In the present study,the complete genomes of four common(4/EV71/Wenzhou/CHN/2014,15/EV71/Wenzhou/CHN/2014,116/EV71/Wenzhou/CHN/2014,and 120/EV71/Wenzhou/CHN/2014)and two virulent(11/EV71/Wenzhou/CHN/2014and 109/EV7...In the present study,the complete genomes of four common(4/EV71/Wenzhou/CHN/2014,15/EV71/Wenzhou/CHN/2014,116/EV71/Wenzhou/CHN/2014,and 120/EV71/Wenzhou/CHN/2014)and two virulent(11/EV71/Wenzhou/CHN/2014and 109/EV71/Wenzhou/CHN/2014)enterovirus 71(EV71)isolates were sequenced and described.They are 7405 bp in length and belong to EV71 sub-genotype C4 (C4a cluster).展开更多
Enterovirus (EV71) can cause severe neurological diseases, but the underlying pathogenesis remains unclear. The capsid protein, viral protein 1 (VP1), plays a critical role in the pathogenicity of EVT1. High level...Enterovirus (EV71) can cause severe neurological diseases, but the underlying pathogenesis remains unclear. The capsid protein, viral protein 1 (VP1), plays a critical role in the pathogenicity of EVT1. High level expression and secretion ofVP 1 protein are necessary for structure, function and immunogenicity in its natural conformation. In our previous studies, 5 codon-optimized VP 1 DNA vaccines, including wt-VP 1, tPA-VP 1, VP l-d, VP 1-hFc and VP 1 - mFc, were constructed and analyzed. They expressed VP1 protein, but the levels of secretion and immunogenicity of these VP1 constructs were significantly different (P〈0.05). In this study, we further investigated the protein lev- els of these constructs and determined that all of these constructs expressed VP1 protein. The secretion level was increased by including a tPA leader sequence, which was further increased by fusing human IgG Fc (hFc) to VP1. VP 1-hFc demonstrated the most potent immunogenicity in mice. Furthermore, hFc domain could be used to purify VPI-hFc protein for additional studies.展开更多
Enterovirus 71 (EV71) and Coxsackievirus A16 (CoxA16) are the major pathogens causing hand, foot, and mouth disease (HFMD) that occurs primarily in children and infants with mild clinical manifestations. HFMD ca...Enterovirus 71 (EV71) and Coxsackievirus A16 (CoxA16) are the major pathogens causing hand, foot, and mouth disease (HFMD) that occurs primarily in children and infants with mild clinical manifestations. HFMD caused by EV71 could develop to a fatal neurological complication.展开更多
Current serum neutralization assays based on the inhibition of the cytopathic effect(Nt-CPE) need to ma nipulate live viruses, which are time-consuming, labor-intensive, and have the potential exposure to infectious...Current serum neutralization assays based on the inhibition of the cytopathic effect(Nt-CPE) need to ma nipulate live viruses, which are time-consuming, labor-intensive, and have the potential exposure to infectious agents, so a safe and objective assay via pseudovirus for the fast and efficient detection of enterovirus 71(EV71) neutralizing antibodies was developed. First, we generated EV71 pseudovirus containing firefly luciferase gene in place of the capsid gene P1 in EV71 genome. Vero cells infected with 200 CCID50(50% cell culture infective dose) of EV71 pseudovirus for 24 h were found to have the best performance. Seval sera were measured by EV71 pseudoparticle neutralization assay(Nt-PPN) and the conventional serological method Nt-CPE. Neutralizing antibody titers measured by Nt-PPN and those obtained by Nt-CPE demonstrate a high correlation between the two methods. Overall, the PPN assay represents a valid alternative to conventional serological methods for the evaluation of EV71 neutralizing anti bodies. This method can be used for detecting neutralizing antibodies of other picornaviruses, such as hepatitis A vi rus(HAV) and coxsackievirus 16(CVA16), and make it possible to determine whether there is cross-reactivity be tween EV71 and CVA16.展开更多
Capsid protein enterovirus 71 (EV71) is one of the major viruses that cause the severe encephalitis and thus result in a high mortality in children less than 5 years of age.In an effort to discover new potent inhibi...Capsid protein enterovirus 71 (EV71) is one of the major viruses that cause the severe encephalitis and thus result in a high mortality in children less than 5 years of age.In an effort to discover new potent inhibitors against EV71,a novel three-dimensional pharmacophore model was developed on 24 inhibitors with different molecular structures and bioactivities.The best hypothesis (Hypo1) has a high predictive power and consists of four features,namely,one hydrophobic point (HY) and three hydrogen-bond acceptors (HA).Two key features of the best Hypo1,HY1 and HA3 match well with an important narrow hydrophobic canyon and with the surface of LYS274 in the target EV71 active site,respectively.The more versatile feature,HA1,is firstly found to be very influential on these compounds’ bioactivities,which may interact with the other side of the active site in the EV71 receptor.The application of the model is successful in predicting the activities of 30 known EV71 inhibitors with a correlation coefficient of 0.831.Furthermore,Hypo1 demonstrates a superior screening capability for retrieving inhibitors from the database with a high enrichment factor of 70.This study provides some important clues in search for more potent inhibitors against EV71 infection.展开更多
