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A thermoresponsive molecularly imprinted hydrogel sensor was constructed for the specific selective recognition of enterovirus 71 (EV71). Due to the introduction of the thermosensitive monomer N-isopropylacrylamide (NIPAM), when the imprinted hydrogel is incubated with the virus at 37℃, the surface specific imprinting cavity will specifically recognize and capture the target virus EV71. When the temperature rises to 45℃, the combined EV71 is rapidly released due to changes in the shape and function of the imprinted sites. The MIP hydrogel-based viral sensor developed recognized, captured, and released the target virus in a non-invasive way. The imprinting factor of the target virus was 5.2, suggesting high selectivity, and the detection limit was 7.1 fM, suggesting high sensitivity. Detection was rapid, as adsorption equilibrium was achieved within 30 min. This method provides a new sustainable avenue for the simple and rapid detection of viruses. [ABSTRACT FROM AUTHOR]

Previous studies demonstrated that EV71-infected cells secrete extracellular vesicles (EVs), facilitating the transfer of viral components to recipient cells and thereby promoting virus spread. Considering lipid signaling plays a crucial role in EVs-mediated cell-to-cell communication, we compared the lipid profile of EVs secreted from uninfected and EV71-infected cells (EVs-Mock and EVs-EV71) using the human rhabdomyosarcoma (RD) cell model. These two groups of EVs were purified by using size exclusion chromatography (SEC), respectively, and evaluated by transmission electron microscopy (TEM), nanoparticle tracking technology (NTA), and Western blotting (WB). In-depth lipidomic analysis of EVs identified 1705 lipid molecules belonging to 43 lipid classes. The data showed a significant increase in the lipid content of EVs after EV71 infection. Meanwhile, we deeply analyzed the changes in lipids and screened for lipid molecules with significant differences compared EVs-EV71 with EVs-Mock EVs. Altogether, we report the alterations in the lipid profile of EVs derived from RD-cells after EV71 infection, which may affect the function of the EVs in the recipient cells. [ABSTRACT FROM AUTHOR]

Enterovirus A71 (EV-A71) causes Hand, Foot, and Mouth Disease and has been clinically associated with neurological complications. However, there is a lack of relevant models to elucidate the neuropathology of EV-A71 and its mechanism, as the current models mainly utilize animal models or immortalized cell lines. In this study, we established a human motor neuron model for EV-A71 infection. Single cell transcriptomics of a mixed neuronal population reveal higher viral RNA load in motor neurons, suggesting higher infectivity and replication of EV-A71 in motor neurons. The elevated RNA load in motor neurons correlates with the downregulation of ferritin-encoding genes. Subsequent analysis confirms that neurons infected with EV-A71 undergo ferroptosis, as evidenced by increased levels of labile Fe2+ and peroxidated lipids. Notably, the Fe2+ chelator Deferoxamine improves mitochondrial function and promotes survival of motor neurons by 40% after EV-A71 infection. These findings deepen understanding of the molecular pathogenesis of EV-A71 infection, providing insights which suggest that improving mitochondrial respiration and inhibition of ferroptosis can mitigate the impact of EV-A71 infection in the central nervous system.

Abstract 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.

Introduction: The Hand, Foot and Mouth Disease (HFMD), caused by enterovirus 71 infection, is a global public health emergency. Severe HFMD poses a significant threat to the life and well-being of children. Numerous studies have indicated that the occurrence of severe HFMD is associated with cytokine storm. However, the precise molecular mechanism underlying cytokine storm development remains elusive, and there are currently no safe and effective treatments available for severe HFMD in children. Methods: In this study, we established a mouse model of severe HFMD to investigate the molecular mechanisms driving cytokine storm. We specifically analyzed metabolic disturbances, focusing on arginine/ornithine metabolism, and assessed the potential therapeutic effects of spermine, an ornithine metabolite. Results: Our results identified disturbances in arginine/ornithine metabolism as a pivotal factor driving cytokine storm onset in severe HFMD cases. Additionally, we discovered that spermine effectively mitigated the inflammatory injury phenotype observed in mice with severe HFMD. Discussion: In conclusion, our findings provide novel insights into the molecular mechanisms underlying severe HFMD from a metabolic perspective while offering a promising new strategy for its safe and effective treatment. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Enterovirus 71 (EV71), a typical representative of unenveloped RNA viruses, is the main pathogenic factor responsible for hand, foot, and mouth disease (HFMD) in infants. This disease seriously threatens the health and lives of humans worldwide, especially in the Asia–Pacific region. Numerous animal antimicrobial peptides have been found with protective functions against viruses, bacteria, fungi, parasites, and other pathogens, but there are few studies on the use of scorpion-derived antimicrobial peptides against unenveloped viruses. Here, we investigated the antiviral activities of scorpion venom antimicrobial peptide BmKn2 and five derivatives, finding that BmKn2 and its derivative BmKn2-T5 exhibit a significant inhibitory effect on EV71. Although both peptides exhibit characteristics typical of amphiphilic α-helices in terms of their secondary structure, BmKn2-T5 displayed lower cellular cytotoxicity than BmKn2. BmKn2-T5 was further found to inhibit EV71 in a dose-dependent manner in vitro. Moreover, time-of-drug-addition experiments showed that BmKn2-T5 mainly restricts EV71, but not its virion or replication, at the early stages of the viral cycle. Interestingly, BmKn2-T5 was also found to suppress the replication of the enveloped viruses DENV, ZIKV, and HSV-1 in the early stages of the viral cycle, which suggests they may share a common early infection step with EV71. Together, the results of our study identified that the scorpion-derived antimicrobial peptide BmKn2-T5 showed valuable antiviral properties against EV71 in vitro, but also against other enveloped viruses, making it a potential new candidate therapeutic molecule. [ABSTRACT FROM AUTHOR]

RNA viruses are critically dependent upon virally encoded proteases to cleave the viral polyproteins into functional proteins. Many of these proteases exhibit a similar fold and contain an essential catalytic cysteine, offering the opportunity to inhibit these enzymes with electrophilic small molecules. Here we describe the successful application of quantitative irreversible tethering (qIT) to identify acrylamide fragments that target the active site cysteine of the 3C protease (3Cpro) of Enterovirus 71, the causative agent of hand, foot and mouth disease in humans, altering the substrate binding region. Further, we re-purpose these hits towards the main protease (Mpro) of SARS-CoV-2 which shares the 3C-like fold and a similar active site. The hit fragments covalently link to the catalytic cysteine of Mpro to inhibit its activity. We demonstrate that targeting the active site cysteine of Mpro can have profound allosteric effects, distorting secondary structures to disrupt the active dimeric unit.

