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

Abstract Background Astragaloside IV (AST-IV), as an effective active ingredient of Astragalus membranaceus (Fisch.) Bunge. It has been found that AST-IV inhibits the replication of dengue virus, hepatitis B virus, adenovirus, and coxsackievirus B3. Enterovirus 71 (EV71) serves as the main pathogen in severe hand-foot-mouth disease (HFMD), but there are no specific drugs available. In this study, we focus on investigating whether AST-IV can inhibit EV71 replication and explore the potential underlying mechanisms. Methods The GES-1 or RD cells were infected with EV71, treated with AST-IV, or co-treated with both EV71 and AST-IV. The EV71 structural protein VP1 levels, the viral titers in the supernatant were measured using western blot and 50% tissue culture infective dose (TCID50), respectively. Network pharmacology was used to predict possible pathways and targets for AST-IV to inhibit EV71 replication. Additionally, ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) was used to investigate the potential targeted metabolites of AST-IV. Associations between metabolites and apparent indicators were performed via Spearman’s algorithm. Results This study illustrated that AST-IV effectively inhibited EV71 replication. Network pharmacology suggested that AST-IV inhibits EV71 replication by targeting PI3K-AKT. Metabolomics results showed that AST-IV achieved these effects by elevating the levels of hypoxanthine, 2-ketobutyric acid, adenine, nicotinic acid mononucleotide, prostaglandin H2, 6-hydroxy-1 H-indole-3- acetamide, oxypurinol, while reducing the levels of PC (14:0/15:0). Furthermore, AST-IV also mitigated EV71-induced oxidative stress by reducing the levels of MDA, ROS, while increasing the activity of T-AOC, CAT, GSH-Px. The inhibition of EV71 replication was also observed when using the ROS inhibitor N-Acetylcysteine (NAC). Additionally, AST-IV exhibited the ability to activate the PI3K-AKT signaling pathway and suppress EV71-induced apoptosis. Conclusion This study suggests that AST-IV may activate the cAMP and the antioxidant stress response by targeting eight key metabolites, including hypoxanthine, 2-ketobutyric acid, adenine, nicotinic acid mononucleotide, prostaglandin H2, 6-Hydroxy-1 H-indole-3-acetamide, oxypurinol and PC (14:0/15:0). This activation can further stimulate the PI3K-AKT signaling to inhibit EV71-induced apoptosis and EV71 replication. Graphical Abstract

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

Increasing evidences suggest that the methyltransferase NSUN2 catalyzes 5-methylcytosine (m5C) modifications on viral RNAs, which are essential for the replication of various viruses. Despite the function of m5C deposition is well characterized, other potential roles of NSUN2 in regulating viral replication remain largely unknown. In this study, the m5C modified residues catalyzed by NSUN2 on enterovirus 71 (EV71) RNAs were mapped. NSUN2, along with m5C modifications, played multiple roles during the EV71 life cycle. Functional m5C modified nucleotides increased the translational efficiency and stability of EV71 RNAs. Additionally, NSUN2 was found to target the viral protein VP1 for binding and promote its stability by inhibiting the ubiquitination. Furthermore, both viral replication and pathogenicity in mice were largely attenuated when functional m5C residues were mutated. Taken together, this study characterizes distinct pathways mediated by NSUN2 in regulating EV71 replication, and highlights the importance of its catalyzed m5C modifications on EV71 RNAs for the viral replication and pathogenicity. [ABSTRACT FROM AUTHOR]

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

Background: Astragaloside IV (AST-IV), as an effective active ingredient of Astragalus membranaceus (Fisch.) Bunge. It has been found that AST-IV inhibits the replication of dengue virus, hepatitis B virus, adenovirus, and coxsackievirus B3. Enterovirus 71 (EV71) serves as the main pathogen in severe hand-foot-mouth disease (HFMD), but there are no specific drugs available. In this study, we focus on investigating whether AST-IV can inhibit EV71 replication and explore the potential underlying mechanisms. Methods: The GES-1 or RD cells were infected with EV71, treated with AST-IV, or co-treated with both EV71 and AST-IV. The EV71 structural protein VP1 levels, the viral titers in the supernatant were measured using western blot and 50% tissue culture infective dose (TCID50), respectively. Network pharmacology was used to predict possible pathways and targets for AST-IV to inhibit EV71 replication. Additionally, ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) was used to investigate the potential targeted metabolites of AST-IV. Associations between metabolites and apparent indicators were performed via Spearman's algorithm. Results: This study illustrated that AST-IV effectively inhibited EV71 replication. Network pharmacology suggested that AST-IV inhibits EV71 replication by targeting PI3K-AKT. Metabolomics results showed that AST-IV achieved these effects by elevating the levels of hypoxanthine, 2-ketobutyric acid, adenine, nicotinic acid mononucleotide, prostaglandin H2, 6-hydroxy-1 H-indole-3- acetamide, oxypurinol, while reducing the levels of PC (14:0/15:0). Furthermore, AST-IV also mitigated EV71-induced oxidative stress by reducing the levels of MDA, ROS, while increasing the activity of T-AOC, CAT, GSH-Px. The inhibition of EV71 replication was also observed when using the ROS inhibitor N-Acetylcysteine (NAC). Additionally, AST-IV exhibited the ability to activate the PI3K-AKT signaling pathway and suppress EV71-induced apoptosis. Conclusion: This study suggests that AST-IV may activate the cAMP and the antioxidant stress response by targeting eight key metabolites, including hypoxanthine, 2-ketobutyric acid, adenine, nicotinic acid mononucleotide, prostaglandin H2, 6-Hydroxy-1 H-indole-3-acetamide, oxypurinol and PC (14:0/15:0). This activation can further stimulate the PI3K-AKT signaling to inhibit EV71-induced apoptosis and EV71 replication. [ABSTRACT FROM AUTHOR]

This study was conducted to efficiently produce virus-like particles (VLPs) of enterovirus 71 (EV71), a causative virus of hand, foot, and mouth disease (HFMD). The expression level of the P1 precursor, a structural protein of EV71, was modified to increase VLP production, and the optimal expression level and duration of the 3CD protein for P1 cleavage were determined. The expression level and duration of 3CD were controlled by the p10 promoter, which was weakened by repeated burst sequence (BS) applications, as well as the OpIE2 promoter, which was weakened by the insertion of random untranslated region sequences of various lengths. The cleavage and production efficiency of the P1 precursor were compared based on the expression time and level of 3CD, revealing that the p10-BS5 promoter with four repeated BSs was the most effective. When P1 and 3CD were expressed using the hyperexpression vector and the p10-BS5 promoter, high levels of structural protein production and normal HFMD-VLP formation were observed, respectively. This study suggests that the production efficiency of HFMD-VLPs can be significantly enhanced by increasing the expression of the P1 precursor and controlling the amount and duration of 3CD expression. [ABSTRACT FROM AUTHOR]