Enterovirus 71(EV71)poses a serious threat to human health,with scattered outbreaks worldwide.There are several vaccines against a few EV71 strains but no efficient drug for the treatment of EV71 infection.Therefore,i...Enterovirus 71(EV71)poses a serious threat to human health,with scattered outbreaks worldwide.There are several vaccines against a few EV71 strains but no efficient drug for the treatment of EV71 infection.Therefore,it is urgent and of significance to develop anti-EV71 drugs.Here,we found that PLX8394,a RAF inhibitor,possesses high antiviral activity against EV71 in vitro,being superior to the traditional clinical drug ribavirin.Moreover,PLX8394 exhibits broad-spectrum antiviral activity against enteroviruses.Notably,in a suckling mouse model,PLX8394 provided a 70%protection rate for EV71-infected mice,reduced the viral load in liver and heart tissues,and relieved the inflammatory response.A mechanistic study showed that PLX8394 inhibited EV71 by suppressing the RAF/MEK/ERK signaling pathway.Thus,PLX8394 lays a foundation for the development of new drugs against EV71.展开更多
Background Hand,foot,and mouth disease caused by enterovirus 71(EV71)infection is prevalent in the Asia-Pacific region in recent years.Currently,no drug is available for the prevention and treatment of EV71 infection....Background Hand,foot,and mouth disease caused by enterovirus 71(EV71)infection is prevalent in the Asia-Pacific region in recent years.Currently,no drug is available for the prevention and treatment of EV71 infection.IMB-0523,a N-phenylbenzamide derivative,inhibits hepatitis B virus replication by upregulating the expression of APOBEC3G.In the present study,the effect of IMB-0523 on EV71 replication and related mechanism were investigated.Methods The cytotoxicity of IMB-0523 was determined by cell counting kit.Quantitative real-time polymerase chain reaction and Western blot assay were used to detect the effect of IMB-0523 on EV71 replication and related mechanism.Cytopathic effect assay was used to investigate the effect of IMB-0523 on different EV71 strains,coxsackievirus A16,and coxsackieviruses of group B.Results The results showed that IMB-0523 could dose-dependently inhibit EV71 replication.Preliminary mechanism studies showed that IMB-0523 could activate STAT3 signaling to upregulate the expression of interferon-stimulated genes to play an antiviral role.In addition,IMB-0523 inhibited the replication of different EV71 strains,coxsackievirus A16,and coxsackieviruses of group B.Conclusions IMB-0523 inhibits EV71 replication by activating the STAT3 signaling pathway to upregulate interferon-stimulated gene expression.IMB-0523 has broad-spectrum antiviral potential and may be used as a lead compound for the development of broad spectrum antiviral drugs.展开更多
Background:During Enterovirus type 71(EV71)infection,the structural viral protein 1(VP1)activates endoplasmic reticulum(ER)stress associated with peripheral myelin protein 22(PMP22)accumulation and induces autophagy.H...Background:During Enterovirus type 71(EV71)infection,the structural viral protein 1(VP1)activates endoplasmic reticulum(ER)stress associated with peripheral myelin protein 22(PMP22)accumulation and induces autophagy.However,the specific mechanism behind this process remains elusive.Methods:In this research,we used the VP1-overexpressing mouse Schwann cells(SCs)models co-transfected with a PMP22 silencing or Autocrine motility factor receptor(AMFR/gp78)overexpressing vector to explore the regulation of gp78 on PMP22 and its relationship with autophagy and apoptosis.Results:The activity of gp78 could be influenced by EV71-VP1,leading to a decrease in the ubiquitination and degradation of PMP22,resulting in PMP22 accumulation in ER.In VP1-overexpressing mouse SCs,all three ER stress sensors,including pancreatic endoplasmic reticulum kinase(PERK),activating transcription factor 6(ATF6)and inositol-requiring enzyme 1(IRE1)and the related downstream signals(C/EBP-homologous protein(CHOP)and Caspase 12)were activated,as well as the ER-resident chaperone Glucose-regulated protein 78(GRP78).In addition,VP1 upregulated the autophagy marker Microtubule-associated protein 1 light chain 3 beta(LC3B),while PMP22 silencing or gp78 overexpression reversed the phenomenon.Meanwhile,PMP22 silencing or gp78 overexpression increased proliferation of EV71-VP1-transfected mouse SCs.Conclusion:Gp78 could regulate PMP22 accumulation through ubiquitination degradation and cause ER stress and autophagy in EV71-VP1-overexpressing mouse SCs.Therefore,the gp78/PMP22/ER stress axis might emerge as a promising therapeutic target for myelin and neuronal damage induced by EV71 infection.展开更多
Background Enterovirus 71 (EV71) and coxsackievirus A16 (Cox A16) are major causative agents for hand, foot and mouth disease (HFMD). Studies indicate that the frequent HFMD outbreaks result in a few hundreds ch...Background Enterovirus 71 (EV71) and coxsackievirus A16 (Cox A16) are major causative agents for hand, foot and mouth disease (HFMD). Studies indicate that the frequent HFMD outbreaks result in a few hundreds children's death in China in recent years. The vaccine and other research for HFMD need to be developed urgently. The aims of our study were: to explore dynamic development of mother-source neutralizing antibodies against EV71 and Cox A16 in infants from Jiangsu Province, China, and to provide the fundamental data for further establishing of corresponding immunization course. Methods Peripheral blood samples were collected from 133 of parturient women once immediately before delivery and their infants at two and seven months of age. Method of micro-dose cytopathogenic effect was used to measure neutralizing antibodies against EV71 and Cox A16, respectively. Results Seropositive rates of anti-EV71 and anti-Cox A16 in prenatal women were 79.7% (106/133) and 92.5% (123/133), respectively; geometric mean titers (GMTs) were 29.0 and 61.9; 75.9% (101/133) prenatal women were both positive in anti-EV71 and anti-Cox A16; seropositive rates of anti-EV71 and anti-Cox A16 were 25.6% (34/133) and 38.3% (51/133) in infants at two months of age; GMTs were 12.3 and 18.0, respectively. GMTs of anti-EV71 were significantly higher for infants at seven months (82.6) compared with that at two months (P 〈0.05), showing infants had inapparently infected by EV71 during two to seven months. Although only one offspring (0.75%) at seven months was found having anti-Cox A16 transfered from maternal, this observation suggested no maternal antibody may remain in infants at seven months. Conclusions The prevalence of EV71 and Cox A16 were relatively high in Jiangsu Province. Bivalent vaccine against both EV71 and Cox A16 should be developed, and the ideal time point for prime immunization for infants is around 2-5 months of age.展开更多