Enterovirus 71 (EV71) is a major causative agent of hand, foot, and mouth disease (HFMD) in children. Nowadays, there are still no effective antiviral drugs for EV71 infection. High mobility group box 1 (HMGB1) is reported to be highly expressed in HFMD patients. However, the role and underlying mechanism of HMGB1 in EV71‐associated HFMD are still unclear. HMGB1 expression was detected using RT‐qPCR and western blot assays. Loss‐ and gain‐function experiments were performed to evaluate the effects of HMGB1 on EV71‐infected cells. The virus titer was examined by TCID50. CCK‐8 and flow cytometry assays were applied to detect the cell viability and cell cycle. Oxidative stress was determined by relative commercial kits. HMGB1 level was elevated in the serum of EV71‐infected patients with HFMD and EV71‐induced RD cells. EV71 infection induced the transfer of HMGB1 from the nucleus into the cytoplasm. HMGB1 knockdown inhibited virus replication, viral protein (VP1) expression and promoted antiviral factor expression. In addition, the inhibition of HMGB1 improved cell viability, protected against S phase arrest, and inhibited EV71‐induced cell injury and oxidative stress, whereas HMGB1 overexpression showed the opposite effects. In terms of mechanism, HMGB1 overexpression activated the TLR4/NF‐κB/NLRP3 signaling pathway and promoted cell pyroptosis. The inhibition of TLR4 and NF‐κB reversed the effects of HMGB1 overexpression on virus replication, oxidative stress, and pyroptosis. In conclusion, HMGB1 knockdown inhibits EV71 replication and attenuates pyroptosis through TLR4/NF‐κB/NLRP3 axis. [ABSTRACT FROM AUTHOR]

The 2A protease (2Apro) encoded by enterovirus 71 (EV71) serves as an accessory protein with significant involvement in the regulation of EV71 infection and viral replication. EV71-2Apro exhibits the ability to suppress various host factors, thereby disrupting the cellular antiviral immune response. However, whether host factors downregulate EV71-2Apro remains largely unknown. Here, we discovered that the speckle-type POZ protein (SPOP), functioning as a host E3 ubiquitin ligase, triggers ubiquitination modifications and subsequent degradation of EV71-2Apro. SPOP interacts with EV71-2Apro and exhibits a dose-dependent downregulation of EV71-2Apro levels. Conversely, knockdown of endogenous SPOP upregulated EV71-2Apro levels. Subsequent investigations revealed that the SPOP facilitates the lysosome-dependent degradation of EV71-2Apro by inducing K48-linked polyubiquitination of EV71-2Apro, which ultimately restricts EV71 replication. These findings shed light on the ubiquitination and lysosome-dependent regulation of the crucial EV71-encoded protease, offering a potential therapeutic target for the treatment of EV71 infection. IMPORTANCE EV71 poses a significant health threat to children aged 5 and below. The process of EV71 infection and replication is predominantly influenced by ubiquitination modifications. Our previous findings indicate that EV71 prompts the activation of host deubiquitinating enzymes, thereby impeding the host interferon signaling pathway as a means of evading the immune response. Nevertheless, the precise mechanisms by which the host employs ubiquitination modifications to hinder EV71 infection remain unclear. The present study demonstrated that the nonstructural protein 2Apro, which is encoded by EV71, exhibits ubiquitination and degradation mediated by the host E3 ubiquitin ligase SPOP. In addition, it is the first report, to our knowledge, that SPOP is involved in the host antiviral response. [ABSTRACT FROM AUTHOR]

Enterovirus 71 (EV71), a typical representative of unenveloped RNA viruses, is the main pathogenic factor responsible for hand, foot, and mouth disease (HFMD) in infants. This disease seriously threatens the health and lives of humans worldwide, especially in the Asia–Pacific region. Numerous animal antimicrobial peptides have been found with protective functions against viruses, bacteria, fungi, parasites, and other pathogens, but there are few studies on the use of scorpion-derived antimicrobial peptides against unenveloped viruses. Here, we investigated the antiviral activities of scorpion venom antimicrobial peptide BmKn2 and five derivatives, finding that BmKn2 and its derivative BmKn2-T5 exhibit a significant inhibitory effect on EV71. Although both peptides exhibit characteristics typical of amphiphilic α-helices in terms of their secondary structure, BmKn2-T5 displayed lower cellular cytotoxicity than BmKn2. BmKn2-T5 was further found to inhibit EV71 in a dose-dependent manner in vitro. Moreover, time-of-drug-addition experiments showed that BmKn2-T5 mainly restricts EV71, but not its virion or replication, at the early stages of the viral cycle. Interestingly, BmKn2-T5 was also found to suppress the replication of the enveloped viruses DENV, ZIKV, and HSV-1 in the early stages of the viral cycle, which suggests they may share a common early infection step with EV71. Together, the results of our study identified that the scorpion-derived antimicrobial peptide BmKn2-T5 showed valuable antiviral properties against EV71 in vitro, but also against other enveloped viruses, making it a potential new candidate therapeutic molecule.

Purpose: EV71 (Enterovirus 71) is a major causative agent of the outbreaks of HFMD (hand, foot, and mouth disease), which is associated with neurological damage caused by permeability disruption of BBB (blood-brain barrier). HMGB1 (high-mobility group box 1) is a widely expressed nuclear protein that triggers host inflammatory responses. Our work aimed to explore the function of HMGB1 in EV71 infection and its contributions to EV71-related BBB damage. Methods: HeLa cells, HT-29 cells and AG6 mice were used to explore the translocation of HMGB1 in EV71 infection in vitro and in vivo. The roles of released HMGB1 on EV71 replication and associated inflammatory cytokines were investigated using recombinant HMGB1 in HeLa cells. The mechanisms of released HMGB1 in EV71-induced BBB injury were explored using recombinant HMGB1 and anti-HMGB1 neutralizing antibodies in monolayer HCMECs (immortalized human brain microvascular endothelial cells) and AG6 mice brain. Results: EV71 induced HMGB1 nucleocytoplasmic translocation and extracellular release in vitro and in vivo. Released HMGB1 acted as an inflammatory mediator in EV71 infection rather than affecting viral replication in vitro. Released HMGB1 disrupted BBB integrity by enhancing VE-cadherin phosphorylation at tyrosine 685 in HCMECs, and reducing total VE-cadherin levels in HCMECs and AG6 mice in EV71 infection. And released HMGB1 induced an increase in activated astrocytes. Neutralization of HMGB1 reversed the increased endothelial hyperpermeability and phosphorylation of VE-cadherin in HCMECs. Conclusion: The inflammatory mediator HMGB1 released by EV71 exacerbated BBB disruption by enhancing VE-cadherin phosphorylation, which in turn aggravated EV71-induced neuroinflammation.

Enterovirus 71 (EV71) commonly causes symptoms such as hand, foot, and mouth disease (HFMD) in infants and children and may lead to neurological disease and even death in severe cases. Appropriate vaccines for the prevention of HFMD are available in the clinic; however, they present different and serious adverse effects that cannot guarantee compliance and efficacy. The purpose of this study was to analyze the potential mechanism of Bryum billardieri Schwaegr. (BBS) against EV71 and analyze its potential active components. A previous in vitro antiviral assay was used to determine the best extraction method for the active site of BBS against EV71, and the results showed that the antiviral activity of BBS was more pronounced in the fraction that was extracted by aqueous extraction and alcoholic precipitation and then obtained by purification on a silica gel column (dichloromethane:methanol = 0:100). In addition, the therapeutic effects of BBS on EV71-infected mice were further investigated by in vivo pharmacological experiments. BBS reduced the lung index, viral titer, and degree of EV71-induced lung, brain, and skeletal muscle damage. Themechanismof anti-EV71 activity of BBSwas also investigated by using ELISA and qRT-PCR, and it was found that BBS exerted its action mainly by regulating the expression of TLR3, TLR4, TNF-α, IL-2, and IFN-γ by modulating the activation of NF-κB and JAK2/STAT1 signaling pathways. Finally, the chemical structures of the active monomers in BBS were determined by using UPLC-MS and NMR techniques. The study revealed that one of the monomers on which BBS exerts its antiviral activity is saponarin. In conclusion, the results of this study suggest that BBS is considered a natural anti-EV71 product with enormous potential, and saponarin would be its non-negligible active monomer. [ABSTRACT FROM AUTHOR]

