challenging. Intracranial MRI has been reported to be a very useful imaging modality that can detect the characteristic white matter lesions around the periventricular regions. In this study, we report a case of a patient with neonatal encephalitis who presented with normal white blood cell counts in the initial cerebrospinal fluid analysis. A lumbar puncture retap identified pleocytosis, and polymerase chain reaction assays detected enterovirus 71 in the blood and stool samples. Furthermore, MRI revealed atypical disseminated cortical and subcortical white matter lesions on diffusion weighted images, and neuroradiological re-evaluation showed necrotic changes 2 weeks later. This unique case expands our knowledge of the spectrum of neurological disorders due to enterovirus 71 infection in neonatal period.

Enterovirus A71 (EV-A71), which belongs to the family Picornaviridae, can invade the central nervous system (CNS) and cause severe CNS complications or death. The EV-A71 antigen has been detected in the neurons in the brains of humans who died from EV-A71 infection. However, the effect of EV-A71 infection on human neuronal cells remains poorly understood. Human neural stem cells (NSCs) and IMR-32 neuroblastoma cells were differentiated into neuronal cells for this study. Although the neuronal cells were permissive to EV-A71 infection, EV-A71 infection did not induce an obvious cytopathic effect on the neuronal cells. EV-A71 infection did not induce apoptosis in neuronal cells. However, autophagy and autophagic flux were induced in EV-A71-infected neuronal cells. The production of autophagosomes was shown to be important for EV-A71 viral RNA (vRNA) replication in neuronal cells.

Based on the etching of gold nanorods (GNRs) and enzyme-linked immunosorbent assay (ELISA), a multicolor immunosensor for visual detection of enterovirus 71 infection is proposed. Once the immunocomplex is formed, the horseradish peroxidase bound to the ELISA plate oxidizes 3,3',5,5'-tetramethylbenzidine (TMB) into TMB 2+ in the presence of hydrogen peroxide. Subsequently, TMB 2+ quantitatively etches GNRs to the short GNRs, leading to a blue shift of longitudinal localized surface plasmon resonance and corresponding color responses. This change is used to develop two types of cut-off standards, which respond to the human anti-enterovirus at a concentration of 71 IgM antibody. The method has been validated with clinical serum samples and showed high sensitivity and specificity . This visual immunosensor has an important application value for point-of-care detection of EV71, especially in areas lacking detection equipment. Graphical abstract.

Aquaporin-4 (AQP4) is a major water channel of the central nervous system. The present study was designed to determine whether AQP4 deletion could ameliorate enterovirus (EV) 71 infection‑induced hand, foot and mouth disease (HFMD) by inhibiting inflammation and apoptosis in mice. EV 71 strains were injected into neonatal BALB/c mice to induce HFMD. Western blotting and ELISA were used to measure the protein expression and cytokine levels. The levels of AQP4 mRNA and protein in the brain were increased in EV 71‑infected mice, while the survival rate and health score were improved in AQP4‑knockout (KO) mice with EV 71 infection. The EV 71 infection‑induced increases of tumor necrosis factor‑α, interleukin (IL)‑1β, IL‑6, monocyte chemotactic protein‑1, interferon (IFN)‑α and IFN‑γ in plasma and brain were inhibited in AQP4‑KO mice. AQP4 deletion reversed the decreased levels of Bcl2 and Bcl2/Bax, and the increased levels of Bax induced by EV 71 infection in the brain. These results demonstrated that AQP4 deletion ameliorated EV 71 induced‑HFMD via inhibiting inflammation and apoptosis in mice.