Human enterovirus 71(EV71)is the main causative pathogen of hand,foot,and mouth disease(HFMD)in children.The epidemic of HFMD has been a public health problem in Asia-Pacific region for decades,and no vaccine and effe...Human enterovirus 71(EV71)is the main causative pathogen of hand,foot,and mouth disease(HFMD)in children.The epidemic of HFMD has been a public health problem in Asia-Pacific region for decades,and no vaccine and effective antiviral medicine are available.Curcumin has been used as a traditional medicine for centuries to treat a diversity of disorders including viral infections.In this study,we demonstrated that curcumin showed potent antiviral effect again EV71.In Vero cells infected with EV71,the addition of curcumin significantly suppressed the synthesis of viral RNA,the expression of viral protein,and the overall production of viral progeny.Similar with the previous reports,curcumin reduced the production of ROS induced by viral infection.However,the antioxidant property of curcumin did not contribute to its antiviral activity,since N-acetyl-L-cysteine,the potent antioxidant failed to suppress viral replication.This study also showed that extracellular signal-regulated kinase(ERK)was activated by either viral infection or curcumin treatment,but the activated ERK did not interfere with the antiviral effect of curcumin,indicating ERK is not involved in the antiviral mechanism of curcumin.Unlike the previous reports that curcumin inhibited protein degradation through ubiquitin–proteasome system(UPS),we found that curcumin had no impact on UPS in control cells.However,curcumin did reduce the activity of proteasomes which was increased by viral infection.In addition,the accumulation of the short-lived proteins,p53 and p21,was increased by the treatment of curcumin in EV71-infected cells.We further probed the antiviral mechanism of curcumin by examining the expression of GBF1 and PI4KB,both of which are required for the formation of viral replication complex.We found that curcumin significantly reduced the level of both proteins.Moreover,the decreased expression of either GBF1 or PI4KB by the application of siRNAs was sufficient to suppress viral replication.We also demonstrated that curcumin showed anti-apoptotic activity at the early stage of viral infection.The results of this study provide solid evidence that curcumin has potent anti-EV71 activity.Whether or not the down-regulated GBF1 and PI4KB by curcumin contribute to its antiviral effect needs further studies.展开更多
Background:Hand,foot,and mouth disease(HFMD)caused by enterovirus 71(EV71)is a potentially life-threatening infectious disease that commonly occurs in children.Diagnosis of HFMD caused by EV71 largely depends on clini...Background:Hand,foot,and mouth disease(HFMD)caused by enterovirus 71(EV71)is a potentially life-threatening infectious disease that commonly occurs in children.Diagnosis of HFMD caused by EV71 largely depends on clinical manifestations and rare serological biomarkers used to identify children suffering from HFMD.Serum cholinesterase(SChE)activity has frequently been reported as a potential biomarker for solid central nervous system tumors,chronic heart failure,and liver cirrhosis.However,its potential value in the diagnosis of neurotropic virus infections,such as HFMD caused by EV71,remains to be determined.Findings:In our study,220 children hospitalized with HFMD caused by EV71,34 inpatients infected with coxsackievirus A16(CVA16),and 43 undefined enterovirus-infected HFMD inpatients were recruited at the Anhui Provincial Children’s Hospital between January 2011 and December 2012.SChE activity was measured.The non-parametric Mann–Whitney U test showed that SChE activity in children diagnosed with HFMD caused by EV71 was significantly higher than in healthy controls(p<0.001),as well as in children with upper respiratory tract infections(p=0.011),bronchopneumonia(p<0.001),septicemia(p<0.001),amygdalitis(p<0.001),and appendicitis(p<0.001).In addition,higher SChE activity was observed in male inpatients with HFMD caused by EV71(47.7%positivity)compared to female inpatients(26.1%positivity)(chi-square test,p=0.002).In our study,no significant differences in SChE levels were observed among different ages(up to 120 months)(r=-0.112,p>0.05).An important finding was that SChE activity declined in the recovery phase of HFMD caused by EV71 compared to the acute phase(p<0.001).Conclusions:Elevated SChE activity was observed in patients with severe HFMD caused by EV71.Therefore,SChE might be a potential assistant biomarker for the diagnosis of HFMD caused by EV71 in children.展开更多