BackgroundEnterovirus 71 (EV71) is a major causative agent of hand, foot, and mouth disease (HFMD), associated with severe manifestations of the disease. Pediatric immunization with inactivated EV71 vaccine was initiated in 2016 in the Asia-Pacific region, including China. We analyzed a time series of HFMD cases attributable to EV71, coxsackievirus A16 (CA16), and other enteroviruses in Chengdu, a major transmission center in China, to assess early impacts of immunization.MethodsReported HFMD cases were obtained from China's notifiable disease surveillance system. We compared observed postvaccination incidence rates during 2017-2018 with counterfactual predictions made from a negative binomial regression and a random forest model fitted to prevaccine years (2011-2015). We fit a change point model to the full time series to evaluate whether the trend of EV71 HFMD changed following vaccination.ResultsBetween 2011 and 2018, 279 352 HFMD cases were reported in the study region. The average incidence rate of EV71 HFMD in 2017-2018 was 60% (95% prediction interval [PI], 41%-72%) lower than predicted in the absence of immunization, corresponding to an estimated 6911 (95% PI, 3246-11 542) EV71 cases averted over 2 years. There were 52% (95% PI, 42%-60%) fewer severe HFMD cases than predicted. However, the incidence rate of non-CA16 and non-EV71 HFMD was elevated in 2018. We identified a significant decline in the trend of EV71 HFMD 4 months into the postvaccine period.ConclusionsWe provide the first real-world evidence that programmatic vaccination against EV71 is effective against childhood HFMD and present an approach to detect early vaccine impact or intended consequences from surveillance data.

RNA viruses are critically dependent upon virally encoded proteases to cleave the viral polyproteins into functional proteins. Many of these proteases exhibit a similar fold and contain an essential catalytic cysteine, offering the opportunity to inhibit these enzymes with electrophilic small molecules. Here we describe the successful application of quantitative irreversible tethering (qIT) to identify acrylamide fragments that target the active site cysteine of the 3C protease (3Cpro) of Enterovirus 71, the causative agent of hand, foot and mouth disease in humans, altering the substrate binding region. Further, we re-purpose these hits towards the main protease (Mpro) of SARS-CoV-2 which shares the 3C-like fold and a similar active site. The hit fragments covalently link to the catalytic cysteine of Mpro to inhibit its activity. We demonstrate that targeting the active site cysteine of Mpro can have profound allosteric effects, distorting secondary structures to disrupt the active dimeric unit.

The same viral infection in different hosts may result in varying levels of clinical symptoms, which is related to the genetic background of the host itself. A total of 406 common cases and 452 severe cases of enterovirus 71 (EV71) infection in Yunnan Province were selected as the research subjects, and SNaPshot technology was used to detect genetic polymorphisms for 25 Tag single-nucleotide polymorphisms (TagSNPs) in the selectin P ligand (SELPLG) and scavenger receptor class B member 2 (SCARB2) genes. Our results demonstrate that SCARB2 polymorphisms (rs74719289, rs3733255 and rs17001551) are related to the severity of EV71 infection (A vs G: OR 0.330; 95% CI 0.115 - 0.947; T vs C: OR 0.336; 95% CI 0.118 - 0.958; and A vs G: OR 0.378; 95% CI 0.145 - 0.984). The SELPLG polymorphisms were not significantly different between common cases and severe cases. Therefore, we conclude that the SCARB2 gene has a protective effect on the course of hand, foot and mouth disease caused by EV71 infection and that SCARB2 gene mutations can reduce the severity of the disease.

Hand, foot, and mouth disease (HFMD) is a common childhood infectious disease caused by enterovirus (EV) infection. EV71 is one of the major pathogens causing hand, foot, andmouth disease and ismore likely to cause exacerbation and death than other enteroviruses. Although a monovalent vaccine for EV71 has been developed, there are no clinically available anti-EV71 specific drugs. Here, we performed virtual screening and biological experiments based on the traditional Chinese medicine monomer library. We identified a traditional Chinese medicine monomer, Salvianolic acid A (SA), a polyphenolic compound isolated fromSalviamiltiorrhiza. Salvianolic acid A inhibits EV71 virus infection in a concentration-dependent manner, and its antiviral activity is higher than that of other reported natural polyphenols and has a high biosafety. Furthermore, molecular dynamics simulations showed that salvianolic acid A can anchor to E71, amember of the enzyme catalytic triad, and cause H40 to move away from the catalytic center. Meanwhile, molecular mechanics generalized born surface area (MMGBSA) and steered molecular dynamics (SMD) results showed that the P1 group of SA wasmost easily unbound to the S1 pocket of 3Cpro, which provided theoretical support to further improve the affinity of salvianolic acid A with 3Cpro. These findings suggest that salvianolic acid A is a novel EV71 3Cpro inhibitor with excellent antiviral activity and is a promising candidate for clinical studies. [ABSTRACT FROM AUTHOR]

AL-471, the leading exponent of a class of potent HIV and enterovirus A71 (EV-A71) entry inhibitors discovered in our research group, contains four l-tryptophan (Trp) units bearing an aromatic isophthalic acid directly attached to the C2 position of each indole ring. Starting from AL-471, we (i) replaced l-Trp with d-Trp, (ii) inserted a flexible linker between C2 and the isophthalic acid, and (iii) substituted a nonaromatic carboxylic acid for the terminal isophthalic acid. Truncated analogues lacking the Trp motif were also synthesized. Our findings indicate that the antiviral activity seems to be largely independent of the stereochemistry (l- or d-) of the Trp fragment and also that both the Trp unit and the distal isophthalic moiety are essential for antiviral activity. The most potent derivative, 23 (AL-534), with the C2 shortest alkyl urea linkage (three methylenes), showed subnanomolar potency against different EV-71 clinical isolates. This finding was only observed before with the early dendrimer prototype AL-385 (12 l-Trp units) but remained unprecedented for the reduced-size prototype AL-471. Molecular modeling showed the feasibility of high-affinity binding of the novel l-Trp-decorated branches of 23 (AL-534) to an alternative site on the VP1 protein that harbors significant sequence variation among EV-71 strains. [ABSTRACT FROM AUTHOR]

ObjectiveTo determine the epidemiological and etiological characteristics of hand，foot，and mouth disease （HFMD） in Pujiang County from 2008 through 2020， so as to provide scientific evidence for prevention and control measures.MethodsDescriptive epidemiological analysis was used to analyze HFMD cases retrieved in the Chinese Information System for Disease Control and Prevention in Pujiang County during 2008‒2020.ResultsFrom 2008 through 2020， a total of 9 393 cases were documented in Pujiang County， with an annual incidence of 182.3 per 100 000， showing a trend of alternating high-incidence and low-incidence years. The seasonal distribution was bimodal， and the incidence peaked in May-July and November-December. The incidence of HFMD in urban areas was significantly higher than that in rural areas， and the incidence was positively correlated with population density. Majority of cases （94.9%） were children under 5 years old， of which boys had higher incidence than girls. The number of documented cases was the highest among preschool children living in families and kindergartens， accounting for 97.3%. Totally， 558 cases were laboratory confirmed in Pujiang County in 2008-2020， of which severe cases were all caused by EV71 infection.ConclusionEnterovirus serotypes in HFMD have continually changed from 2008 through 2020 in Pujiang County. However， severe HFMD remains principally attributable to EV71 infection， suggesting that it is necessary to strengthen the surveillance on the etiology of HFMD. In addition， it warrants further promotion of EV71 vaccination.