BACKGROUND: It is reported that anti‐enterovirus 71 (EV71) drugs have some side effects on human health. Notably, fungi plays a crucial role in promoting human health and anti‐virus. Grifola frondosa is a type of large medicinal and edible fungi, rich in active substances. The present study aimed to investigate the anti‐EV71 effect of G. frondosa and the potential active substances. RESULTS: In the present study, the water extract of G. frondosa was subjected to ethanol precipitation to obtain the water‐extracted supernatant of G. frondosa (GFWS) and water‐extracted precipitation of G. frondosa. Their inhibitory effects on EV71 virus were studied based on a cell model. The results showed that GFWS had stronger security and anti‐EV71 effects. In addition, the chemical constituents of GFWS were identified by ultra‐high performance liquid chromatography‐tandem mass spectrometry, which were selected for further separation and purification. Three compounds, N‐butylaniline, succinic acid and l‐tryptophan, were isolated from GFWS by NMR spectroscopy. It is noteworthy that N‐butylaniline and l‐tryptophan were isolated and identified from the G. frondosa fruiting bodies for the first time. Our study found that l‐tryptophan has anti‐EV71 virus activity, which reduced EV71‐induced apoptosis and significantly inhibited the replication process after virus adsorption. Furthermore, it could also bind to capsid protein VP1 to prevent the virus from attaching to the cells. CONCLUSION: l‐tryptophan was an inhibitor of the EV71 virus, which could be used in infant nutrition and possibly provide a new drug to treat hand, foot and mouth disease. © 2024 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Purpose: Enterovirus 71, a pathogen that causes hand-foot and mouth disease (HFMD) is currently regarded as an increasing neurotropic virus in Asia and can cause severe complications in pediatric patients with blister-like sores or rashes on the hand, feet, and mouth. Notwithstanding the significant burden of the disease, no authorized vaccine is available. Previously identified attenuated and inactivated vaccines are worthless over time owing to changes in the viral genome. Materials and Methods: A novel vaccine construct using B-cell derived T-cell epitopes from the virulent polyprotein found the induction of possible immune response. In order to boost the immune system, a beta-defensin 1 preproprotein adjuvant with EAAAK linker was added at the N-terminal end of the vaccine sequence. Results: The immunogenicity of the designed, refined, and verified prospective threedimensional-structure of the multi-epitope vaccine was found to be quite high, exhibiting nonallergenic and antigenic properties. The vaccine candidates bound to toll-like receptor 3 in a molecular docking analysis, and the efficacy of the potential vaccine to generate a strong immune response was assessed through in silico immunological simulation. Conclusion: Computational analysis has shown that the proposed multi-epitope vaccine is possibly safe for use in humans and can elicit an immune response. [ABSTRACT FROM AUTHOR]

This study aimed to investigate the inhibitory effect of astragaloside IV (AS-IV) on enterovirus 71 (EV71)-induced pyroptosis. The results indicated that AST-IV inhibited EV71-induced pyroptosis in normal human gastric epithelial (GES-1) cells. Therefore, we investigated the mechanisms underlying pyroptosis inhibition by AST-IV. We found that EV71 inhibited the Nrf2-Keap1 pathway, whereas AST-IV treatment activated it. Overexpression of Nrf2 led to the inhibition of EV71-induced pyroptosis. EV71 infection and AST-IV treatment upregulated and downregulated thioredoxin interacting protein (TXNIP), respectively. Downregulation of TXNIP activated the Keap1/Nrf2 signaling pathway and inhibited EV71-induced pyroptosis. Furthermore, transcription factor 12 (TCF12) reduced the protein and mRNA levels of TXNIP, and AST-IV treatment upregulated TCF12. These results demonstrate that the TCF12-TXNIP-Keap1/Nrf2 axis participates in the inhibition of pyroptosis by AST-IV in EV71-infected GES-1 cells, suggesting that AST-IV could be used as a dietary supplement to alleviate EV71-induced pyroptosis.

Enterovirus 71 (EV71) is the common causative agent of hand-foot-mouth disease (HFMD). Despite evidence in mice model suggested that the interferon (IFN) signaling pathways play a role in defending against this virus, knowledge on the IFN-mediated antiviral response is still limited. Here we identified an IFN-stimulated gene (ISG) called L3HYPDH, whose expression inhibits EV71 replication. Mapping assay indicated that amino acids 61–120 and 295–354 are critical for its optimal antiviral activity. Mechanismly, L3HYPDH specifically inhibits protein translation mediated by EV71 internal ribosome entry site (IRES). Our data thus uncovered a new mechanism utilized by the host cell to restrict EV71 replication.

EV71, a significant pathogen causing hand-foot-mouth disease, is associated with severe neurological complications such as brain stem encephalitis, aseptic meningitis, and acute flaccid paralysis. While the role of mitochondrial dynamics in regulating the replication of numerous viruses is recognized, its specific involvement in EV71 remains unclear. This study aimed to elucidate the role of mitochondrial dynamics in human neuroblastoma SK-N-SH cells during EV71 infection. Utilizing laser confocal microscopy and transmission electron microscopy, we observed that EV71 infection induced mitochondrial elongation and damage to cristae structures, concurrently accelerating mitochondrial movement. Furthermore, we identified the reduction in the expression of dynamin-related protein 1 (Drp1) and optic atrophy protein 1 (Opa1) and the increased expression of Mitofusion 2 (Mfn2) upon EV71 infection. Notably, EV71 directly stimulated the generation of mitochondrial reactive oxygen species (ROS), leading to a decline in mitochondrial membrane potential and ATP levels. Remarkably, the application of melatonin, a potent mitochondrial protector, inhibited EV71 replication by restoring Drp1 expression. These findings collectively indicate that EV71 induces alterations in mitochondrial morphology and dynamics within SK-N-SH cells, potentially impairing mitochondrial function and contributing to nervous system dysfunction. The restoration of proper mitochondrial dynamics may hold promise as a prospective approach to counteract EV71 infection.

The transition time during which a virus leaves its host and infects the next susceptible host is critical for virus survival. Enterovirus 71 (EV71) is stable in aqueous environments, but its molecular interactions with bacteria and their biofilms are not well-established. Helicobacter pylori is a highly successful gut bacterial pathogen, with its capacity to form biofilms being linked to its transmission. Given that both are gut-associated microbes, we hypothesized that biofilms formed by H. pylori may play a significant role in the survival of EV71 in the external environment. In this study, we examine the interactions of EV71 with the preformed biofilm of H. pylori to mimic its natural state in the environment. Immunofluorescence confocal microscopy and scanning electron microscopy revealed that EV71 particles persisted for up to 10 days when incubated with the H. pylori biofilm. Furthermore, the presence of the H. pylori biofilm significantly augmented viral viability, as verified through virus plaque assays. Interestingly, the viability of EV71 was dependent on the quantity of H. pylori biofilm formation. Thus, two H. pylori strains able to generate large amounts of biofilm could facilitate EV71 viability for up to 17 days, whereas two other H. pylori strains that produced moderate or low quantities of biofilm could not prolong virus viability. It is interesting that biofilm contains N-acetyl-glucosamine and glycosaminoglycan, and that EV71 has binding affinity to cell-surface heparan sulfate glycosaminoglycan, which acts as an EV71 attachment receptor. The synergistic ability of H. pylori biofilm to promote EV71 viability for extended periods implies that H. pylori biofilm may serve as an additional pathway of EV71 transmission. [ABSTRACT FROM AUTHOR]

This study was conducted to efficiently produce virus-like particles (VLPs) of enterovirus 71 (EV71), a causative virus of hand, foot, and mouth disease (HFMD). The expression level of the P1 precursor, a structural protein of EV71, was modified to increase VLP production, and the optimal expression level and duration of the 3CD protein for P1 cleavage were determined. The expression level and duration of 3CD were controlled by the p10 promoter, which was weakened by repeated burst sequence (BS) applications, as well as the OpIE2 promoter, which was weakened by the insertion of random untranslated region sequences of various lengths. The cleavage and production efficiency of the P1 precursor were compared based on the expression time and level of 3CD, revealing that the p10-BS5 promoter with four repeated BSs was the most effective. When P1 and 3CD were expressed using the hyperexpression vector and the p10-BS5 promoter, high levels of structural protein production and normal HFMD-VLP formation were observed, respectively. This study suggests that the production efficiency of HFMD-VLPs can be significantly enhanced by increasing the expression of the P1 precursor and controlling the amount and duration of 3CD expression.