Enterovirus 71 (EV71) is the major causative pathogen of human hand, foot, and mouth disease (hHFMD) and has evolved to use various cellular receptors for infection. However, the relationship between receptor preference and EV71 virulence has not been fully revealed. By using reverse genetics, we identified that a single E98K mutation in VP1 is responsible for rapid viral replication in vitro. The E98K mutation enhanced binding of EV71-GZCII to cells in a heparan sulfate (HS)-dependent manner, and it attenuated the virulence of EV71-GZCII in BALB/c mice, indicating that the HS-binding property is negatively associated with viral virulence. HS is widely expressed in vascular endothelial cells in different mouse tissues, and weak colocalization of HS with scavenger receptor B2 (SCARB2) was detected. The cGZCII-98K virus bound more efficiently to mouse tissue homogenates, and the cGZCII-98K virus titers in mouse tissues and blood were much lower than the cGZCII virus titers. Together, these findings suggest that the enhanced adsorption of the cGZCII-98K virus, which likely occurs through HS, is unable to support the efficient replication of EV71 in vivo. Our study confirmed the role of HS-binding sites in EV71 infection and highlighted the importance of the HS receptor in EV71 pathogenesis.

Enterovirus A71 (EVA71) and Coxsackievirus A10 (CVA10) are representative types of Enterovirus A. Dependent on the host cell types, the EVA71 entry may utilize clathrin-, caveola-, and endophilin-A2-mediated endocytosis. However, the cell-entry and intracellular trafficking pathways of CVA10, using KREMEN1 as its receptor, are unclear. Here, we tested the relevant mechanisms through RNA interference (RNAi) and chemical inhibitors. We found that endocytosis of EVA71 and CVA10 in rhabdomyosarcoma (RD) cells engaged multiple pathways, and both viruses required Rac1. Interestingly, while CDC42 and Pak1 participated in EVA71 infection, PI3K played a role in CVA10 infection. The functions of Rab proteins in intracellular trafficking of CVA10 and EVA71 were examined by RNAi. Knockdown of Rab5 and Rab21 significantly reduced CVA10 infectivity, while knockdown of Rab5, Rab7 and Rab9 reduced EVA71 infectivity. Confocal microscopy confirmed the colocalization of CVA10 virions with Rab5 or Rab21, and colocalization of EVA71 virions with Rab5 or Rab7. Additionally, we observed that both CVA10 and EVA71 infections were inhibited by endosome acidification inhibitors, bafilomycin-A1 and NH 4 Cl. Together, our findings comparatively illustrate the entry and intracellular trafficking processes of representative Enterovirus A types and revealed novel enterovirus intervention targets., Competing Interests: Declaration of competing interest The authors declare that there is no conflict of interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Coxsackievirus A10 (CVA10) is one of the major etiological agents of hand, foot, and mouth disease. There are no vaccine and antiviral drugs for controlling CVA10 infection. Reverse genetic tools for CVA10 will benefit its mechanistic study and development of vaccines and antivirals. Here, two infectious clones for the prototype and a Myc-tagged CVA10 were constructed. Viable CVA10 viruses were harvested by transfecting the viral mRNA into human rhabdomyosarcoma (RD) cells. Rescued CVA10 was further confirmed by next generation sequencing and characterized experimentally. We also constructed the vectors for CVA10 subgenomic replicon with luciferase reporter and viral capsid with EGFP reporter, respectively. Co-transfection of the viral replicon RNA and capsid expresser in human embryonic kidney 293T (HEK293T) cells led to the production of single round infectious particles (SRIPs). Based on CVA10 replicon RNA, SRIPs with either the enterovirus A71 (EVA71) capsid or the CVA10 capsid were generated. Infection by EVA71 SRIPs required SCARB2, while CVA10 SRIPs did not. Finally, we showed great improvement of the replicon activity and SRIPs production by insertion of a cis-active hammerhead ribozyme (HHRib) before the 5'-untranslated region (UTR). In summary, reverse genetic tools for prototype strain of CVA10, including both the infectious clone and the SRIPs system, were successfully established. These tools will facilitate the basic and translational study of CVA10.