Similar to that of other enteroviruses, the replication of enterovirus 71(EV71) occurs on rearranged membranous structures called replication organelles(ROs). Phosphatidylinositol 4-kinase Ⅲ(PI4KB), which is required...Similar to that of other enteroviruses, the replication of enterovirus 71(EV71) occurs on rearranged membranous structures called replication organelles(ROs). Phosphatidylinositol 4-kinase Ⅲ(PI4KB), which is required by enteroviruses for RO formation, yields phosphatidylinositol-4-phosphate(PI4P) on ROs. PI4P then binds and induces conformational changes in the RNA-dependent RNA polymerase(Rd Rp) to modulate Rd Rp activity. Here, we targeted 3D polymerase, the core enzyme of EV71 ROs, and found that the host factor Annexin A2(ANXA2) can interact with 3 D polymerase and promote the replication of EV71. Then, an experiment showed that the annexin domain of ANXA2, which possesses membranebinding capacity, mediates the interaction of ANXA2 with EV71 3 D polymerase. Further research showed that ANXA2 is localized on ROs and interacts with PI4KB. Overexpression of ANXA2 stimulated the formation of PI4P, and the level of PI4P was decreased in ANXA2-knockout cells. Furthermore, ANXA2, PI4KB, and 3D were shown to be localized to the viral RNA replication site, where they form a higher-order protein complex, and the presence of ANXA2 promoted the PI4 KB-3D interaction. Altogether, our data provide new insight into the role of ANXA2 in facilitating formation of the EV71 RNA replication complex.展开更多
In this paper, cinobufagin and resibufogenin were found to inhibit enterovirus 71(EV71) infection in vitro in cell viability and plaque reduction assays. The 50% inhibitory concentrations(lCs0) of einobufagin and ...In this paper, cinobufagin and resibufogenin were found to inhibit enterovirus 71(EV71) infection in vitro in cell viability and plaque reduction assays. The 50% inhibitory concentrations(lCs0) of einobufagin and resibufoge- nin were (10.94±2.36) and (218±31) nmol/L, respectively, the 50% cytotoxic concentrations(CCs0) of them were (1277±223) and (1385±254) nmol/L, respectively, and the anti-EV71 selectivity index(SI50) of cinobufagin was 116.7, which are promisingly developed into drug. Using a VP1 detection assay and a constructed reporter luciferase, we found that cinobufagin and resibufogenin disrupted the synthesis of EV71 protein. However, neither of them inhibited EV71 RNA replication. Our study suggests that cinobufagin and resibufogenin are the promising candidates that should he fllrther investigated for the treatment of EV71 caused disease.展开更多
Five overlapping clones covering the full genome of Enterovirus71 China strain SHZH98 were obtained and then the sequences were determined by the chain termination method. It showed that the full length of EV71 SHZH98...Five overlapping clones covering the full genome of Enterovirus71 China strain SHZH98 were obtained and then the sequences were determined by the chain termination method. It showed that the full length of EV71 SHZH98 genome (not including Poly A tail) is 7408 bp. There are some diversities on the lengths and sequences of 5′ UTR and 3′ UTR between SHZH98 and the other EV71 strains. In P1 capsid region, which is closely associated with viral immunogenicity, EV71 strain SHZH98 shares the highest homology with Taiwan strains; but in P2 and P3 non-structural gene regions there are higher identities with Coxsakievirus A16 and EV71 strains MS, BrCr than with Taiwan strains. Phylogenetic tree constructed by structural gene region indicates that China strain SHZH98 has a closer relationship with Taiwan strains, however, in the non-coding region it has a closer relationship with Coxsakievirus A16, EV71 strains MS and BrCr. EV71 China strain was analyzed at the molecular level. The results will contribute to the basic study on enteroviruses and the EV71 prevention in China.展开更多
Objective:To explore which pattern recognition receptors(PRRs)play a key role in the development of hand,foot,and mouth disease(HFMD)by analyzing PRR-associated genes.Methods:We conducted a comparative analysis of PRR...Objective:To explore which pattern recognition receptors(PRRs)play a key role in the development of hand,foot,and mouth disease(HFMD)by analyzing PRR-associated genes.Methods:We conducted a comparative analysis of PRR-associated gene expression in human peripheral blood mononuclear cells(PBMCs)infected with enterovirus 71(EV-A71)which were derived from patients with HFMD of different severities and at different stages.A total of 30 PRR-associated genes were identified as significantly upregulated both over time and across different EV-A71 isolates.Subsequently,ELISA was employed to quantify the expression of the six most prominent genes among these 30 identified genes,specifically,BST2,IRF7,IFI16,TRIM21,MX1,and DDX58.Results:Compared with those at the recovery stage,the expression levels of BST2(P=0.027),IFI16(P=0.016),MX1(P=0.046)and DDX58(P=0.008)in the acute stage of infection were significantly upregulated,while no significant difference in the expression levels of IRF7(P=0.495)and TRIM21(P=0.071)was found between different stages of the disease.The expression levels of BST2,IRF7,IFI16 and MX1 were significantly higher in children infected with single pathogen than those infected with mixed pathogens,and BST2,IRF7,IFI16 and MX1 expression levels were significantly lower in coxsackie B virus(COXB)positive patients than the negative patients.Expression levels of one or more of BST2,IRF7,IFI16,TRIM21,MX1 and DDX58 genes were correlated with PCT levels,various white blood cell counts,and serum antibody levels that reflect disease course of HFMD.Aspartate aminotransferase was correlated with BST2,MX1 and DDX58 expression levels.Conclusions:PRR-associated genes likely initiate the immune response in patients at the acute stage of HFMD.展开更多