Introduction: This study was designed to explore the association between the TMEM173 polymorphism (rs7447927) and the severity of enterovirus 71 (EV71) infection among Chinese children. Methods: The TMEM173 polymorphism was identified in EV71‐infected patients (n = 497) and healthy controls (n = 535) using the improved multiplex ligation detection reaction (iMLDR). The interferon‐α (IFN‐α) serum levels were detected using enzyme linked immunosorbent assay (ELISA). Results: The frequencies of the GG genotype and G allele of TMEM173 rs7447927 in the mild EV71 infection and severe EV71 infection groups were markedly higher than those in the control group. The GG genotype and G allele frequencies in severely infected EV71 patients were significantly higher than those in mildly infected EV71 patients. Severely infected EV71 patients with the GG genotype had higher white blood cell counts (WBC), and C‐reactive proteins (CRP), and blood glucose (BG) levels, longer fever duration, higher vomiting frequency, spirit changes, and electroencephalography (EEG) abnormalities. IFN‐α serum concentration in severely infected patients was significantly higher than in the mildly infected group. The IFN‐α concentration in the GG genotype was significantly higher compared with those in the GC and CC genotypes in severe cases. Conclusions: The TMEM173 rs7447927 polymorphism was associated with EV71 infection susceptibility and severity. The G allele and GG genotype are susceptibility factors in the development of severe EV71 infection in Chinese children. [ABSTRACT FROM AUTHOR]

Enterovirus 71 (EV71) is the major pathogen causing fatal neurological complications of hand, foot, and mouth disease (HFMD) in young children. Currently no effective antiviral therapy is available. In the present study, we found that natural compound Berberine (BBR) displayed potent inhibitory effects on EV71 replication in various neural cells (IC50 of 2.79-4.03 µM). In a newborn mouse model of lethal EV71 infection, Berberine at 2-5mg/kg markedly reduced mortality and clinical scores. Consistently, the replication of EV71 and pathological changes were attenuated in various infected organs including brain and lung with BBR treatment. Interestingly, EV71 infection in the brain mainly localized in the peripheral zone of brainstemand largely in astrocytes. Primary culture of astrocytes from newbornmouse brain confirmed the efficient EV71 replication that wasmostly inhibited by BBR treatment at 5 µM. Further investigations revealed remarkably elevated cellular reactive oxygen species (ROS) levels that coincided with EV71 replication in primary cultured astrocytes and various cell lines. BBR largely abolished the virus-elevated ROS production and greatly diminished EV71 replication by up-regulating NFE2 like bZIP transcription factor 2 (Nrf2) via the kelch like ECH associated protein 1 (Keap)-Nrf2 axis. The nuclear localization of Nrf2 and expression of downstream antioxidant enzymes heme oxygenase 1 (HO-1) and NAD(P)H quinone dehydrogenase 1 (NQO1) were increased significantly by BBR treatment. Collectively, our findings revealed that BBR prevents lethal EV71 neurological infection via inhibiting virus replication through regulating Keap-Nrf2 axis and ROS generation in astrocytes of brainstem, thus providing a potential antiviral treatment for severe EV71 infection associated with neurological complications. [ABSTRACT FROM AUTHOR]

Poliovirus (PV) can spread through neural pathway to the central nervous system and replicates in motor neurons, which leads to poliomyelitis. Enterovirus 71 (EV71), which is closely related to PV, is one of the causative agents of hand-foot-and-mouth disease and can cause severe neurological diseases similar to poliomyelitis. Since PV is similar to EV71 in its motor neurotoxicity, we tried to understand if the results obtained with PV are of general applicability to EV71 and other viruses with similar characteristics. Using microfluidic devices, we demonstrated that both PV capsid and the PV genome undergo axonal retrograde transport with human PV receptor (hPVR), and the transported virus replicated in the soma of hPVR-expressing motor neurons. Similar to PV in hPVR-transgenic (Tg) mice, neural pathway ensuring spreading of EV71 has been shown in adult human scavenger receptor class B, member 2 (hSCARB2)-Tg mice. We have validated this finding in microfluidic devices by showing that EV71 is retrogradely transported together with hSCARB2 to the cell body where it replicates in an hSCARB2-dependent manner. • PV capsid and genome undergo axonal retrograde transport with human PVR (hPVR). • Retrogradely transported PV replicates in the soma of hPVR-expressing motor neurons. • Neural pathway of non-mouse-adapted EV71 is present in adult hSCARB2-Tg mice. • Retrogradely transported EV71 replicates in the soma in a hSCARB2-dependent manner. • hSCARB2 selectively undergo retrograde transport. [ABSTRACT FROM AUTHOR]

Enterovirus A71 (EV-A71) causes Hand, Foot, and Mouth Disease and has been clinically associated with neurological complications. However, there is a lack of relevant models to elucidate the neuropathology of EV-A71 and its mechanism, as the current models mainly utilize animal models or immortalized cell lines. In this study, we established a human motor neuron model for EV-A71 infection. Single cell transcriptomics of a mixed neuronal population reveal higher viral RNA load in motor neurons, suggesting higher infectivity and replication of EV-A71 in motor neurons. The elevated RNA load in motor neurons correlates with the downregulation of ferritin-encoding genes. Subsequent analysis confirms that neurons infected with EV-A71 undergo ferroptosis, as evidenced by increased levels of labile Fe 2+ and peroxidated lipids. Notably, the Fe 2+ chelator Deferoxamine improves mitochondrial function and promotes survival of motor neurons by 40% after EV-A71 infection. These findings deepen understanding of the molecular pathogenesis of EV-A71 infection, providing insights which suggest that improving mitochondrial respiration and inhibition of ferroptosis can mitigate the impact of EV-A71 infection in the central nervous system.

Enterovirus 71 (EV71) is a major pathogen that causes hand, foot, and mouth disease (HFMD) in infants and children. Notably, no clinically approved drugs specifically target EV71. The EV71 2A protease (2A pro ), a cysteine protease produced by the virus, is essential for the virus' replication and has a significant impact on the functioning of host cells. Thus, it presents a valuable target for the discovery of antiviral medications. In this study, based on the monomers and their derivatives in the Library of Traditional Chinese Medicine (TCM), we performed virtual screening and biological experiments. We identified a derivative of a traditional herbal monomer, Etoposide, commonly isolated from the roots and rhizomes of Podophyllum spp. Etoposide inhibited replication of EV71 A, B, C, and CVA16 viruses in a concentration-dependent manner in a variety of cell lines with minimal cytotoxicity. Furthermore, both molecular dynamics simulations and site-directed mutagenesis assays revealed that Etoposide inhibited the activity of the EV71 2A protease by mainly binding to two residues, Y89 and P107. The findings indicate that Etoposide serves as a promising inhibitor of the EV71 2A pro , demonstrating strong antiviral properties and positioning itself as a formidable candidate for clinical trials against EV71.IMPORTANCEWe first used a drug screening approach focused on monomeric compounds and their derivatives from traditional Chinese medicine to identify an EV71 2A pro inhibitor-Etoposide. We then performed biological experiments to validate that Etoposide suppresses the replication of the EV71 virus in a concentration-dependent manner with minimal cytotoxicity to various cell lines. Remarkably, it shows inhibitory activity against EV71 A, B, C, and CVA16, suggesting that Etoposide may be a potential broad-spectrum inhibitor. We revealed a novel mechanism that Etoposide inhibits EV71 proliferation by targeting 2A pro , and the interactions with Y89 and P107 are of great importance. The findings suggest that Etoposide serves as a promising inhibitor of EV71 2A pro , demonstrating significant antiviral properties. It stands out as a strong candidate for broad-spectrum applications in clinical research.