Enterovirus A71 (EV-A71) is the major pathogen of hand, foot, and mouth disease (HFMD); in some severe cases, it could develop into central nervous system (CNS) disease such as aseptic meningitis, encephalitis, and neurogenic pulmonary edema in children under 5 years. The EV-A71 pathogenesis which is involved with the CNS is unclear due to the lack of a simple and reliable mouse model thus far. Most clinical EV-A71 isolates could not effectively infect the neonatal mouse, which used to be an EV-A71 infection model. The small extracellular vesicles (sEVs) released from clinical EV-A71 isolate-infected cells were infectious in cell lines and could cause a high viral replication in mice. Neonatal ICR mice were injected intraperitoneally with these infectious sEVs and showed more weight loss and higher mortality than those mice injected with the clinical EV-A71 isolate. By using these sEVs, we provided a simple and effective method by which we can generate a stable and valuable animal model for the studies of EV-A71 pathogenesis and therapy. IMPORTANCE EV-A71 was supposed to infect the CNS through the neural pathway and the circulation of the blood in previous studies. Reverse axon transport had been confirmed as an important pathway for EV-A71 to infect the CNS; however, it is still unknown how EV-A71 infects the CNS through the circulation of the blood. Combined with the infectivity of sEVs secreted from EV-A71-infected cells and the characteristic that sEVs could cross the blood-brain barrier, we considered that sEVs may play a vital role in EV-A71 pathogenesis of the CNS., (Copyright © 2020 Gu et al.)

Viral plaque morphologies in human cell lines are markers for growth capability and they have been used to assess the viral fitness and selection of attenuated mutants for live-attenuated vaccine development. In this study, we investigate whether the naturally occurring plaque size variation reflects the virulence of the variants of EV-A71. Variants of two different plaque sizes (big and small) from EV-A71 sub-genotype B4 strain 41 were characterized. The plaque variants displayed different in vitro growth kinetics compared to the parental wild type. The plaque variants showed specific mutations being present in each variant strain. The big plaque variants showed four mutations I97L, N104S, S246P and N282D in the VP1 while the small plaque variants showed I97T, N237T and T292A in the VP1. No other mutations were detected in the whole genome of the two variants. The variants showed stable homogenous small plaques and big plaques, respectively, when re-infected in rhabdomyosarcoma (RD) and Vero cells. The parental strain showed faster growth kinetics and had higher viral RNA copy number than both the big and small plaque variants. Homology modelling shows that both plaque variants have differences in the structure of the VP1 protein due to the presence of unique spontaneous mutations found in each plaque variant This study suggests that the EV-A71 sub-genotype B4 strain 41 has at least two variants with different plaque morphologies. These differences were likely due to the presence of spontaneous mutations that are unique to each of the plaque variants. The ability to maintain the respective plaque morphology upon passaging indicates the presence of quasi-species in the parental population.