Enterovirus 71 (EV71) infection is one of the main causes of severe hand, foot and mouth disease (HFMD), which is usually accompanied by a marked inflammatory response. The excessive inflammatory response has been implicated to serve an important role in EV71-caused HFMD. Pyroptosis is a type of inflammatory programmed cell death. Therefore, a novel treatment strategy against EV71 infection could aim to alleviate the inflammatory response through inhibition of EV71-induced pyroptosis. The present study revealed that metformin had this therapeutic potential. A cell model of EV71 infection was established, cell viability was measured by CCK8 assay, cell damage was measured by LDH release kit, and the dead and dying cells were excluded by propidium iodide staining. The intracellular levels of DEP domain-containing mTOR interacting protein (DEPTOR) and pyroptosis-associated molecules were measured by western blot analysis, the NLRP3 expression was assessed by immunofluorescence labeling, and virus titers in cell culture supernatants were determined by a cell culture infectious dose 50 assay. The results demonstrated that EV71 infection could induce pyroptosis in a time- and dose-dependent manner, and metformin could inhibit EV71-induced pyroptosis. The mechanism of metformin inhibiting EV71-induced pyroptosis was explored next. Subsequent experiments indicated that metformin could increase the levels of DEPTOR, which were decreased by EV71. Finally, overexpression of DEPTOR in cells could reduce EV71-induced pyroptosis. Overall, the present study demonstrated that metformin could exert a novel pharmacodynamic anti-pyroptosis effect in the treatment of EV71 infection by upregulating DEPTOR expression. [ABSTRACT FROM AUTHOR]

Colorectal cancer (CRC) is an intestinal malignant tumor with high morbidity and mortality worldwide. Inoperability or resistanance to radiation and chemotherapy occur in the conventional treatments against CRC. Oncolytic viruses (OVs) are one kind of virus that selectively infects and lyses cancer cells, which is considered to be a new anticancer therapy with biological and immune‐based approaches. Enterovirus 71 (EV71), belonging to the enterovirus genus in the family Picornaviridae, is a single positive‐stranded RNA virus. EV71 is transmitted in a fetal‐oral route and infects gastrointestinal tract in infants. Here, EV71 is exploited to be a novel oncolytic virus in colorectal cancer. It is revealed that EV71 infection can selectively cause colorectal cancer cells cytotoxicity but not primary intestinal epithelial cells. Consistently, EV71 injection significantly inhibits tumor growth in nude mice xenografted colorectal cancer cells. In detail, EV71 infects colorectal cancer cells to repress the expression of Ki67 and B‐cell leukemia 2 (Bcl‐2) leading to the inhibition of cell proliferation, while activating the cleavage of poly‐adenosine diphosphatase‐ribose polymerase and Caspase‐3 protein resulting in the promotion of cell apoptosis. The findings demonstrate the oncolytic feature of EV71 in CRC treatment and may provide a potential clue for clinical anticancer therapy. [ABSTRACT FROM AUTHOR]

Infection of the central nervous system caused by enterovirus 71 (EV71) remains the main cause of death in hand‐foot‐and‐mouth disease. However, the mechanism responsible for how EV71 breaks through the blood‐brain barrier to infect brain cells has yet to be elucidated. By performing a high‐throughput small interfering RNA (siRNA) screening and validation, we found that the infection of human brain microvascular endothelial cells (HBMECs) by EV71 was independent of the endocytosis pathways mediated by caveolin, clathrin, and macropinocytosis but dependent on ADP‐ribosylation factor 6 (ARF6), a small guanosinetriphosphate (GTP)‐binding protein of the Ras superfamily. The specific siRNA targeting ARF6 markedly inhibited HBMECs susceptibility to EV71. EV71 infectivity was inhibited by NAV‐2729, a specific inhibitor of ARF6, in a dose‐dependent manner. The subcellular analysis demonstrated the co‐localization of the endocytosed EV71 and ARF6, while knockdown of ARF6 with siRNA remarkably influenced EV71 endocytosis. By immunoprecipitation assays, we found a direct interaction of ARF6 with EV71 viral protein. Furthermore, ARF1, another small GTP‐binding protein, was also found to participate in ARF6‐mediated EV71 endocytosis. Murine experiments demonstrated that NAV‐2729 significantly alleviated mortality caused by EV71 infection. Our study revealed a new pathway by which EV71 enters the HBMECs and provides new targets for drug development. [ABSTRACT FROM AUTHOR]

ObjectiveTo analyze prevalence characteristics of hand, foot and mouth disease (HFMD) in Shenzhen city from 2008 to 2020 and to evaluate the impact of inactivated enterovirus 71 (EV71) vaccination on HFMD incidence for providing evidence to promoting inactivated EV71 vaccination and formulating strategies on HFMD prevention and control. MethodsThe data on HFMD incidence for Shenzhen municipality during 2008 – 2020 were retrieved from China′s Infectious Disease Information System and that on inactivated EV71 vaccination were extracted from Childhood Immunization Planning Information System of Shenzhen Municipality; the information on prevalent strains related to HFMD epidemics in the city was collected simultaneously from pathogenic surveillance dataset. Descriptive epidemiological statistics was conducted to analyze prevalence characteristics of HFMD and the effect of inactivated EV71 vaccination on HFMD incidence and prevalent pathogenic strains. ResultsTotally 469 686 HFMD cases were reported during the 13-year period in the city, with an average annual incidence rate of 312.25/100 000. During a year, HFMD incidence was obviously higher in summer season than in winter season, with two incidence peaks in April – July and September – October. Higher incidence rate were observed in the regions with a large number of migrant workers or high population density. The majority of the reported cases were males, children aged 4 years and younger, and homebound children, accounting for 60.54%, 89.69%, and 76.58% of total cases, respectively. The HFMD incidence declined evidently from 516.13/100 000 in 2016 to 84.42/100 000 in 2020 with the yearly increasing of EV71 vaccination rate; the proportion of HFMD cases due to the infection of EV71 also decreased from 16.19% in 2016 to zero in 2020. ConclusionInactivated EV71 vaccination can effectively prevent and control HFMD epidemic, especially for the HFMD epidemic caused by EV71 infection.

Objective To prepare polyclonal antisera against enterovirus 71(EV71) capsid proteins. Methods Five B cell epitope prediction softwares were used to analyze capsid proteins of EV71 and four peptides against viral protein were synthesized, VP1(aa.204-223), VP2(aa.133-163), VP3(aa.139-148+aa.173-191) and VP4(aa.1-23). They were conjugated with keyhole limpet hemocyanin(KLH). BALB/c mice were subcutaneously immunized with six doses of KLH-peptides adjuvanted with MF59/CpGC274 at two-week interval. Serum sample was collected and examined with ELISA and Western blot. Results The four antisera were bound to both EV71 virus and denatured virus effectively. VP2 antiserum showed activity with denatured virus and the other three had similar binding activity to EV71 virus and denatured virus. The four antiserum samples were bound to both EV71 virus-like particle (VLP) and denatured VLP effectively. VP1 and VP2 antiserum could bind to VP1, VP0 of EV71 VLP separately. VP2 antiserum showed the highese binding activity and VP1 antiserum showed a inperior binding activity as compared to the formers. VP1 and VP2 antiserum were bound specifically and efficiently to EV71 virus. VP0 of empty particles and VP2 of intact EV71 virus particles could be simultaneously identified by VP2 antiserum. VP3 and VP4 antiserum failed to binding to no obvious binding to VLP and virus. Conclusions Four epitope peptide polyclonal antisera against EV71 are successfully prepared, which can provide ootential detection tools used in ELISA and Western blot and may be used for QC evaluation of EV71 vaccine.