Objective:To investigate the relationship between the levels of plasma adrenaline and norepinephrine and gene polymorphism of β1 adrenergic receptor G1165 C in children with enterovirus 71(EV71) infection in hand foo...Objective:To investigate the relationship between the levels of plasma adrenaline and norepinephrine and gene polymorphism of β1 adrenergic receptor G1165 C in children with enterovirus 71(EV71) infection in hand foot and mouth disease(HFMD). Methods:The polymerase chain reaction(PCR) was used to detect the expression of gene polymorphism of β1 adrenergic receptor G1165 C in vitro. The levels of plasma adrenaline and norepinephrine were measured by enzyme-linked immunosorbent assay(ELISA). Results:The plasma norepinephrine level of severe group was significantly higher than the mild group in children with EV71 infection in HFMD(P<0.05); however,the levels of plasma adrenalinein in two groups had no statistical differences(P>0.05); There was no significant difference in the distribution of β1 adrenergic receptor G1165 C genotype and allele between EV71 infection group and healthy control group(P> 0.05). Further analysis of EV71 infection group by dividing it into mild and severe groups showed that there was no significant difference in the distribution of genotype and allele between these two groups as well(P> 0.05). There was no significant difference in the levels of epinephrine and norepinephrine in different genotypes of EV71 infection group(P> 0.05),and in the levels of plasma epinephrine and norepinephrine in the mild and severe groups(P> 0.05). Conclusions:As the disease gets worse,the plasma norepinephrine level has a rising trend in children with EV71 infection in HFMD,which is an important indicator to evaluate the progress of the disease. However,the gene polymorphism of eptor G1165 C have no significant correlation,not only with the susceptibility and severit β1 adrenergic recy of EV71 infection in hand,foot and mouth disease,but also with the levels of catecholamine.展开更多
Objective To eliminate the side effects of aluminum adjuvant and His-tag,we constructed chimeric VLPs displaying the epitope of EV71(SP70) without His-tagged.Then evaluating whether the VLPs could efficiently evoke ...Objective To eliminate the side effects of aluminum adjuvant and His-tag,we constructed chimeric VLPs displaying the epitope of EV71(SP70) without His-tagged.Then evaluating whether the VLPs could efficiently evoke not only humoral but also cellular immune responses against EV71 without adjuvant.Methods The fusion protein was constructed by inserting SP70 into the MIR of truncated HBc Ag sequence,expressed in E.Coli,and purified through ion exchange chromatography and density gradient centrifugation.Mice were immunized with the VLPs and sera were collected afterwards.The specific antibody titers,Ig G subtypes and neutralizing efficacy were detected by ELISA,neutralization assay,and EV71 lethal challenge.IFN-γ and IL-4 secreted by splenocytes were tested by ELISPOT assay.Results HBc-SP70 proteins can self-assemble into empty VLPs.After immunization with HBc-SP70 VLPs,the detectable anti-EV71 antibodies were effective in neutralizing EV71 and protected newborn mice from EV71 lethal challenge.There was no significant difference for the immune efficacy whether the aluminum adjuvant was added or not.The specific Ig G subtypes were mainly IgG1 and IgG2 b and splenocytes from the mice immunized produced high levels of IFN-γ and IL-4.Conclusion The fusion proteins without His-tagged was expressed and purified as soluble chimeric HBc-SP70 VLPs without renaturation.In the absence of adjuvant,they were efficient to elicit high levels of Th1/Th2 mixed immune response as well as assisted by aluminum adjuvant.Furthermore,the chimeric VLPs have potential to prevent HBV and EV71 infection simultaneously.展开更多
基金funded by Natural Science Foundation of Zhejiang(LQ14C010006)National Natural Science Foundation of China(81501363)Planned Science and Technology Project of Zhejiang(2014C33261)
文摘In the present study,the complete genomes of four common(4/EV71/Wenzhou/CHN/2014,15/EV71/Wenzhou/CHN/2014,116/EV71/Wenzhou/CHN/2014,and 120/EV71/Wenzhou/CHN/2014)and two virulent(11/EV71/Wenzhou/CHN/2014and 109/EV71/Wenzhou/CHN/2014)enterovirus 71(EV71)isolates were sequenced and described.They are 7405 bp in length and belong to EV71 sub-genotype C4 (C4a cluster).
基金supported by the National Natural Science Foundation of China(Grant No.81000725 and 31470889)the Priority Academic Program of Basic Medical Science of Nanjing Medical University(Grant No.JX10131801060)
文摘Enterovirus (EV71) can cause severe neurological diseases, but the underlying pathogenesis remains unclear. The capsid protein, viral protein 1 (VP1), plays a critical role in the pathogenicity of EVT1. High level expression and secretion ofVP 1 protein are necessary for structure, function and immunogenicity in its natural conformation. In our previous studies, 5 codon-optimized VP 1 DNA vaccines, including wt-VP 1, tPA-VP 1, VP l-d, VP 1-hFc and VP 1 - mFc, were constructed and analyzed. They expressed VP1 protein, but the levels of secretion and immunogenicity of these VP1 constructs were significantly different (P〈0.05). In this study, we further investigated the protein lev- els of these constructs and determined that all of these constructs expressed VP1 protein. The secretion level was increased by including a tPA leader sequence, which was further increased by fusing human IgG Fc (hFc) to VP1. VP 1-hFc demonstrated the most potent immunogenicity in mice. Furthermore, hFc domain could be used to purify VPI-hFc protein for additional studies.
基金supported by the National Natural Science of China(81371833)Shandong Province Key Research and Development Plan(2015GGB2400)
文摘Enterovirus 71 (EV71) and Coxsackievirus A16 (CoxA16) are the major pathogens causing hand, foot, and mouth disease (HFMD) that occurs primarily in children and infants with mild clinical manifestations. HFMD caused by EV71 could develop to a fatal neurological complication.