Enterovirus A71 (EV71) remains the most common causative agent of hand, foot, and mouth disease (HFMD), and the neurological complications induced by EV71 are usually the leading cause of death in children with HFMD. However, the mechanism of nervous system changes caused by EV71 infection is still unclear. Therefore, in the current study, EV71 was inoculated into the human neuroblastoma cell line SH-SY5Y and subsequent transcriptome sequencing was used to examine the alterations of the transcriptome in infected SH-SY5Y cells. It is expected to determine the underlying mechanism of neurological diseases in response to EV71 infection. As a result, a total of 82,406,974, 112,410,808 and 87,780,371 clean reads were found in the control, EV71-12 h and EV71-24 h groups, respectively. Moreover, 160 and 745 differentially expressed genes were identified in the EV71-12 h and EV71-24 h groups, respectively, as compared to the control group. Next, to further explore the pathogenic mechanism triggered by EV71 infection, we mainly focused on the common differentially expressed genes at different time points of EV71 infection. And it was discovered that there were 95 common differentially expressed genes, which were used to conduct GO and pathway analysis. GO enrichment analysis demarcated related biological processes, molecular functions and cellular components, and KEGG pathway analysis enabled annotations of metabolic pathways and revealed interactions among the significantly enriched pathways. The results showed that the enriched GO term "Nervous system development" and enriched pathway "CCKR signaling map" might be important contributors to EV71-induced neuropathological mechanisms. In addition, we also screened 10 up- and down-regulated non-protein coding genes with significantly different expression in our transcriptome profiling, which suggested that these abnormally regulated non-protein-encoding genes might also play important roles in the pathogenesis of EV71 infection. Eventually, RT-qPCR technology was adopted to validate the transcriptome sequencing data and the experiment demonstrated that the RT-qPCR and transcriptome sequencing results were basically consistent. In summary, this is the first transcriptome analysis of SH-SY5Y cells in response to EV71 infection and provides valuable cues for further exploring the mechanism of nervous system changes caused by EV71 infection., Competing Interests: Declaration of Competing Interest The authors have declared that no competing interests exist., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Purpose: SCN1A variants cause a spectrum of epilepsy syndromes from Dravet Syndrome, a severe epileptic encephalopathy of early infancy to the milder disorder of genetic epilepsy with febrile seizures plus (GEFS+). These genetic epilepsies are associated with increased risk of poor outcome including complications of status epilepticus and early mortality. Individualised management of young children known to be at increased risk should be considered, such as around vaccination management., Methods: We describe two siblings with a novel pathogenic SCN1A variant, their management and clinical outcomes following routine childhood vaccinations., Results: The index case who had a family history of epilepsy of unknown genetic aetiology, died from hypoxic ischemic encephalopathy following his 12-month vaccinations, in the context of status epilepticus and enterovirus 71 infection. The sibling of the index case with the same SCN1A variant was subsequently managed with prophylactic regular sodium valproate and additional clobazam post vaccination to reduce the risk of seizure. She has successfully completed the childhood immunisations to 18 months with no seizures and normal neurodevelopmental progress., Conclusion: As the aetiology of genetic epilepsies is increasingly known in early childhood, opportunities to personalise care, minimise risks and optimise outcomes are changing. Further research is needed on the risks and benefits of symptomatic and preventative management of seizures around vaccinations in young children with genetic epilepsies., Competing Interests: Declaration of Competing Interest The authors have no conflict of interest to declare., (Crown Copyright © 2020. Published by Elsevier Ltd. All rights reserved.)

Enterovirus A71 (EV-A71) is a major pathogen that causes severe and fatal cases of hand-foot-and-mouth disease (HFMD). HFMD caused by EV-A71 seriously endangers children's health. Although autophagy is an important antiviral defense mechanism, some viruses have evolved strategies to utilize autophagy to promote self-replication. EV-A71 can utilize autophagy vesicles as replication scaffolds, indicating that EV-A71 infection is closely related to its autophagy induction mechanism. VP1, a structural protein of EV-A71, has been reported to induce autophagy, but the underlying mechanism is still unclear. In this study, we found that the C-terminus (aa 251-297) of VP1 induces autophagy. Mass spectrometry analysis suggested that prohibitin 2 (PHB2) interacts with the C-terminus of the EV-A71 VP1 protein, and this was further verified by coimmunoprecipitation assays. After PHB2 knockdown, EV-A71 replication, viral particle release, and viral protein synthesis were reduced, and autophagy was inhibited. The results suggest that PHB2 interaction with VP1 is essential for induction of autophagy and the infectivity of EV-A71. Furthermore, we confirmed that EV-A71 induced complete autophagy that required autolysosomal acidification, thus affecting EV-A71 infection. In summary, this study revealed that the host protein PHB2 is involved in an autophagy mechanism during EV-A71 infection.