Host immune activation is critical for enterovirus 71 (EV71) clearance and immunopathogenesis. However, the mechanism of innate immune activation, especially of cell membrane-bound toll-like receptors (TLRs), against EV71 remains unknown. We previously demonstrated that TLR2 and its heterodimer inhibit EV71 replication. In this study, we systematically investigated the effects of TLR1/2/4/6 monomers and TLR2 heterodimer (TLR2/TLR1, TLR2/TLR6, and TLR2/TLR4) on EV71 replication and innate immune activation. We found that the overexpression of human- or mouse-derived TLR1/2/4/6 monomers and TLR2 heterodimer significantly inhibited EV71 replication and induced the production of interleukin (IL)-8 via activation of the phosphoinositide 3-kinase/ protein kinase B (PI3K/AKT) and mitogen-activated protein kinase (MAPK) pathways. Furthermore,human–mouse chimeric TLR2 heterodimer inhibited EV71 replication and activated innate immunity. Dominant-negative TIR-less (DN)-TLR1/2/4/6 did not exert any inhibitory effects, whereas DN-TLR2 heterodimer inhibited EV71 replication. Prokaryotic expression of purified recombinant EV71 capsid proteins (VP1, VP2, VP3, and VP4) or overexpression of EV71 capsid proteins induced the production of IL-6 and IL-8 via activation of the PI3K/AKT and MAPK pathways. Notably, two types of EV71 capsid proteins served as pathogen-associated molecular patterns for TLR monomers (TLR2 and TLR4) and TLR2 heterodimer (TLR2/TLR1, TLR2/TLR6, and TLR2/TLR4) and activated innate immunity. Collectively, our results revealed that membrane TLRs inhibited EV71 replication via activation of the antiviral innate response, providing insights into the EV71 innate immune activation mechanism. [ABSTRACT FROM AUTHOR]

Hand-foot-and-mouth disease (HFMD) is the most common enteroviral infection in South-East Asia. When evaluating the role of enterovirus 71 (EVA71) as an etiological agent of infectious disease in South Vietnam, we revealed a high proportion of EVA71 among identified species A enteroviruses found in 3542 samples from HFMD cases; 125 samples from cases of enteroviral meningitis; and 130 samples from acute flaccid paralysis (AFP) cases. These represent 50%, 54.8%, and 51.5%, respectively. According to molecular analysis, 90% of EVA71 were attributed to genotype C4 and 10% were attributed to genotype B5. The predominance of EVA71 circulation among the population proves the need to strengthen surveillance (with monitoring of enterovirus circulation for facilitation of HFMD outbreak prediction) and to increase the effectiveness of preventative measures by the implementation of vaccination against EVA71-associated infections. A phase III trial of a Taiwanese vaccine (EV71vac) in Taiwan and South Vietnam showed its safety, tolerability, and efficacy in children aged 2–71 months. This B4 genotype-based vaccine, which features cross-protection against B5 and C4 genotypes, and other existing EV71 vaccines can serve as a good approach to solving the HFMD problem, which is so important for Vietnam. [ABSTRACT FROM AUTHOR]

Mucosal surfaces are in contact with the external environment and protect the body from infection by various microbes. To prevent infectious diseases at the first line of defense, the establishment of pathogen-specific mucosal immunity by mucosal vaccine delivery is needed. Curdlan, a 1,3-β-glucan has a strong immunostimulatory effect when delivered as a vaccine adjuvant. Here, we investigated whether intranasal administration of curdlan and antigen (Ag) could induce sufficient mucosal immune responses and protect against viral infections. Intranasal co-administration of curdlan and OVA increased OVA-specific IgG and IgA Abs in both serum and mucosal secretions. In addition, intranasal co-administration of curdlan and OVA induced the differentiation of OVA-specific Th1/Th17 cells in the draining lymph nodes. To investigate the protective immunity of curdlan against viral infection, intranasal co-administration of curdlan with recombinant VP1 of EV71 C4a was administered and showed enhanced protection against enterovirus 71 in a passive serum transfer model using neonatal hSCARB2 mice, although intranasal administration of VP1 plus curdlan increased VP1-specific helper T cells responses but not mucosal IgA. Next, Mongolian gerbils were intranasally immunized with curdlan plus VP1, and they had effective protection against EV71 C4a infection, while decreasing viral infection and tissue damage by inducing Th17 responses. These results indicated that intranasal curdlan with Ag improved Ag-specific protective immunity by enhancing mucosal IgA and Th17 against viral infection. Our results suggest that curdlan is an advantageous candidate as a mucosal adjuvant and delivery vehicle for the development of mucosal vaccines. [ABSTRACT FROM AUTHOR]

Enterovirus A71, a non-enveloped single-stranded (+) RNA virus, enters host cells through three stages: attachment, endocytosis and uncoating. In recent years, receptors/co-receptors anchored on the host cell membrane and involved in this process have been continuously identified. Among these, hSCARB-2 was the first receptor revealed to specifically bind to a definite site of the EV-A71 viral capsid and plays an indispensable role during viral entry. It actually acts as the main receptor due to its ability to recognize all EV-A71 strains. In addition, PSGL-1 is the second EV-A71 receptor discovered. Unlike hSCARB-2, PSGL-1 binding is strain-specific; only 20% of EV-A71 strains isolated to date are able to recognize and bind it. Some other receptors, such as sialylated glycan, Anx 2, HS, HSP90, vimentin, nucleolin and fibronectin, were discovered successively and considered as "co-receptors" because, without hSCARB-2 or PSGL-1, they are not able to mediate entry. For cypA, prohibitin and hWARS, whether they belong to the category of receptors or of co-receptors still needs further investigation. In fact, they have shown to exhibit an hSCARB-2-independent entry. All this information has gradually enriched our knowledge of EV-A71's early stages of infection. In addition to the availability of receptors/co-receptors for EV-A71 on host cells, the complex interaction between the virus and host proteins and various intracellular signaling pathways that are intricately connected to each other is critical for a successful EV-A71 invasion and for escaping the attack of the immune system. However, a lot remains unknown about the EV-A71 entry process. Nevertheless, researchers have been continuously interested in developing EV-A71 entry inhibitors, as this study area offers a large number of targets. To date, important progress has been made toward the development of several inhibitors targeting: receptors/co-receptors, including their soluble forms and chemically designed compounds; virus capsids, such as capsid inhibitors designed on the VP1 capsid; compounds potentially interfering with related signaling pathways, such as MAPK-, IFN- and ATR-inhibitors; and other strategies, such as siRNA and monoclonal antibodies targeting entry. The present review summarizes these latest studies, which are undoubtedly of great significance in developing a novel therapeutic approach against EV-A71. [ABSTRACT FROM AUTHOR]

EV-A71 is a common viral pathogen that causes hand, foot and mouth disease. It is a single-stranded RNA virus that has a low fidelity RNA polymerase and, as a result, spontaneous mutations frequently occur in the EV-A71 genome. The mutations within the genome give rise to quasispecies within the viral population that could be further defined by haplotypes. In vitro virulence of EV-A71 was shown by plaque size in Rhabdomyosarcoma (RD) cells, which was substantiated by in vitro characterizations of growth, RNA replication, binding, attachment and host cell internalization. Viruses could exhibit different host cell adaptations in different cell lines during viral passaging. The EV-A71/WT (derived from EV-A71 subgenotype B4) was shown to comprise six haplotypes through next-generation sequencing, where only EV-A71/Hap2 was found to be cultivable in RD cells, while EV-A71/Hap4 was the only cultivable haplotype in Vero cells. The EV-A71/WT produced plaques of four different sizes (small, medium, big, huge) in RD cells, while only two plaque variants (small, medium) were present in Vero cells. The small plaque variant isolated from RD cells displayed lower RNA replication rates, slower in vitro growth kinetics, higher TCID50 and lower attachment, binding and entry ability when compared against EV-A71/WT due to the mutation at 3D-S228P that disrupted the active site of the RNA polymerase, resulting in low replication and growth of the variant. [ABSTRACT FROM AUTHOR]