基金Supported by the National Natural Science Foundation of China(No.20872048)
文摘Current serum neutralization assays based on the inhibition of the cytopathic effect(Nt-CPE) need to ma nipulate live viruses, which are time-consuming, labor-intensive, and have the potential exposure to infectious agents, so a safe and objective assay via pseudovirus for the fast and efficient detection of enterovirus 71(EV71) neutralizing antibodies was developed. First, we generated EV71 pseudovirus containing firefly luciferase gene in place of the capsid gene P1 in EV71 genome. Vero cells infected with 200 CCID50(50% cell culture infective dose) of EV71 pseudovirus for 24 h were found to have the best performance. Seval sera were measured by EV71 pseudoparticle neutralization assay(Nt-PPN) and the conventional serological method Nt-CPE. Neutralizing antibody titers measured by Nt-PPN and those obtained by Nt-CPE demonstrate a high correlation between the two methods. Overall, the PPN assay represents a valid alternative to conventional serological methods for the evaluation of EV71 neutralizing anti bodies. This method can be used for detecting neutralizing antibodies of other picornaviruses, such as hepatitis A vi rus(HAV) and coxsackievirus 16(CVA16), and make it possible to determine whether there is cross-reactivity be tween EV71 and CVA16.
基金Sponsored by the National Natural Science Foundation of China (No. 30800719)
文摘Capsid protein enterovirus 71 (EV71) is one of the major viruses that cause the severe encephalitis and thus result in a high mortality in children less than 5 years of age.In an effort to discover new potent inhibitors against EV71,a novel three-dimensional pharmacophore model was developed on 24 inhibitors with different molecular structures and bioactivities.The best hypothesis (Hypo1) has a high predictive power and consists of four features,namely,one hydrophobic point (HY) and three hydrogen-bond acceptors (HA).Two key features of the best Hypo1,HY1 and HA3 match well with an important narrow hydrophobic canyon and with the surface of LYS274 in the target EV71 active site,respectively.The more versatile feature,HA1,is firstly found to be very influential on these compounds’ bioactivities,which may interact with the other side of the active site in the EV71 receptor.The application of the model is successful in predicting the activities of 30 known EV71 inhibitors with a correlation coefficient of 0.831.Furthermore,Hypo1 demonstrates a superior screening capability for retrieving inhibitors from the database with a high enrichment factor of 70.This study provides some important clues in search for more potent inhibitors against EV71 infection.
基金supported by grants from the National Key Research and Development Plan of China(Grant No.2021YFC2300700)the China Postdoctoral Science Foundation(No.2021M693363)the grants from Hubei Health Commission(No.WJ2021M027).
文摘Enterovirus 71(EV71)poses a serious threat to human health,with scattered outbreaks worldwide.There are several vaccines against a few EV71 strains but no efficient drug for the treatment of EV71 infection.Therefore,it is urgent and of significance to develop anti-EV71 drugs.Here,we found that PLX8394,a RAF inhibitor,possesses high antiviral activity against EV71 in vitro,being superior to the traditional clinical drug ribavirin.Moreover,PLX8394 exhibits broad-spectrum antiviral activity against enteroviruses.Notably,in a suckling mouse model,PLX8394 provided a 70%protection rate for EV71-infected mice,reduced the viral load in liver and heart tissues,and relieved the inflammatory response.A mechanistic study showed that PLX8394 inhibited EV71 by suppressing the RAF/MEK/ERK signaling pathway.Thus,PLX8394 lays a foundation for the development of new drugs against EV71.
基金CAMS Innovation Fund for Medical Sciences(2021-I2M-1-030)National Science and Technology Major Projects for"Major New Drugs Innovation and Development"(2018ZX09711003).
文摘Background Hand,foot,and mouth disease caused by enterovirus 71(EV71)infection is prevalent in the Asia-Pacific region in recent years.Currently,no drug is available for the prevention and treatment of EV71 infection.IMB-0523,a N-phenylbenzamide derivative,inhibits hepatitis B virus replication by upregulating the expression of APOBEC3G.In the present study,the effect of IMB-0523 on EV71 replication and related mechanism were investigated.Methods The cytotoxicity of IMB-0523 was determined by cell counting kit.Quantitative real-time polymerase chain reaction and Western blot assay were used to detect the effect of IMB-0523 on EV71 replication and related mechanism.Cytopathic effect assay was used to investigate the effect of IMB-0523 on different EV71 strains,coxsackievirus A16,and coxsackieviruses of group B.Results The results showed that IMB-0523 could dose-dependently inhibit EV71 replication.Preliminary mechanism studies showed that IMB-0523 could activate STAT3 signaling to upregulate the expression of interferon-stimulated genes to play an antiviral role.In addition,IMB-0523 inhibited the replication of different EV71 strains,coxsackievirus A16,and coxsackieviruses of group B.Conclusions IMB-0523 inhibits EV71 replication by activating the STAT3 signaling pathway to upregulate interferon-stimulated gene expression.IMB-0523 has broad-spectrum antiviral potential and may be used as a lead compound for the development of broad spectrum antiviral drugs.
基金The study was supported by Guangdong Natural Science Foundation(Grant Numbers 2020A1515010014,2022A1515012411)Science and Technology Key Project for People’s Livelihood of Guangzhou,China(Grant Number 202206010060)+1 种基金Guangzhou Science and Technology Bureau Basic Research Project(SL2024A03J01288)Innovative Project of Children’s Research Institute,Guangzhou Women and Children’s Medical Center,China(Grant Numbers Pre-NSFC-2019-002,NKE PRE-2019-015).