Coxsackievirus A16 (CV-A16) and enterovirus A71 (EV-A71) are the major causes of hand, foot and mouth disease in young children. Although less so with CV-A16, both viruses are associated with serious neurological syndromes, but the differences between their central nervous system infections remain unclear. We conducted a comparative infection study using clinically-isolated CV-A16 and EV-A71 strains in a 1-day-old mouse model to better understand the neuropathology and neurovirulence of the viruses. New serotype-specific probes for in situ hybridization were developed and validated to detect CV-A16 and EV-A71 RNA in infected tissues. Demonstration of CV-A16 virus antigens/RNA, mainly in the brainstem and spinal cord neurons, confirmed neurovirulence, but showed lower densities than in EV-A71 infected animals. A higher lethal dose 50 for CV-A16 suggested that CV-A16 is less neurovirulent. Focal virus antigens/RNA in the anterior horn white matter and adjacent efferent motor nerves suggested that neuroinvasion is possibly via retrograde axonal transport in peripheral motor nerves., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Enteroviruses are positive sense single-stranded RNA viruses. Most infections from enteroviruses are asymptomatic and can circulate "silently", increasing the risk of an outbreak. For preventing such outbreaks, a rapid, cost-effective, and simple assay for the detection of enteroviruses is of great importance. In this study, we developed a rapid, simple, sensitive, and specific isothermal reverse transcription assay (RT-Loop-Mediated Amplification, RT-LAMP) for the detection of EV-A, B, C, and D species of enteroviruses, by targeting the highly conserved 5'UTR region. The assay was designed and validated based on reference sequences of the four species and on clinical and environmental isolates. The limit of detection of the assay is 0.75 CCID 50 /assay for Enterovirus A, B, and D and 0.075 CCID 50 /assay for Enterovirus C. LAMP allows immediate diagnosis in just 30-50 min, instead of a minimum of 120 min needed for PCR with an equal or better sensitivity. Moreover, due to its isothermal nature, there is no need for expensive equipment, thus decreasing the cost of each reaction. Therefore, this assay is ideal for use in resource-limited settings such as primary care facilities and environmental and clinical laboratories in developing countries.

Enterovirus A71 (EV-A71) infection is primarily responsible for fatal hand, foot, and mouth disease (HFMD) cases. Infants and younger children are more likely to suffer central nervous system damage as a result of EV-A71 infection, but this virus mostly does not affect older children and adults. This study investigated the possible mechanism underlying the age-dependent lethal effect of EV-A71 infection by comparing neonatal and adult mouse models of EV-A71 infection. Although viral proliferation is absent in both neonatal and adult mice, we observed that EV-A71, as a stimulus for astrocytes, elevates the levels of cytokines and monoamine neurotransmitters in neonatal mice. Then, we selected IL-6 and adrenaline as targets in a pharmacological approach to further validate the roles of these factors in mediating the mortality of neonatal mice after EV-A71 infection. Intracerebral injection of IL-6 and adrenaline enhanced the severity of EV-A71 infection, while treatment with an anti-IL-6-neutralizing antibody or the adrenergic-antagonist phenoxybenzamine reversed the lethal effect of EV-A71 in neonatal mice. These results suggest that the central nervous system (CNS) damage in neonatal cases of EV-A71 infection might be caused by an activated fetal cerebral immune response to the virus, including the disruption of brainstem function through increased levels of cytokines and neurotransmitters, rather than the typical cytopathic effect (CPE) of viral infection.