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

Abstract Introduction This study was designed to explore the association between the TMEM173 polymorphism (rs7447927) and the severity of enterovirus 71 (EV71) infection among Chinese children. Methods The TMEM173 polymorphism was identified in EV71‐infected patients (n = 497) and healthy controls (n = 535) using the improved multiplex ligation detection reaction (iMLDR). The interferon‐α (IFN‐α) serum levels were detected using enzyme linked immunosorbent assay (ELISA). Results The frequencies of the GG genotype and G allele of TMEM173 rs7447927 in the mild EV71 infection and severe EV71 infection groups were markedly higher than those in the control group. The GG genotype and G allele frequencies in severely infected EV71 patients were significantly higher than those in mildly infected EV71 patients. Severely infected EV71 patients with the GG genotype had higher white blood cell counts (WBC), and C‐reactive proteins (CRP), and blood glucose (BG) levels, longer fever duration, higher vomiting frequency, spirit changes, and electroencephalography (EEG) abnormalities. IFN‐α serum concentration in severely infected patients was significantly higher than in the mildly infected group. The IFN‐α concentration in the GG genotype was significantly higher compared with those in the GC and CC genotypes in severe cases. Conclusions The TMEM173 rs7447927 polymorphism was associated with EV71 infection susceptibility and severity. The G allele and GG genotype are susceptibility factors in the development of severe EV71 infection in Chinese children.

Enterovirus (EV) 71 caused episodes of outbreaks in China and Southeast Asia during the last few decades. We have previously reported that EV71 induces reactive oxygen species (ROS). However, the underlying mechanism remains elusive. Co-immunoprecipitation-proteomic analysis revealed that enteroviral 2B protein interacted with mitochondrial voltage-dependent anion channel 3 (VDAC3). Knockdown (KD) of VDAC3 expression specifically inhibited enteroviral replication. Single-round viral replication was also inhibited in KD cells, suggesting that VDAC3 plays an essential role in replication. Consistent with this, VDAC3 gene KD significantly reduced the EV71-induced mitochondrial ROS generation. Exogenous 2B expression could induce the mitochondrial ROS generation that was significantly reduced in VDAC3-KD cells or in the Mito-TEMPO-treated cells. Moreover, VDAC3 appears to be necessary for regulation of antioxidant metabolism. VDAC3 gene KD led to the enhancement of such pathways as hypotaurine/taurine synthesis in the infected cells. Taken together, these findings suggest that 2B and VDAC3 interact to enhance mitochondrial ROS generation, which promotes viral replication. [ABSTRACT FROM AUTHOR]

Abstract Fluorescent labeling and dynamic tracking is a powerful tool for exploring virus infection mechanisms. However, for small-sized viruses, virus tracking studies are usually hindered by a lack of appropriate labeling methods that do not dampen virus yield or infectivity. Here, we report a universal strategy for labeling viruses with chemical dyes and Quantum dots (QDs). Enterovirus 71 (EV71) was produced in a cell line that stably expresses a mutant methionyl-tRNA synthetase (MetRS), which can charge azidonorleucine (ANL) to the methionine sites of viral proteins during translation. Then, the ANL-containing virus was easily labeled with DBCO-AF647 and DBCO-QDs. The labeled virus shows sufficient yield and no obvious decrease in infectivity and can be used for imaging the virus entry process. Using the labeled EV71, different functions of scavenger receptor class B, member 2 (SCARB2), and heparan sulfate (HS) in EV71 infection were comparatively studied. The cell entry process of a strong HS-binding EV71 strain was investigated by real-time dynamic visualization of EV71-QDs in living cells. Taken together, our study described a universal biocompatible virus labeling method, visualized the dynamic viral entry process, and reported details of the receptor usage of EV71. Graphic Abstract

Enterovirus 71 (EV71) is one of the causative agents of hand-foot-and-mouth disease, which in some circumstances could lead to severe neurological diseases. Despite of its importance for human health, little is known about the early stages of EV71 infection. EV71 starts uncoating with its receptor, human scavenger receptor B2 (hSCARB2), at low pH. We show that EV71 was not targeted to lysosomes in human rhabdomyosarcoma cells overexpressing hSCARB2 and that the autophagic pathway is not essential for EV71 productive uncoating. Instead, EV71 was efficiently uncoated 30 min after infection in late endosomes (LEs) containing hSCARB2, mannose-6-phosphate receptor (M6PR), RAB9, bis(monoacylglycero)phosphate and lysosomal associated membrane protein 2 (LAMP2). Furthering the notion that mature LEs are crucial for EV71 uncoating, cation-dependent (CD)-M6PR knockdown impairs EV71 infection. Since hSCARB2 interacts with cation-independent (CI)-M6PR through M6P-binding sites and CD-M6PR also harbor a M6P-binding site, CD-M6PR is likely to play important roles in EV71 uncoating in LEs.

• Hand, foot and mouth disease (HFMD) is frequently caused by enterovirus 71 (EV71) • HFMD is emerging as a major public health concern in the Asia-Pacific region. • TAK-021 is a candidate inactivated virus vaccine based on EV71 B2 sub-genogroup. • SCARB2-transgenic (SCARB2-tg) mice are susceptible to infection by multiple EV71 sub-genogroups. • TAK-021 is immunogenic and can protect against heterologous EV71 sub-genogroups in SCARB2-tg mice. Enterovirus 71 (EV71) is a major cause of outbreaks of hand, foot and mouth disease, most frequently in children, and is a public health concern in the Asia-Pacific region. Takeda is developing TAK-021, an inactivated EV71 vaccine candidate based on sub-genogroup B2 strain MS87. In a phase I clinical trial, TAK-021 was safe, well tolerated, and immunogenic in healthy adults and elicited cross-neutralizing antibodies against heterologous EV71 sub-genogroup viruses. TAK-021 confers protection from lethal challenge with a mouse-adapted homologous strain in AG129 mice. However, it has not been determined whether TAK-021 can provide cross-protection against heterologous EV71 sub-genogroups. We examined the efficacy of TAK-021 against challenge with EV71 sub-genogroups B4, B5, C1, C2, and C4 on day 42 (short-term) and sub-genogroups B5 and C4 on day 120 (long-term) after immunization of human scavenger receptor B2 transgenic (hSCARB2-tg) mice with TAK-021 on days 0 and 28. Antibody titers were monitored over 120 days using plaque reduction neutralization test of the homologous vaccine virus. TAK-021 elicited neutralizing antibody (nAb) in greater than 90% of the mice and nAb persisted through day 120. Challenge of control animals led to weight loss and death, as well as virus detection in various organs and histopathological lesions in the brain. All mice that received two doses of TAK-021 developed nAb and survived a short-term challenge given on day 42, while more than 80% survived a long-term challenge given on day 120. EV71 was detected less frequently and at lower levels in organs of immunized mice compared to non-immunized control mice. The results show that TAK-021 can confer protection in mice against the EV71 sub-genogroups tested. [ABSTRACT FROM AUTHOR]