Abstract Background The enterovirus 71 (EV-A71) vaccine has been used in Hefei for several years, and the epidemiological significance of vaccination in this area is unclear. We aims to explore the spatial–temporal-demographic and virological changes of hand, foot and mouth disease (HFMD) after vaccination in China. Methods The data for HFMD from 2012 to 2020 were downloaded with the help of HFMD reporting system of Hefei Center for Disease Control and Prevention and combined with the EV-A71 vaccination status in Hefei. The study defined the period between 2012 to 2016 as the pre-vaccination period and explored the effect of vaccination on the incidence of HFMD by comparing the changes of HFMD before and after vaccination in terms of spatial, temporal, demographic and virological aspects. Results During the study period, a higher incidence occurred in urban area and the random distribution changed to a slight cluster after vaccination. HFMD incidence had inconsistent seasonality over years, with one or two incidence peaks in varying years. The morbidity decreased from 215.22/105 in 2012–2016 to 179.81/105 in 2017–2020 (p

Abstract Background Hand, foot, and mouth disease (HFMD) is a viral infection caused by a virus from the enterovirus genus of picornavirus family that majorly affects children. Though most cases of HFMD do not cause major problems, the outbreaks of Enterovirus 71 (EV71) can produce a high risk of neurological sequelae, including meningoencephalitis, lung difficulties, and mortality. In Asia, HFMD caused by EV71 has emerged as an acutely infectious disease of highly pathogenic potential, which demands the attention of the international medical community. Main body of the abstract Some online databases including NCBI, PubMed, Google Scholar, ProQuest, Scopus, and EBSCO were also accessed using keywords relating to the topic for data mining. The paid articles were accessed through the Centre Library facility of Siksha O Anusandhan University. This work describes the structure, outbreak, molecular epidemiology of Enterovirus 71 along with different EV71 vaccines. Many vaccines have been developed such as inactivated whole-virus live attenuated, subviral particles, and DNA vaccines to cure the patients. In Asia–Pacific nations, inactivated EV71 vaccination still confronts considerable obstacles in terms of vaccine standardization, registration, price, and harmonization of pathogen surveillance and measurements. Short conclusion HFMD has emerged as a severe health hazard in Asia–Pacific countries in recent decades. In Mainland China and other countries with high HFMD prevalence, the inactivated EV71 vaccination will be a vital tool in safeguarding children's health. When creating inactivated EV71 vaccines, Mainland China ensured maintaining high standards of vaccine quality. The Phase III clinical studies were used to confirm the safety and effectiveness of vaccinations. Graphical Abstract

The authors reported a rare case of ectopic atrial tachycardia (EAT) with reversible myocardial dysfunction associated with enterovirus 71 (EV71) infection. A previously healthy 11-year-old boy presented with progressive heart failure. An initial ECG revealed a regular narrow QRS tachycardia with abnormal P wave morphology. Echocardiography showed severely impaired left ventricular function (LVEF 20%). Viral study (PCR) in stool was positive for EV71. Treatment with inotropic support, amiodarone, and carvedilol resulted in gradual restoration of sinus rhythm over 10 days. Cardiac function returned to normal within three months. EV71 can cause EAT along with other complications and should be considered in patients with persistent tachycardia and cardiac dysfunction. Recognizing this arrhythmia led to proper management to control ventricular rate, then termination of the tachycardia and gradual restoration of ventricular function to normal. [ABSTRACT FROM AUTHOR]

Abstract Background Little research has been conducted on the spatio-temporal relationship between the severe cases and the enteroviruses infections of hand, foot and mouth disease (HFMD). This study aimed to investigate epidemic features and spatial clusters of HFMD incidence rates and assess the relationship between Enterovirus 71 (EV71) and Coxsackievirus A16 (CoxA16) and severe cases of HMFD in Gansu province, China. Methods Weekly county-specific data on HFMD between 1st January and 31st December 2018 were collected from the China Infectious Disease Information System (CIDIS), including enterovirus type (EV71 and CoxA16), severe and non-severe cases in Gansu province, China. Temporal risk [frequency index (α), duration index (β) and intensity index (γ)] and spatial cluster analysis were used to assess epidemic features and identify high-risk areas for HFMD. Time-series cross-correlation function and regression model were used to explore the relationship between the ratios of two types of viruses (i.e. EV71/Cox16) (EC) and severe cases index (i.e. severe cases/non-severe cases) (SI) of HFMD. Results Some counties in Dingxi City, Gansu were identified as a hot spot for the temporal risk indices. Time-series cross-correlation analysis showed that SI was significantly associated with EC (r = 0.417, P

Enterovirus 71 (EV71) was the major causative agent of hand, foot, and mouth disease, resulting in serious complications in children. It was worth noting that viruses must be provided with energy and raw materials for synthesis by hijacking the host cell metabolism. Lacto- N -fucopentaose I (LNFPI), as one of representative oligosaccharide in human milk, had been demonstrated to inhibit the EV71 infection. However, the regulatory mechanisms of LNFPI for metabolic changes caused by EV71 remained largely unknown. This work aimed to reveal the regulatory effects of LNFPI on metabolic abnormalities induced by EV71. The results showed that LNFPI induced an increase in the expression of glucose-6-phosphate transporter by down-regulating GLUT2, thereby reducing energy hijacking and alleviating metabolic abnormalities caused by EV71 infection. This intervention also hindered the transcription and metabolism of nucleotides, consequently inhibiting EV71 replication. [ABSTRACT FROM AUTHOR]

The transition time during which a virus leaves its host and infects the next susceptible host is critical for virus survival. Enterovirus 71 (EV71) is stable in aqueous environments, but its molecular interactions with bacteria and their biofilms are not well-established. Helicobacter pylori is a highly successful gut bacterial pathogen, with its capacity to form biofilms being linked to its transmission. Given that both are gut-associated microbes, we hypothesized that biofilms formed by H. pylori may play a significant role in the survival of EV71 in the external environment. In this study, we examine the interactions of EV71 with the preformed biofilm of H. pylori to mimic its natural state in the environment. Immunofluorescence confocal microscopy and scanning electron microscopy revealed that EV71 particles persisted for up to 10 days when incubated with the H. pylori biofilm. Furthermore, the presence of the H. pylori biofilm significantly augmented viral viability, as verified through virus plaque assays. Interestingly, the viability of EV71 was dependent on the quantity of H. pylori biofilm formation. Thus, two H. pylori strains able to generate large amounts of biofilm could facilitate EV71 viability for up to 17 days, whereas two other H. pylori strains that produced moderate or low quantities of biofilm could not prolong virus viability. It is interesting that biofilm contains N-acetyl-glucosamine and glycosaminoglycan, and that EV71 has binding affinity to cell-surface heparan sulfate glycosaminoglycan, which acts as an EV71 attachment receptor. The synergistic ability of H. pylori biofilm to promote EV71 viability for extended periods implies that H. pylori biofilm may serve as an additional pathway of EV71 transmission.