Objective: To investigate the effects of enterovirus 71 (EV71) on mitochondrial dynamics in human Glioma U251 cells., Methods: The EV71 was replicated in Vero cells and the 50% tissue culture infective dose (TCID 50 ) was calculated based on the Reed-Muench formula. After the U251 cells were infected with EV71, the cellular morphology was assessed through the light microscope. The mitochondrial morphology was detected by MitoTracker Deep Red staining under laser confocal microscopy and the mitochondrial ultrastructure was visualized by transmission electron microscopy. The expressions of mitochondrial fission proteins Drp1, p-Drp1 and fusion protein Opa1 were examined by Western blot. The level of ATP was measured by a commercial ATP assay kit. The generation of mitochondrial superoxide was detected by MitoSOX staining., Results: The TCID 50 of EV71 was 10 －5.4 /0.1 mL. Twenty-four or 48 h after EV71 infection, the U251 cells appeared shrunken, round and dead. The laser confocal microscopy and transmission electron microscopy images showed that the EV71 infection induced mitochondrial elongation and cristae damage. Moreover, Western blot analysis demonstrated that the protein expressions of Drp1 and Opa1 were downregulated at both 24 and 48 h after EV71 infection in U251 cells, companied with a significant increase in Drp1 phosphorylation at 48 h after infection ( P <0.05). In addition, a decreased ATP level and elevated mitochondrial superoxide generation were observed in the EV71 infected group, as compared to the control group., Conclusion: Our study demonstrated that infection with EV71 led to changes of mitochondrial morphology and dynamics in U251 cells, which may impair mitochondrial function and contribute to nervous system dysfunction., (Copyright© by Editorial Board of Journal of Sichuan University (Medical Science Edition).)

Enterovirus 71 (EV71) is a causative agent of hand-foot-mouth disease, and it sometimes causes severe neurological disease. Development of effective vaccines and animal models to evaluate vaccine candidates are needed. However, the animal models currently used for vaccine efficacy testing, monkeys and neonatal mice, have economic, ethical, and practical drawbacks. In addition, EV71 strains prepared for lethal challenge often develop decreased virulence during propagation in cell culture. To overcome these problems, we used a mouse model expressing human scavenger receptor B2 (hSCARB2) that showed lifelong susceptibility to EV71. We selected virulent EV71 strains belonging to the subgenogroups B4, B5, C1, C2, and C4 and propagated them using a culture method for EV71 without an apparent reduction in virulence. Here, we describe a novel EV71 vaccine efficacy test based on these hSCARB2 transgenic (Tg) mice and these virulent viruses. Adult Tg mice were immunized subcutaneously with formalin-inactivated EV71. The vaccine elicited sufficient levels of neutralizing antibodies in the immunized mice. The mice were subjected to lethal challenge with virulent viruses via intravenous injection. Survival, clinical signs, and body weight changes were observed for 2 weeks. Most immunized mice survived without clinical signs or histopathological lesions. The viral replication in immunized mice was much lower than that in nonimmunized mice. Mice immunized with the EV71 vaccine were only partially protected against lethal challenge with coxsackievirus A16. These results indicate that this new model is useful for in vivo EV71 vaccine efficacy testing. IMPORTANCE The development of new vaccines for EV71 relies on the availability of small animal models suitable for in vivo efficacy testing. Monkeys and neonatal mice have been used, but the use of these animals has several drawbacks, including high costs, limited susceptibility, and poor experimental reproducibility. In addition, the related ethical issues are considerable. The new efficacy test based on hSCARB2 Tg mice and virulent EV71 strains propagated in genetically modified cell lines presented here can overcome these disadvantages and is expected to accelerate the development of new EV71 vaccines., (Copyright © 2020 American Society for Microbiology.)

Enterovirus 71 (EV71) is a major pathogen that causes hand, foot and mouth disease (HFMD), which is a life threatening disease in certain children. The pathogenesis of EV71-caused HFMD is poorly defined due to the lack of simple and robust animal models with severe phenotypes that recapitulate symptoms observed in humans. Here, we generated the infectious clone of a clinical isolate from a severe HFMD patient. Virus rescued from the cDNA clone was infectious in cell lines. When administrated intraperitoneally to neonatal ICR, BALB/c and C57 immune competent mice at a dosage of1.4 × 10 4  pfu per mouse, the virus caused weight loss, paralysis and death in the infected mice after 4-5 days of infection. In the infected mice, detectable viral replication was detected in various tissues such as heart, liver, brain, lung, kidney, small intestine, leg skeletal muscle and medulla oblongata. The histology of the infected mice included massive myolysis, glomerular atrophy, villous blunting in small intestine, widened alveolar septum, diminished alveolar spaces and lymphocytes infiltration into the lung. By using the UV-inactivated virus as a control, we elucidated that the virus first amplified in the leg skeletal muscle tissue and the muscle tissue served as a primary viral replication site. In summary, we generated a stable EV71 infectious clone that is capable of infecting neonatal immune competent mice without adaptive mutations and provide a simple, valuable animal model for the studies of EV71pathogenesis and therapy.