Posttranslational modifications (PTMs) of viral proteins play critical roles in virus infection. The role of neddylation in enterovirus 71 (EV71) replication remains poorly defined. Here, we showed that the structural protein VP2 of EV71 can be modified by neural precursor cell-expressed developmentally downregulated protein 8 (NEDD8) in an E3 ligase X-linked inhibitor of apoptosis protein (XIAP)-dependent manner. Mutagenesis and biochemical analyses mapped the neddylation site at lysine 69 (K69) of VP2 and demonstrated that neddylation reduced the stability of VP2. In agreement with the essential role of VP2 in viral replication, studies with EV71 reporter viruses with wild-type VP2 (enhanced green fluorescent protein [EGFP]-EV71) and a K69R mutant VP2 (EGFP-EV71-VP2 K69R) showed that abolishment of VP2 neddylation increased EV71 replication. In support of this finding, overexpression of NEDD8 significantly inhibited the replication of wild-type EV71 and EGFP-EV71, but not EGFP-EV71-VP2 K69R, whereas pharmacologic inhibition of neddylation with the NEDD8-activating enzyme inhibitor MLN4924 promoted the replication of EV71 in biologically relevant cell types. Our results thus support the notion that EV71 replication can be negatively regulated by host cellular and pathobiological cues through neddylation of VP2 protein. [ABSTRACT FROM AUTHOR]

Hand foot and mouth disease (HFMD) caused by Enterovirus 71 (EV71) infection is still a major infectious disease threatening children's life and health in the absence of effective antiviral drugs due to its high prevalence and neurovirulence. A study of EV71-specific host response might shed some light on the reason behind its unique epidemiologic features and help to find means to conquer EV71 infection. We reported that host heat shock protein A6 (HSPA6) was induced by EV71 infection and involved infection in both Rhabdomyosarcoma (RD) cells and neurogliocytes. Most importantly, we found that EV71 did not induce the expression of other heat shock proteins HSPA1, HSPA8, and HSPB1 under the same conditions, and other HFMD-associated viruses including CVA16, CVA6, CVA10, and CVB1-3 did not induce the upregulation of HSPA6. In addition, EV71 infection enhanced the cytoplasmic aggregation of HSPA6 and its colocalization with viral capsid protein VP1. These findings suggest that HSPA6 is a potential EV71-specific host factor worthy of further study. [ABSTRACT FROM AUTHOR]

Enterovirus 71 (EV71) is a non-enveloped virus and it can be released from host cells through a traditional cytolytic manner. Now, we showed EV71 could be spread non-lytically between cells during early viral infection. In order to explain this phenomenon, we separated supernatant fluids of rhabdomyosarcoma (RD) cells cultures infected with EV71 by isopycnic gradient centrifugation. Two populations of virus particles were morphology indistinguishable by transmission electron microscope (TEM). It showed that some EV71 particles were wrapped inside extracellular vesicles which were verified to be exosomes by immunoassay and morphologic analysis. In addition, exosomes containing viral RNA were shed in plasma of EV71-infected encephalitis in children. Our findings indicate that the “non-enveloped” EV71 virions could be wrapped within exosomes which promote their spread in the absence of cell lysis. Abbreviation: EV71: enterovirus 71; EXO: exosome; RD: rhabdomyosarcoma; TEM: transmission electron microscope; HFMD: hand, foot, and mouth disease; HIV: immunodeficiency virus; HCV: hepatitis C virus; HTLV: Human T-cell lymphotropic virus; HAV: hepatitis A virus; MOI: multiplicity of infection; EVs: extracellular vesicles; VP1: viral capsid protein 1; NTA: nanoparticle tracking analysis; CNS: central nervous system

Background: Enterovirus 71 (EV71) is one of the major causative pathogens of hand, foot, and mouth disease (HFMD). Immune cells play a critical role in determining the outcomes of virus infection. We aimed to characterize the lymphocyte subsets and transcriptional levels of T lymphocytes-associated transcription factors in peripheral blood cells of children with EV71 infection. Methods: Peripheral blood samples from 32 children with EV71 infection and 32 control subjects were included in this study. The frequencies of T-, B-lymphocytes, and their subsets were determined by flow cytometry. The expression of transcription factors, including T-bet, Gata3, ROR γ t, Foxp3, TCF-1, and BCL-6 in the whole blood cells were evaluated by real-time reverse-transcription quantitative polymerase chain reaction (RT-qPCR). Results: The frequencies of T cells, helper T cells (Th), cytotoxic T cells (Tc), IFN-γ+ Th1, IFN-γ+ Tc1, and regulatory T (Treg) cells were significantly decreased (P

Hand foot and mouth disease (HFMD) caused by Enterovirus 71 (EV71) infection is still a major infectious disease threatening children’s life and health in the absence of effective antiviral drugs due to its high prevalence and neurovirulence. A study of EV71-specific host response might shed some light on the reason behind its unique epidemiologic features and help to find means to conquer EV71 infection. We reported that host heat shock protein A6 (HSPA6) was induced by EV71 infection and involved infection in both Rhabdomyosarcoma (RD) cells and neurogliocytes. Most importantly, we found that EV71 did not induce the expression of other heat shock proteins HSPA1, HSPA8, and HSPB1 under the same conditions, and other HFMD-associated viruses including CVA16, CVA6, CVA10, and CVB1-3 did not induce the upregulation of HSPA6. In addition, EV71 infection enhanced the cytoplasmic aggregation of HSPA6 and its colocalization with viral capsid protein VP1. These findings suggest that HSPA6 is a potential EV71-specific host factor worthy of further study.

Enterovirus 71 (EV71) is the major pathogen causing severe neurological complications and hand, foot, and mouth disease. The intestinal mucosal immune system has a complete immune response and immune regulation mechanism, consisting of densely arranged monolayer intestinal epithelial cells (IECs) and intestinal intraepithelial lymphocytes (iIELs) distributed among the IECs, which constitute the first line of intestinal mucosa against infection of foreign pathogens. As an enterovirus, EV71 is transmitted by the intestinal tract; however, the mechanisms it uses to evade the immunosurveillance of the intestinal mucosal immune system are still incompletely clarified. The present study investigated how EV71 evades from recognizing and eliminating IECs, iIELs, and iNK cells. We found that EV71 infection induced a higher level of type III interferons (IFN-λ) than type I interferons (IFN-β) in IECs, and the addition of IFN-λ markedly restricted EV71 replication in IECs. These results indicate that IFN-λ plays a more important role in anti-EV71 intestinal infection. However, EV71 infection could markedly attenuate the antiviral responses of IFN-λ. Mechanistically, 2A protease (2Apro) and 3C protease (3Cpro) of EV71 inhibited the IFN-λ production and IFN-λ receptor expression and further decreased the response of IECs to IFN-λ. In addition, we found that EV71-infected IECs were less susceptible to the lysis of intestinal NK (iNK) cells and CD3+iIELs. We revealed that the viral 2Apro and 3Cpro could significantly reduce the expression of the ligands of natural killer group 2D (NKG2D) and promote the expression of PD-L1 on IECs, rendering them to evade the recognition and killing of iNK and CD3+iIELs. These results provide novel evasion mechanisms of EV71 from intestinal mucosal innate immunity and may give new insights into antiviral therapy.