文摘Background:During Enterovirus type 71(EV71)infection,the structural viral protein 1(VP1)activates endoplasmic reticulum(ER)stress associated with peripheral myelin protein 22(PMP22)accumulation and induces autophagy.However,the specific mechanism behind this process remains elusive.Methods:In this research,we used the VP1-overexpressing mouse Schwann cells(SCs)models co-transfected with a PMP22 silencing or Autocrine motility factor receptor(AMFR/gp78)overexpressing vector to explore the regulation of gp78 on PMP22 and its relationship with autophagy and apoptosis.Results:The activity of gp78 could be influenced by EV71-VP1,leading to a decrease in the ubiquitination and degradation of PMP22,resulting in PMP22 accumulation in ER.In VP1-overexpressing mouse SCs,all three ER stress sensors,including pancreatic endoplasmic reticulum kinase(PERK),activating transcription factor 6(ATF6)and inositol-requiring enzyme 1(IRE1)and the related downstream signals(C/EBP-homologous protein(CHOP)and Caspase 12)were activated,as well as the ER-resident chaperone Glucose-regulated protein 78(GRP78).In addition,VP1 upregulated the autophagy marker Microtubule-associated protein 1 light chain 3 beta(LC3B),while PMP22 silencing or gp78 overexpression reversed the phenomenon.Meanwhile,PMP22 silencing or gp78 overexpression increased proliferation of EV71-VP1-transfected mouse SCs.Conclusion:Gp78 could regulate PMP22 accumulation through ubiquitination degradation and cause ER stress and autophagy in EV71-VP1-overexpressing mouse SCs.Therefore,the gp78/PMP22/ER stress axis might emerge as a promising therapeutic target for myelin and neuronal damage induced by EV71 infection.
文摘Background Enterovirus 71 (EV71) and coxsackievirus A16 (Cox A16) are major causative agents for hand, foot and mouth disease (HFMD). Studies indicate that the frequent HFMD outbreaks result in a few hundreds children's death in China in recent years. The vaccine and other research for HFMD need to be developed urgently. The aims of our study were: to explore dynamic development of mother-source neutralizing antibodies against EV71 and Cox A16 in infants from Jiangsu Province, China, and to provide the fundamental data for further establishing of corresponding immunization course. Methods Peripheral blood samples were collected from 133 of parturient women once immediately before delivery and their infants at two and seven months of age. Method of micro-dose cytopathogenic effect was used to measure neutralizing antibodies against EV71 and Cox A16, respectively. Results Seropositive rates of anti-EV71 and anti-Cox A16 in prenatal women were 79.7% (106/133) and 92.5% (123/133), respectively; geometric mean titers (GMTs) were 29.0 and 61.9; 75.9% (101/133) prenatal women were both positive in anti-EV71 and anti-Cox A16; seropositive rates of anti-EV71 and anti-Cox A16 were 25.6% (34/133) and 38.3% (51/133) in infants at two months of age; GMTs were 12.3 and 18.0, respectively. GMTs of anti-EV71 were significantly higher for infants at seven months (82.6) compared with that at two months (P 〈0.05), showing infants had inapparently infected by EV71 during two to seven months. Although only one offspring (0.75%) at seven months was found having anti-Cox A16 transfered from maternal, this observation suggested no maternal antibody may remain in infants at seven months. Conclusions The prevalence of EV71 and Cox A16 were relatively high in Jiangsu Province. Bivalent vaccine against both EV71 and Cox A16 should be developed, and the ideal time point for prime immunization for infants is around 2-5 months of age.
基金This work was supported by the grants of National Natural Science Foundation of China to Zhaohua Zhong(Grant No.81271825)Wenran Zhao(Grant No.31270198).
文摘Human enterovirus 71(EV71)is the main causative pathogen of hand,foot,and mouth disease(HFMD)in children.The epidemic of HFMD has been a public health problem in Asia-Pacific region for decades,and no vaccine and effective antiviral medicine are available.Curcumin has been used as a traditional medicine for centuries to treat a diversity of disorders including viral infections.In this study,we demonstrated that curcumin showed potent antiviral effect again EV71.In Vero cells infected with EV71,the addition of curcumin significantly suppressed the synthesis of viral RNA,the expression of viral protein,and the overall production of viral progeny.Similar with the previous reports,curcumin reduced the production of ROS induced by viral infection.However,the antioxidant property of curcumin did not contribute to its antiviral activity,since N-acetyl-L-cysteine,the potent antioxidant failed to suppress viral replication.This study also showed that extracellular signal-regulated kinase(ERK)was activated by either viral infection or curcumin treatment,but the activated ERK did not interfere with the antiviral effect of curcumin,indicating ERK is not involved in the antiviral mechanism of curcumin.Unlike the previous reports that curcumin inhibited protein degradation through ubiquitin–proteasome system(UPS),we found that curcumin had no impact on UPS in control cells.However,curcumin did reduce the activity of proteasomes which was increased by viral infection.In addition,the accumulation of the short-lived proteins,p53 and p21,was increased by the treatment of curcumin in EV71-infected cells.We further probed the antiviral mechanism of curcumin by examining the expression of GBF1 and PI4KB,both of which are required for the formation of viral replication complex.We found that curcumin significantly reduced the level of both proteins.Moreover,the decreased expression of either GBF1 or PI4KB by the application of siRNAs was sufficient to suppress viral replication.We also demonstrated that curcumin showed anti-apoptotic activity at the early stage of viral infection.The results of this study provide solid evidence that curcumin has potent anti-EV71 activity.Whether or not the down-regulated GBF1 and PI4KB by curcumin contribute to its antiviral effect needs further studies.