Enterovirus 71 (EV71) is one of the most common intestinal virus that causes hand, foot, and mouth disease (HFMD) in infants and young children (mostly ≤5 years of age). Generally, children with EV71-infected HFMD have mild symptoms that resolve spontaneously within 7-14 days without complications. However, some EV71-infected HFMD cases lead to severe complications such as aseptic meningitis, encephalitis, acute flaccid paralysis, pulmonary edema, cardiorespiratory complication, circulatory disorders, poliomyelitis-like paralysis, myocarditis, meningoencephalitis, neonatal sepsis, and even death. The mechanism of EV71 pathogenesis has been studied extensively, and the regulation of host immune responses is suspected to aggravate EV71-induced severe complications. Recently, several cytokines or chemokines such as TNF- α , IFN- γ , IL-1 β , IL-18, IL-33, IL-37, IL-4, IL-13, IL-6, IL-12, IL-23, IL-27, IL-35, IL-10, IL-22, IL-17F, IL-8, IP-10, MCP-1, G-CSF, and HMGB1 have been reported to be associated with severe EV71 infection by numerous research teams, including our own. This review is aimed at summarizing the pathophysiology of the cytokines and chemokines with severe EV71 infection., Competing Interests: The authors have no conflicts of interest to declare., (Copyright © 2020 Weijian Zhang et al.)

Background: The most recent documented Australian outbreak of enterovirus A71 (EV-A71) occurred in Sydney from 2012 to 2013. Over a four-month period more than 100 children presented to four paediatric hospitals with encephalitic presentations including fever and myoclonic jerks. The heterogeneous presentations included typical encephalomyelitis, and cardiopulmonary complications., Objectives: To characterise the genomes of enterovirus strains circulating during the 2013 Sydney EV-A71 outbreak and determine their phylogeny, phylogeography and association between genome and clinical phenotype., Study Design: We performed an analysis of enterovirus (EV) positive specimens from children presenting to hospitals in the greater Sydney region of Australia during the 2013 outbreak. We amplified near full-length genomes of EV, and used next generation sequencing technology to sequence the virus. We used phylogenetic/phylogeographic analysis to characterize the outbreak viruses., Results: We amplified and sequenced 23/63 (37 %) genomes, and identified the majority (61 %) as EV-A71. The EV-A71 sequences showed high level sequence homology to C4a genogroups of EV-A71 circulating in China and Vietnam during 2012-13. Phylogenetic analysis showed EV-A71 strains associated with more severe symptoms, including encephalitis or cardiopulmonary failure, grouped together more closely than those from patients with hand, foot and mouth disease. Amongst the non-EV-A71 sequences were five other EV subtypes (representing enterovirus subtypes A and B), reflecting the diversity of EV co-circulation within the community., Conclusions: This is the first Australian study investigating the near full-length genome of EV strains identified during a known outbreak of EV-A71. EV-A71 sequences were very similar to strains circulating in Asia during the same time period. Whole genome sequencing offers additional information over routine diagnostic testing such as characterisation of emerging recombinant strains and inform vaccine design., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Enterovirus A71 (EV-A71) and coxsackievirus A16 (CV-A16) are the most common causes of hand, foot, and mouth disease. Severe EV-A71 and CV-A16 infections may be associated with life-threatening complications. However, the pathogenic mechanisms underlying these severe clinical and pathological features remain incompletely understood. Lipids are known to play critical roles in multiple stages of the virus replication cycle. The specific lipid profile induced upon virus infection is required for optimal virus replication. The perturbations in the host cell lipidomic profiles upon enterovirus infection have not been fully characterized. To this end, we performed ultra-high performance liquid chromatography-electrospray ionization-quadrupole-time of flight-mass spectrometry (UPLC-ESI-Q-TOF-MS)-based lipidomics to characterize the change in host lipidome upon EV-A71 and CV-A16 infections. Our results revealed that 47 lipids within 11 lipid classes were significantly perturbed after EV-A71 and CV-A16 infection. Four polyunsaturated fatty acids (PUFAs), namely, arachidonic acid (AA), docosahexaenoic acid (DHA), docosapentaenoic acid (DPA), and eicosapentaenoic acid (EPA), were consistently upregulated upon EV-A71 and CV-A16 infection. Importantly, exogenously supplying three of these four PUFAs, including AA, DHA, and EPA, in cell cultures significantly reduced EV-A71 and CV-A16 replication. Taken together, our results suggested that enteroviruses might specifically modulate the host lipid pathways for optimal virus replication. Excessive exogenous addition of lipids that disrupted this delicate homeostatic state could prevent efficient viral replication. Precise manipulation of the host lipid profile might be a potential host-targeting antiviral strategy for enterovirus infection.