Background: The aim of this study was to evaluate the long-term sequelae and cognitive profiles resulting from severe hand, foot, and mouth disease (HFMD) with central nervous system (CNS) involvement. Methods: 294 HFMD cases were included in a retrospective follow-up study. Physical examinations were conducted. The Chinese Wechsler Preschool and Primary Scale of Intelligence, Fourth Edition (WPPSI-IV) was used to assess intelligence. Results: 58 mild HFMD cases and 99 severe HFMD cases with mild CNS involvement did not present any neurological sequelae. In comparison, the sequelae incidence for severe HFMD with more severe CNS complications was 50.0%. The proportion of full-scale intelligence quotient (FSIQ) impairment was 45.0%. In the 2:6–3:11 age group, severe HFMD with more severe CNS complications and lower maternal education level were risk factors for verbal comprehension disorder. Urban–rural residence and lower paternal education level were risk factors for FSIQ disorder. Furthermore, in the 4:0–6:11 age group, severe HFMD with more severe CNS complication was a risk factor for visual spatial disorder and fluid reasoning disorder. Lower paternal education level was a risk factor for FSIQ disorder. Conclusion: Early assessment and intervention among severe HFMD patients with more severe CNS involvement at a very young age will prove beneficial for their future performance.

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.

Herbal medicines contain single herb or many herbal ingredients derived from the plant and their byproducts. The beneficial effects of medicinal plants in the healthcare system are mainly due to the presence of secondary metabolites which have numerous biological applications in medicine. Chrysosplenetin is a polymethoxylated flavonoid class phytochemical found to be present in Artemisia annua L. and Dracocephalum heterophyllum Benth. Scientific research data of chrysosplenetin has been collected from various literature databases in the present work and analyzed for their medicinal properties and pharmacological activities. Medicinal importance and pharmacological activity data of chrysosplenetin has been collected from Google, Science Direct, PubMed, and Scopus and analyzed in the present work to know the therapeutic benefit of chrysosplenetin. However, the analytical data of chrysosplenetin has been also collected and analyzed in the present work to know the importance of modern analytical techniques in the separation, isolation, and identification of chrysosplenetin. Scientific data analysis of collected research work from various literature databases revealed the biological importance of chrysosplenetin in medicine. Pharmacological data of chrysosplenetin revealed the anti-malarial, antiproliferative, and antibacterial effect of chrysosplenetin. Chrysosplenetin has a significant effect on bone loss, enterovirus 71, trichomoniasis, tyrosinase enzyme, multidrug resistance, inflammatory, and neurological disorders. Pharmacokinetic parameters of chrysosplenetin have been also studied in the present work. Literature data analysis signified the potential role of different analytical techniques in medicine for their isolation, separation, and quantification. Scientific data analysis of the collected data of chrysosplenetin in the present work signified the therapeutic benefit of chrysosplenetin in medicine for the treatment of numerous diseases and associated complications.

Background: The enterovirus 71 (EV-A71) vaccine has been used in Hefei for several years, and the epidemiological significance of vaccination in this area is unclear. We aims to explore the spatial-temporal-demographic and virological changes of hand, foot and mouth disease (HFMD) after vaccination in China.Methods: The data for HFMD from 2012 to 2020 were downloaded with the help of HFMD reporting system of Hefei Center for Disease Control and Prevention and combined with the EV-A71 vaccination status in Hefei. The study defined the period between 2012 to 2016 as the pre-vaccination period and explored the effect of vaccination on the incidence of HFMD by comparing the changes of HFMD before and after vaccination in terms of spatial, temporal, demographic and virological aspects.Results: During the study period, a higher incidence occurred in urban area and the random distribution changed to a slight cluster after vaccination. HFMD incidence had inconsistent seasonality over years, with one or two incidence peaks in varying years. The morbidity decreased from 215.22/105 in 2012-2016 to 179.81/105 in 2017-2020 (p < 0.001). Boys, 0-4 years old children and Scattered children were more susceptible to HFMD compared with the others, the proportions decreased after vaccination except in Scattered children. The main pathogenic enterovirus gradually changed from EV-A71 to Other Enteroviruses, especially coxsackieviruses A6 (CV-A6) after the implementation of EV-A71 vaccination.Conclusions: The EV-A71 vaccine was effective in reducing the incidence of HFMD and changing the spatial, temporal, demographic, and virological characteristic. These changes should be considered during the vaccination implementation to further reduce the disease burden of HFMD. [ABSTRACT FROM AUTHOR]

Enterovirus 71 (EV71) is a dominant pathogenic agent that may cause severe central nervous system (CNS) diseases among infants and young children in the Asia-pacific. The inflammasome is closely implicated in EV71-induced CNS injuries through a series of signaling pathways. However, the activation pathway of NLRP3 inflammasome involved in EV71-mediated CNS injuries remains poorly defined. In the studies, EV71 infection, ERK1/2 phosphorylation, and activation of NLRP3 are abolished in glioblastoma cells with low vimentin expression by CRISPR/Cas9-mediated knockdown. PD098059, an inhibitor of p-ERK, remarkably blocks the vimentin-mediated ERK1/2 phosphorylation in EV71-infected cells. Nuclear translocation of NF-κB p65 is dependent on p-ERK in a time-dependent manner. Moreover, NLRP3 activation and caspase-1 production are limited in EV71-infected cells upon the caffeic acid phenethyl ester (CAPE) administration, an inhibitor of NF-κB, which contributes to the inflammasome regulation. In conclusion, these results suggest that EV71-mediated NLRP3 inflammasome could be activated via the VIM-ERK-NF-κB pathway, and the treatment of the dephosphorylation of ERK and NF-κB inhibitors is beneficial to host defense in EV71-infected CNS. [ABSTRACT FROM AUTHOR]

Biotransformation of specific saponins in the valuable medical plants to increase their bioavailability and pharmaceutical activities has attracted more and more attention. A gene encoding a thermophilic glycoside hydrolase from Fervidobaterium pennivorans DSM9078 was cloned and expressed in Escherichia coli. The purified recombinant enzyme, exhibiting endoglucanase cellulase activity, was used to transform gypenoside XLIX into gylongiposide I via highly selective and efficient hydrolysis of the glucose moiety linked to the C21 position in gypenoside XLIX. Under the optimal reaction conditions for large scale production of gylongiposide I, 35 g gypenoside XLIX was transformed by using 20 g crude enzyme at pH 6.0 and 80 °C for 4 h with a molar yield of 100%. Finally, 11.51 g of gylongiposide I was purified using a silica gel column with 91.84% chromatographic purity. Furthermore, inhibitory activities of gypenoside XLIX and gylongiposide I against Enterovirus 71 (EV71) were investigated. Importantly, the EC50 of gypenoside XLIX and gylongiposide I calculated from viral titers in supernatants was 3.53 μM and 1.53 μM, respectively. Moreover, the transformed product gylongiposide I has better anti-EV71 activity than the glycosylated precursor. In conclusion, this enzymatic method would be useful in the large-scale production of gylongiposide I, which would be a novel potent anti-EV71 candidate. [ABSTRACT FROM AUTHOR]

Enterovirus 71 (EV71) is one of the major pathogens of hand, foot, and mouth disease, which poses a major risk to public health and infant safety. 3C protease (3Cpro), a non-structural protein of EV71, promotes viral protein maturation by cleaving polyprotein precursors and facilitates viral immune escape by cleaving host proteins. In this study, we screened for human proteins that could interact with EV71 3Cpro using a yeast two-hybrid assay. Immune-associated protein TRAF3 Interacting Protein 3 (TRAF3IP3) was selected for further study. The results of co-immunoprecipitation and immunofluorescence demonstrated the interaction between TRAF3IP3 and EV71 3Cpro. A cleavage band was detected, indicating that both transfected 3Cpro and EV71 infection could cleave TRAF3IP3. 87Q-88G was identified as the only 3Cpro cleavage site in TRAF3IP3. In Jurkat and rhabdomyosarcoma (RD) cells, TRAF3IP3 inhibited EV71 replication, and 3Cpro cleavage partially resisted TRAF3IP3-induced inhibition. Additionally, the nuclear localization signal (NLS) and nuclear export signal (NES) of TRAF3IP3 were identified. The NES contributed to TRAF3IP3 alteration of 3Cpro localization and inhibition of EV71 replication. Together, these results indicate that TRAF3IP3 inhibits EV71 replication and 3Cpro resists such inhibition via proteolytic cleavage, providing a new example of virus-host interaction. [ABSTRACT FROM AUTHOR]