ABSTRACT Enterovirus 71 (EV71) is the major pathogen of hand, foot, and mouth disease. In severe cases, it can cause life-threatening neurological complications, such as aseptic meningitis and polio-like paralysis. There are no specific antiviral treatments for EV71 infections. In a previous study, the host protein growth arrest and DNA damage-inducible protein 34 (GADD34) expression was upregulated during EV71 infection determined by ribosome profiling and RNA-sequencing. Here, we investigated the interactions of host protein GADD34 and EV71 during infections. Rhabdomyosarcoma (RD) cells were infected with EV71 resulting in a significant increase in expression of GADD34 mRNA and protein. Through screening of EV71 protein we determined that the non-structural precursor protein 3CD is responsible for upregulating GADD34. EV71 3CD increased the RNA and protein levels of GADD34, while the 3CD mutant Y441S could not. 3CD upregulated GADD34 translation via the upstream open reading frame (uORF) of GADD34 5'untranslated regions (UTR). EV71 replication was attenuated by the knockdown of GADD34. The function of GADD34 to dephosphorylate eIF2α was unrelated to the upregulation of EV71 replication, but the PEST 1, 2, and 3 regions of GADD34 were required. GADD34 promoted the EV71 internal ribosome entry site (IRES) activity through the PEST repeats and affected several other viruses. Finally, GADD34 amino acids 563 to 565 interacted with 3CD, assisting GADD34 to target the EV71 IRES. Our research reveals a new mechanism by which GADD34 promotes viral IRES and how the EV71 non-structural precursor protein 3CD regulates host protein expression to support viral replication. IMPORTANCE Identification of host factors involved in viral replication is an important approach in discovering viral pathogenic mechanisms and identifying potential therapeutic targets. Previously, we screened host proteins that were upregulated by EV71 infection. Here, we report the interaction between the upregulated host protein GADD34 and EV71. EV71 non-structural precursor protein 3CD activates the RNA and protein expression of GADD34. Our study reveals that 3CD regulates the uORF of the 5′-UTR to increase GADD34 translation, providing a new explanation for how viral proteins regulate host protein expression. GADD34 is important for EV71 replication, and the key functional domains of GADD34 that promote EV71 are PEST 1, 2, and 3 regions. We report that GADD34 promotes viral IRES for the first time and this process is independent of its eIF2α phosphatase activity.

Previous studies demonstrated that EV71-infected cells secrete extracellular vesicles (EVs), facilitating the transfer of viral components to recipient cells and thereby promoting virus spread. Considering lipid signaling plays a crucial role in EVs-mediated cell-to-cell communication, we compared the lipid profile of EVs secreted from uninfected and EV71-infected cells (EVs-Mock and EVs-EV71) using the human rhabdomyosarcoma (RD) cell model. These two groups of EVs were purified by using size exclusion chromatography (SEC), respectively, and evaluated by transmission electron microscopy (TEM), nanoparticle tracking technology (NTA), and Western blotting (WB). In-depth lipidomic analysis of EVs identified 1705 lipid molecules belonging to 43 lipid classes. The data showed a significant increase in the lipid content of EVs after EV71 infection. Meanwhile, we deeply analyzed the changes in lipids and screened for lipid molecules with significant differences compared EVs-EV71 with EVs-Mock EVs. Altogether, we report the alterations in the lipid profile of EVs derived from RD-cells after EV71 infection, which may affect the function of the EVs in the recipient cells., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Mao, Gao, Shen, Bao, Xiang, Chen, Gao, Huang, He and Wang.)

ATG13 and FIP200 are two subunits of the ULK kinase complex, a key regulatory component of the autophagy machinery. We have previously found that the FIP200-ATG13 subcomplex controls picornavirus replication outside its role in the ULK kinase complex and autophagy. Here, we characterized HSBP1, a very small cytoplasmic coiled-coil protein, as a novel interactor of FIP200 and ATG13 that binds these two proteins via FIP200. HSBP1 is a novel pro-picornaviral host factor since its knockdown or knockout, inhibits the replication of various picornaviruses. The anti-picornaviral function of the FIP200-ATG13 subcomplex was abolished when HSBP1 was depleted, inferring that this subcomplex negatively regulates HSBP1's pro-picornaviral function during infections. HSBP1depletion also reduces the stability of ULK kinase complex subunits, resulting in an impairment in autophagy induction. Altogether, our data show that HSBP1 interaction with FIP200-ATG13-containing complexes is involved in the regulation of different cellular pathways. [ABSTRACT FROM AUTHOR]

ABSTRACT The fatal pathogen enterovirus 71 (EV71) is a major cause of hand-foot-and-mouth disease (HFMD), which leads to serious neurological syndromes. While there are no effective clinical agents available for EV71 treatment thus far, EV71 3C protease (3Cpro), a cysteine protease encoded by the virus, has become a promising drug target for discovery of antiviral drugs, given that it plays a crucial role in virus proliferation and interferes with host cell function. Here, we report two inhibitors of EV71 3Cpro, FOPMC and FIOMC, that were developed from previously reported cyanohydrin derivative (R)-1 by replacing the acyl cyanohydrin group with 4-iminooxazolidin-2-one. FOPMC and FIOMC have potent antiviral activity and dramatically improved metabolic stability. These two inhibitors demonstrated broad anti-EV effects on various cell lines and five epidemic viral strains. We further illuminated the binding models between 3Cpro and FOPMC/FIOMC through molecular docking and molecular dynamics simulations. The substitution of an acyl cyanohydrin group with 4-iminooxazolidin-2-one does make FOPMC and FIOMC potent anti-EV71 drug candidates as universal nonclassical bioisosteres with a cyanohydrin moiety. IMPORTANCE EV71 is one of the most epidemic agents of HFMD. Thus far, there are no antiviral drugs available for clinical usage. The conserved EV71 3Cpro plays pivotal roles in virus proliferation and defense host immunity, as well as having no homology in host cells, making it a most promising antiviral target. In this work, we identified that propyl- and isopropyl-substituted 4-iminooxazolidin-2-one moieties (FOPMC and FIOMC) effectively inhibited five epidemic viral strains in rhabdomyosarcoma (RD), HEK-293T, and VeroE6 cell lines. The inhibition mechanism was also illustrated with molecular docking and molecular dynamics (MD) simulations. The successful replacement of the labile cyanohydrin greatly improved the stability and pharmacokinetic properties of (R)-1, making 4-iminooxazolidin-2-one a nonclassical bioisosteric moiety of cyanohydrin. This discovery addressed a critical issue of the primitive structural scaffold of these promising anti-EV71 inhibitors and could lead to their development as broad-spectrum anti-EV agents.

ATG13 and FIP200 are two subunits of the ULK kinase complex, a key regulatory component of the autophagy machinery. We have previously found that the FIP200-ATG13 subcomplex controls picornavirus replication outside its role in the ULK kinase complex and autophagy. Here, we characterized HSBP1, a very small cytoplasmic coiled-coil protein, as a novel interactor of FIP200 and ATG13 that binds these two proteins via FIP200. HSBP1 is a novel pro-picornaviral host factor since its knockdown or knockout, inhibits the replication of various picornaviruses. The anti-picornaviral function of the FIP200-ATG13 subcomplex was abolished when HSBP1 was depleted, inferring that this subcomplex negatively regulates HSBP1’s pro-picornaviral function during infections. HSBP1depletion also reduces the stability of ULK kinase complex subunits, resulting in an impairment in autophagy induction. Altogether, our data show that HSBP1 interaction with FIP200-ATG13-containing complexes is involved in the regulation of different cellular pathways.