Enterovirus and Coxsackievirus are the major viruses that cause hand, foot, and mouth disease (HFMD) outbreaks worldwide. Several studies have shown the potential of viral envelope protein 1 (VP1) on providing protective effects from viral strains of different genotypes. However, whether VP1 has the cross-protection in Enteroviruses or Coxsackievirus has not been studied in-depth. In this study, the vp1 gene of Enterovirus 71 (EV71) and Coxsackievirus B3 (CB3) was inserted into the vector pET22b (+) to form the respective expression plasmids pEVP1 or pCVP1, and then transformed into Escherichia coli strain BL21 (DE3). The recombinant EVP1 or CVP1 protein was overexpressed successfully and effectively purified to homogeneity. Then, we identified that EVP1 and CVP1 protein could generate effectively specific humoral immunity and cellular immunity in mice, what's more, we determined the cross-protection of VP1 between EV71 and CB3 in a murine model. The results showed that immunization with EVP1 could effectively induce specific IgG and secretory IgA against CVP1 and the sera from EVP1-immunized mice could neutralize CB3 with mean titers 1:440. In contrast, no measurable neutralizing antibodies to EV71 were detected in CVP1-immunized mice. Then, newborn BALB/C mice, whose mother was immunized with EVP1 or CVP1, were administered with different lethal doses of EV71 or CB3. The EVP1 immunized group showed a 90% protective efficacy for a CB3 dosage of 120 LD 50 , but the CVP1 immunized group showed no significantly different protective efficacy against 15 LD 50 of EV71 compared with the BSA immunized group. Hence, EVP1 is a promising subunit vaccine candidate against Enterovirus 71 and Coxsackievirus B3 caused HFMD., (Copyright © 2019 Chen, Liu, Fang, Nan, Li, Ning, Luo, Li and Wang.)

As a neurotropic virus, enterovirus A71 (EV-A71) emerge and remerge in the Asia-Pacific region since the 1990s, and has continuously been a threat to global public health, especially in children. Annually, EV-A71 results in hand-foot-and-mouth disease (HFMD) and occasionally causes severe neurological disease. Here we reviewed the global epidemiology and genotypic evolution of EV-A71 since 1997. The natural selection, mutation and recombination events observed in the genetic evolution were described. In addition, we have updated the antigenicity and virulence determinants that are known to date. Understanding EV-A71 epidemiology, genetic evolution, antigenicity, and virulence determinants can expand our insights of EV-A71 pathogenesis, which may benefit us in the future.