Abstract Background To investigate the relationship between polymorphism of scavenger receptor class B member 2 (SCARB2) gene and clinical severity of enterovirus (EV)-71 associated hand-foot-mouth disease (HFMD). Methods Among the 100 recruited cases, 56 were in the severe HFMD group (case group) and 44 were in the general HFMD group (control group). By screening functional single nucleotide polymorphisms (SNPs) and hot SNPs, and performing SNP site optimization, some SNP sites of SCARB2 gene were selected for analysis. Genotyping was performed using a MassArray platform. PLINK software was used for statistical processing and analysis of the correlation differences between the mutant genotypes in the severe and general HFMD groups. The relationship between the SNPs and clinical severity of enterovirus (EV)-71 associated HFMD was assessed. Results 28 SNPs in SCARB2 were selected by site optimization. Then three loci were not in agreement with the minor allele frequency (MAF) in the 1000 Han Chinese in Beijing (CHB) dataset. Another three loci could not be detected. Nine loci were not suitable for further analysis (MAF 1). Logistic regression analysis of rs6812193 T alleles between the severe and general HFMD groups, respectively (P 0.023 1, L95 1.210 > 1). Genotype logistic regression analysis of the rs6812193 alleles CT + TT versus CC gave an OR of 4.56 (95% confidence interval [95% CI] 1.22–17.04, P = 0.012). Conclusion The rs6812193 T allele was a susceptibility SNP for SHFMD, and the rs6812193 polymorphism might be significantly associated with the susceptibility to EV-71 infection.

Enterovirus 71 (EV71) is recognized as a major causative agent of hand, foot, and mouth disease (HFMD), posing a significant global public health concern due to its widespread impact and resulting in a major public health issue worldwide. Despite its prevalence, current clinical therapy lacks effective antiviral agents. Fucosylated chondroitin sulfates (FCS) derived from sea cucumber exhibits a range of biological activities including potent antiviral effects. This study provides compelling evidence of the potent antiviral efficacy of FCS against EV71. To further elucidate the impact of structural variations on the anti-EV71 activity, native FCSs with diverse sulfation patterns and a varity of FCS derivatives were prepared and analyzed. Notably, this study presents the detailed structural characterization of FCSs from the sea cucumbers Holothuria scabra Jaege and Holothuria fuscopunctata. Analysis of the structure-activity relationships revealed that molecular weight, sulfated fucose branches, and sulfation pattern were all crucial factors contributing to the potent inhibitory effects of FCS against EV71. Interestingly, molecular weight emerged as the most significant structural determinant of the antiviral potency. These findings suggest the promising potential of utilizing FCS as an innovative EV71 entry inhibitor for the treatment of HFMD. [Display omitted] [ABSTRACT FROM AUTHOR]

Enterovirus 71 (EV71) is one of the major pathogens of hand, foot, and mouth disease, which poses a major risk to public health and infant safety. 3C protease (3Cpro), a non-structural protein of EV71, promotes viral protein maturation by cleaving polyprotein precursors and facilitates viral immune escape by cleaving host proteins. In this study, we screened for human proteins that could interact with EV71 3Cpro using a yeast two-hybrid assay. Immune-associated protein TRAF3 Interacting Protein 3 (TRAF3IP3) was selected for further study. The results of co-immunoprecipitation and immunofluorescence demonstrated the interaction between TRAF3IP3 and EV71 3Cpro. A cleavage band was detected, indicating that both transfected 3Cpro and EV71 infection could cleave TRAF3IP3. 87Q-88G was identified as the only 3Cpro cleavage site in TRAF3IP3. In Jurkat and rhabdomyosarcoma (RD) cells, TRAF3IP3 inhibited EV71 replication, and 3Cpro cleavage partially resisted TRAF3IP3-induced inhibition. Additionally, the nuclear localization signal (NLS) and nuclear export signal (NES) of TRAF3IP3 were identified. The NES contributed to TRAF3IP3 alteration of 3Cpro localization and inhibition of EV71 replication. Together, these results indicate that TRAF3IP3 inhibits EV71 replication and 3Cpro resists such inhibition via proteolytic cleavage, providing a new example of virus-host interaction.

Background: Enterovirus 71 (EV71) infects millions of children every year in China and has become a challenge to public health. However, there is no effective treatment for EV71 infection. Long noncoding RNAs (lncRNAs) have been found to play various roles in virus replication and infection. Objective: We aimed to explore the role of a novel long noncoding RNA AK097647 (lncRNA-AK097647) during EV71 infection. Methods: To assess the role of lncRNA-AK097647 during EV71 infection, siRNAs were used to silence lncRNA-K097647 expression. RT-qPCR assay and Western blotting were applied to measure the mRNA and protein levels of EV71 VP1 and the phosphorylation of NF-κB. ELISA was used to detect the level of IFN-λ1 expression. Results: The novel lncRNA-AK097647 was upregulated in human rhabdomyosarcoma cells and the blood of hand, foot, and mouth disease patients infected with EV71, as demonstrated by RT-qPCR. Interestingly, RNAi-mediated knockdown of lncRNA-AK097647 dramatically increased the level of IFN-λ1 expression, resulting in the suppression of EV71 replication. In contrast, overexpression of lncRNA-AK097647 decreased the level of IFN-λ1 expression and resulted in increased EV71 replication. In addition, we found that lncRNA-AK097647 could inhibit the phosphorylation of NF-κB. Conclusion: These results suggest a novel mechanism by which EV71 evades the IFN-mediated host antiviral response by increasing lncRNA-AK097647 expression.

Objectives: To explore the mechanisms of interferon-induced transmembrane protein 3 (IFITM3) in response to enterovirus-71-associated hand, foot and mouth disease (EV71-HFMD), in terms of DNA methylation, single-nucleotide polymorphism (SNP) genotype and gene expression. Methods: In total, 120 patients with EV71-HFMD (60 with mild EV71-HFMD and 60 with severe EV71-HFMD) and 60 healthy controls were enrolled in this study. SNP genotype, IFITM3 promoter methylation and mRNA expression of peripheral blood mononuclear cells were examined using the improved multi-temperature ligase detection reaction, quantitative reverse transcriptase polymerase chain reaction and MiSeq, respectively. Results: The distribution of methylation in patients with EV71-HFMD was significantly lower compared with healthy controls, and the severe EV71-HFMD group showed the lowest frequency of IFITM3 promoter methylation. The average level of IFITM3 promoter CpG methylation was negatively correlated with IFITM3 mRNA expression, and hypermethylation of several specific CpG units contributed to IFITM3 downregulation. IFITM3 expression and promoter methylation correlated with EV71 infection progression, especially in the severe EV71-HFMD group. Compared with mild cases, genotype GG and the G allele of rs12252 were over-represented in patients with severe EV71-HFMD. Conclusions: IFITM3 methylation status and SNP genotyping may help clinicians to choose the correct treatment strategy for patients with EV71-HFMD.