A microfluidic platform integrated with two generated monoclonal antibodies (mAbs) was innovatively fabricated for ultrasensitive detection of enterovirus 71 (EV71). To date, this is the first report on EV71 detection using a microfluidic chip-based immunoassay. The mAbs 1F4 and 2H2 against the major capsid protein VP1 of EV71 were conjugated with carboxyl-functionalized magnetic beads (MBs) and carboxyl-functionalized quantum dots (QDs), respectively. MB-conjugated mAbs separated VP1 from samples, while QD-conjugated mAbs provided fluorescence detection signals and formed a sandwich structure. The monolayer of MBs and QDs captured the VP1 sandwich structure showed stronger fluorescence signal in the microfluidic channel, which could be directly evaluated by fluorescence intensity in response to 365 nm ultraviolet (UV) excitation. The limit of detection (LOD) of the microfluidic platform for VP1 was 10 pg mL−1, which was a 31-fold increase compared to sandwich enzyme-linked immunosorbent assay (ELISA, 310 pg mL−1) using identical antibodies. The microfluidic platform performed well with clinical samples, especially for fecal samples with complex compositions and impurities. The assay is quick to perform (within 30 min), and apparently requires just one syringe without other complex liquid flow system, making it is easy to operate and more suitable for on-site detection and future commercial application.

Previous studies have shown that DEAD (Asp-Glu-Ala-Asp)-box RNA helicases play important roles in viral infection, either as cytosolic sensors of pathogenic molecules or as essential host factors against viral infection. In the current study, we found that DDX6, an RNA helicase belonging to the DEAD-box family of helicase, exhibited anti-Enterovirus 71 activity through augmenting RIG-I-mediated type-I IFN response. Moreover, DDX6 binds viral RNA to form an RNA-protein complex to positively regulate the RIG-I-mediated interferon response; however, EV71 has evolved a strategy to antagonize the antiviral effect of DDX6 by proteolytic degradation of the molecule through its non-structural protein 2A, a virus-encoded protease.

Enterovirus 71 (EV71) is a positive single-stranded RNA virus from the enterovirus genus of the Picornaviridae family. Most young children infected with EV71 develop mild symptoms of hand, foot and mouth disease, but some develop severe symptoms with neurological involvement. Limb paralysis from EV71 infection is presumed to arise mainly from dysfunction of motor neurons in the spinal cord. However, EV71 also targets and damages skeletal muscle, which may also contribute to the debilitating symptoms. In this study, we have delineated the impacts of EV71 infection on skeletal muscle using a mouse model. Mouse pups infected with EV71 developed limb paralysis, starting at day 3 post-infection and peaking at day 5-7 post-infection. At later times, mice recovered gradually but not completely. Notably, severe disease was associated with high levels of myositis accompanied by muscle calcification and persistent motor end plate abnormalities. Interestingly, macrophages exhibited a dynamic change in phenotype, with inflammatory macrophages (CD45+CD11b+Ly6Chi) appearing in the early stage of infection and anti-inflammatory/restorative macrophages (CD45+CD11b+Ly6Clow/-) appearing in the late stage. The presence of inflammatory macrophages was associated with severe inflammation, while the restorative macrophages were associated with recovery. Altogether, we have demonstrated that EV71 infection causes myositis, muscle calcification and structural defects in motor end plates. Subsequent muscle regeneration is associated with a dynamic change in macrophage phenotype.

Fluorescent labeling and dynamic tracking is a powerful tool for exploring virus infection mechanisms. However, for small-sized viruses, virus tracking studies are usually hindered by a lack of appropriate labeling methods that do not dampen virus yield or infectivity. Here, we report a universal strategy for labeling viruses with chemical dyes and Quantum dots (QDs). Enterovirus 71 (EV71) was produced in a cell line that stably expresses a mutant methionyl-tRNA synthetase (MetRS), which can charge azidonorleucine (ANL) to the methionine sites of viral proteins during translation. Then, the ANL-containing virus was easily labeled with DBCO-AF647 and DBCO-QDs. The labeled virus shows sufficient yield and no obvious decrease in infectivity and can be used for imaging the virus entry process. Using the labeled EV71, different functions of scavenger receptor class B, member 2 (SCARB2), and heparan sulfate (HS) in EV71 infection were comparatively studied. The cell entry process of a strong HS-binding EV71 strain was investigated by real-time dynamic visualization of EV71-QDs in living cells. Taken together, our study described a universal biocompatible virus labeling method, visualized the dynamic viral entry process, and reported details of the receptor usage of EV71. [ABSTRACT FROM AUTHOR]

Previous studies have shown that DEAD (Glu-Asp-Ala-Glu)-box RNA helicases play important roles in viral infection, either as cytosolic sensors of pathogenic molecules or as essential host factors against viral infection. In the current study, we found that DDX6, an RNA helicase belonging to the DEAD-box family of helicase, exhibited anti-Enterovirus 71 activity through augmenting RIG-I-mediated type-I IFN response. Moreover, DDX6 binds viral RNA to form an RNA-protein complex to positively regulate the RIG-I-mediated interferon response; however, EV71 has evolved a strategy to antagonize the antiviral effect of DDX6 by proteolytic degradation of the molecule through its non-structural protein 2A, a virus-encoded protease. [ABSTRACT FROM AUTHOR]

Unprecedented 3D hexa‐adducts of [60]fullerene peripherally decorated with twelve tryptophan (Trp) or tyrosine (Tyr) residues have been synthesized. Studies on the antiviral activity of these novel compounds against HIV and EV71 reveal that they are much more potent against HIV and equally active against EV71 than the previously described dendrimer prototypes AL‐385 and AL‐463, which possess the same number of Trp/Tyr residues on the periphery but attached to a smaller and more flexible pentaerythritol core. These results demonstrate the relevance of the globular 3D presentation of the peripheral groups (Trp/Tyr) as well as the length of the spacer connecting them to the central core to interact with the viral envelopes, particularly in the case of HIV, and support the hypothesis that [60]fullerene can be an alternative and attractive biocompatible carbon‐based scaffold for this type of highly symmetrical dendrimers. In addition, the functionalized fullerenes here described, which display twelve peripheral negatively charged indole moieties on their globular surface, define a new and versatile class of compounds with a promising potential in biomedical applications. [ABSTRACT FROM AUTHOR]

Enterovirus 71 (EV71) is a positive single-stranded RNA virus from the enterovirus genus of the Picornaviridae family. Most young children infected with EV71 develop mild symptoms of hand, foot and mouth disease, but some develop severe symptoms with neurological involvement. Limb paralysis from EV71 infection is presumed to arise mainly from dysfunction of motor neurons in the spinal cord. However, EV71 also targets and damages skeletal muscle, which may also contribute to the debilitating symptoms. In this study, we have delineated the impacts of EV71 infection on skeletal muscle using a mouse model. Mouse pups infected with EV71 developed limb paralysis, starting at day 3 post-infection and peaking at day 5-7 post-infection. At later times, mice recovered gradually but not completely. Notably, severe disease was associated with high levels of myositis accompanied by muscle calcification and persistent motor end plate abnormalities. Interestingly, macrophages exhibited a dynamic change in phenotype, with inflammatory macrophages (CD45+CD11b+Ly6Chi) appearing in the early stage of infection and anti-inflammatory/restorative macrophages (CD45+CD11b+Ly6Clow/-) appearing in the late stage. The presence of inflammatory macrophages was associated with severe inflammation, while the restorative macrophages were associated with recovery. Altogether, we have demonstrated that EV71 infection causes myositis, muscle calcification and structural defects in motor end plates. Subsequent muscle regeneration is associated with a dynamic change in macrophage phenotype. [ABSTRACT FROM AUTHOR]