Hand-foot-and-mouth disease (HFMD) is the most common enteroviral infection in South-East Asia. When evaluating the role of enterovirus 71 (EVA71) as an etiological agent of infectious disease in South Vietnam, we revealed a high proportion of EVA71 among identified species A enteroviruses found in 3542 samples from HFMD cases; 125 samples from cases of enteroviral meningitis; and 130 samples from acute flaccid paralysis (AFP) cases. These represent 50%, 54.8%, and 51.5%, respectively. According to molecular analysis, 90% of EVA71 were attributed to genotype C4 and 10% were attributed to genotype B5. The predominance of EVA71 circulation among the population proves the need to strengthen surveillance (with monitoring of enterovirus circulation for facilitation of HFMD outbreak prediction) and to increase the effectiveness of preventative measures by the implementation of vaccination against EVA71-associated infections. A phase III trial of a Taiwanese vaccine (EV71vac) in Taiwan and South Vietnam showed its safety, tolerability, and efficacy in children aged 2–71 months. This B4 genotype-based vaccine, which features cross-protection against B5 and C4 genotypes, and other existing EV71 vaccines can serve as a good approach to solving the HFMD problem, which is so important for Vietnam.

[Display omitted] • AST-IV is a food or dietary ingredient with excellent disease prevention potential. • AST-IV has been used to treat inflammation. • AST-IV inhibited EV71 induced-pyroptosis. • AST-IV inhibited EV71 induced-pyroptosis through the TCF12-TXNIP-Keap1/Nrf2 axis. This study aimed to investigate the inhibitory effect of astragaloside IV (AS-IV) on enterovirus 71 (EV71)-induced pyroptosis. The results indicated that AST-IV inhibited EV71-induced pyroptosis in normal human gastric epithelial (GES-1) cells. Therefore, we investigated the mechanisms underlying pyroptosis inhibition by AST-IV. We found that EV71 inhibited the Nrf2-Keap1 pathway, whereas AST-IV treatment activated it. Overexpression of Nrf2 led to the inhibition of EV71-induced pyroptosis. EV71 infection and AST-IV treatment upregulated and downregulated thioredoxin interacting protein (TXNIP), respectively. Downregulation of TXNIP activated the Keap1/Nrf2 signaling pathway and inhibited EV71-induced pyroptosis. Furthermore, transcription factor 12 (TCF12) reduced the protein and mRNA levels of TXNIP, and AST-IV treatment upregulated TCF12. These results demonstrate that the TCF12-TXNIP-Keap1/Nrf2 axis participates in the inhibition of pyroptosis by AST-IV in EV71-infected GES-1 cells, suggesting that AST-IV could be used as a dietary supplement to alleviate EV71-induced pyroptosis. [ABSTRACT FROM AUTHOR]

Enterovirus A71, a non-enveloped single-stranded (+) RNA virus, enters host cells through three stages: attachment, endocytosis and uncoating. In recent years, receptors/co-receptors anchored on the host cell membrane and involved in this process have been continuously identified. Among these, hSCARB-2 was the first receptor revealed to specifically bind to a definite site of the EV-A71 viral capsid and plays an indispensable role during viral entry. It actually acts as the main receptor due to its ability to recognize all EV-A71 strains. In addition, PSGL-1 is the second EV-A71 receptor discovered. Unlike hSCARB-2, PSGL-1 binding is strain-specific; only 20% of EV-A71 strains isolated to date are able to recognize and bind it. Some other receptors, such as sialylated glycan, Anx 2, HS, HSP90, vimentin, nucleolin and fibronectin, were discovered successively and considered as “co-receptors” because, without hSCARB-2 or PSGL-1, they are not able to mediate entry. For cypA, prohibitin and hWARS, whether they belong to the category of receptors or of co-receptors still needs further investigation. In fact, they have shown to exhibit an hSCARB-2-independent entry. All this information has gradually enriched our knowledge of EV-A71’s early stages of infection. In addition to the availability of receptors/co-receptors for EV-A71 on host cells, the complex interaction between the virus and host proteins and various intracellular signaling pathways that are intricately connected to each other is critical for a successful EV-A71 invasion and for escaping the attack of the immune system. However, a lot remains unknown about the EV-A71 entry process. Nevertheless, researchers have been continuously interested in developing EV-A71 entry inhibitors, as this study area offers a large number of targets. To date, important progress has been made toward the development of several inhibitors targeting: receptors/co-receptors, including their soluble forms and chemically designed compounds; virus capsids, such as capsid inhibitors designed on the VP1 capsid; compounds potentially interfering with related signaling pathways, such as MAPK-, IFN- and ATR-inhibitors; and other strategies, such as siRNA and monoclonal antibodies targeting entry. The present review summarizes these latest studies, which are undoubtedly of great significance in developing a novel therapeutic approach against EV-A71.

EV-A71 is a common viral pathogen that causes hand, foot and mouth disease. It is a single-stranded RNA virus that has a low fidelity RNA polymerase and, as a result, spontaneous mutations frequently occur in the EV-A71 genome. The mutations within the genome give rise to quasispecies within the viral population that could be further defined by haplotypes. In vitro virulence of EV-A71 was shown by plaque size in Rhabdomyosarcoma (RD) cells, which was substantiated by in vitro characterizations of growth, RNA replication, binding, attachment and host cell internalization. Viruses could exhibit different host cell adaptations in different cell lines during viral passaging. The EV-A71/WT (derived from EV-A71 subgenotype B4) was shown to comprise six haplotypes through next-generation sequencing, where only EV-A71/Hap2 was found to be cultivable in RD cells, while EV-A71/Hap4 was the only cultivable haplotype in Vero cells. The EV-A71/WT produced plaques of four different sizes (small, medium, big, huge) in RD cells, while only two plaque variants (small, medium) were present in Vero cells. The small plaque variant isolated from RD cells displayed lower RNA replication rates, slower in vitro growth kinetics, higher TCID50 and lower attachment, binding and entry ability when compared against EV-A71/WT due to the mutation at 3D-S228P that disrupted the active site of the RNA polymerase, resulting in low replication and growth of the variant.

As an important neurotropic enterovirus, enterovirus 71 (EV71) is occasionally associated with severe neurological diseases and high mortality rates in infants and young children. Understanding the interaction between host factors and EV71 will play a vital role in developing antivirals and optimizing vaccines. Here, we performed a genome-wide CRISPR-Cas9 knockout screen and revealed that scavenger receptor class B member 2 (SCARB2), solute carrier family 35 member B2 (SLC35B2), and beta-1,3-glucuronyltransferase 3 (B3GAT3) are essential in facilitating EV71 replication. Subsequently, the exploration of molecular mechanisms suggested that the knockout of SLC35B2 or B3GAT3, not SCARB2, led to a remarkable decrease in the binding of EV71 to cells and internalization into cells. Furthermore, we found that the infection efficiency for EV71 was positively correlated with the level of host cell sulfation, not simply with the amount of heparan sulfate, suggesting that an unidentified sulfated protein(s) must contribute to EV71 infection. In support of this idea, we screened possible sulfated proteins among the proteinous receptors for EV71 and confirmed that SCARB2 could uniquely interact with both tyrosyl protein sulfotransferases in humans. We then performed mass spectrometric analysis of SCARB2, identifying five sites with tyrosine sulfation. The function verification test indicated that there were more than five tyrosine-sulfated sites on SCARB2. Finally, we constructed a model for EV71 entry in which both heparan sulfate and SCARB2 are regulated by SLC35B2 and act cooperatively to support viral binding, internalization, and uncoating. Taken together, this is the first time that we performed the pooled CRISPRCas9 genetic screening to investigate the interplay of host cells and EV71. Furthermore, we found that a novel host factor, SLC35B2, played a dual role in regulating the overall sulfation comprising heparan sulfate sulfation and protein tyrosine sulfation, which are critical for EV71 entry. [ABSTRACT FROM AUTHOR]