基金supported by the National Natural Science Foundation of China(No:81271826).
文摘Background:Hand,foot,and mouth disease(HFMD)caused by enterovirus 71(EV71)is a potentially life-threatening infectious disease that commonly occurs in children.Diagnosis of HFMD caused by EV71 largely depends on clinical manifestations and rare serological biomarkers used to identify children suffering from HFMD.Serum cholinesterase(SChE)activity has frequently been reported as a potential biomarker for solid central nervous system tumors,chronic heart failure,and liver cirrhosis.However,its potential value in the diagnosis of neurotropic virus infections,such as HFMD caused by EV71,remains to be determined.Findings:In our study,220 children hospitalized with HFMD caused by EV71,34 inpatients infected with coxsackievirus A16(CVA16),and 43 undefined enterovirus-infected HFMD inpatients were recruited at the Anhui Provincial Children’s Hospital between January 2011 and December 2012.SChE activity was measured.The non-parametric Mann–Whitney U test showed that SChE activity in children diagnosed with HFMD caused by EV71 was significantly higher than in healthy controls(p<0.001),as well as in children with upper respiratory tract infections(p=0.011),bronchopneumonia(p<0.001),septicemia(p<0.001),amygdalitis(p<0.001),and appendicitis(p<0.001).In addition,higher SChE activity was observed in male inpatients with HFMD caused by EV71(47.7%positivity)compared to female inpatients(26.1%positivity)(chi-square test,p=0.002).In our study,no significant differences in SChE levels were observed among different ages(up to 120 months)(r=-0.112,p>0.05).An important finding was that SChE activity declined in the recovery phase of HFMD caused by EV71 compared to the acute phase(p<0.001).Conclusions:Elevated SChE activity was observed in patients with severe HFMD caused by EV71.Therefore,SChE might be a potential assistant biomarker for the diagnosis of HFMD caused by EV71 in children.
基金This study was supported by the National Science Foundation of China(81971976,81772236)Major Project of Technology Innovation Program of Hubei Province(2018ACA123)。
文摘Similar to that of other enteroviruses, the replication of enterovirus 71(EV71) occurs on rearranged membranous structures called replication organelles(ROs). Phosphatidylinositol 4-kinase Ⅲ(PI4KB), which is required by enteroviruses for RO formation, yields phosphatidylinositol-4-phosphate(PI4P) on ROs. PI4P then binds and induces conformational changes in the RNA-dependent RNA polymerase(Rd Rp) to modulate Rd Rp activity. Here, we targeted 3D polymerase, the core enzyme of EV71 ROs, and found that the host factor Annexin A2(ANXA2) can interact with 3 D polymerase and promote the replication of EV71. Then, an experiment showed that the annexin domain of ANXA2, which possesses membranebinding capacity, mediates the interaction of ANXA2 with EV71 3 D polymerase. Further research showed that ANXA2 is localized on ROs and interacts with PI4KB. Overexpression of ANXA2 stimulated the formation of PI4P, and the level of PI4P was decreased in ANXA2-knockout cells. Furthermore, ANXA2, PI4KB, and 3D were shown to be localized to the viral RNA replication site, where they form a higher-order protein complex, and the presence of ANXA2 promoted the PI4 KB-3D interaction. Altogether, our data provide new insight into the role of ANXA2 in facilitating formation of the EV71 RNA replication complex.
文摘In this paper, cinobufagin and resibufogenin were found to inhibit enterovirus 71(EV71) infection in vitro in cell viability and plaque reduction assays. The 50% inhibitory concentrations(lCs0) of einobufagin and resibufoge- nin were (10.94±2.36) and (218±31) nmol/L, respectively, the 50% cytotoxic concentrations(CCs0) of them were (1277±223) and (1385±254) nmol/L, respectively, and the anti-EV71 selectivity index(SI50) of cinobufagin was 116.7, which are promisingly developed into drug. Using a VP1 detection assay and a constructed reporter luciferase, we found that cinobufagin and resibufogenin disrupted the synthesis of EV71 protein. However, neither of them inhibited EV71 RNA replication. Our study suggests that cinobufagin and resibufogenin are the promising candidates that should he fllrther investigated for the treatment of EV71 caused disease.
基金the National Natural Science Foundation of China (Grant No.39823002).
文摘Five overlapping clones covering the full genome of Enterovirus71 China strain SHZH98 were obtained and then the sequences were determined by the chain termination method. It showed that the full length of EV71 SHZH98 genome (not including Poly A tail) is 7408 bp. There are some diversities on the lengths and sequences of 5′ UTR and 3′ UTR between SHZH98 and the other EV71 strains. In P1 capsid region, which is closely associated with viral immunogenicity, EV71 strain SHZH98 shares the highest homology with Taiwan strains; but in P2 and P3 non-structural gene regions there are higher identities with Coxsakievirus A16 and EV71 strains MS, BrCr than with Taiwan strains. Phylogenetic tree constructed by structural gene region indicates that China strain SHZH98 has a closer relationship with Taiwan strains, however, in the non-coding region it has a closer relationship with Coxsakievirus A16, EV71 strains MS and BrCr. EV71 China strain was analyzed at the molecular level. The results will contribute to the basic study on enteroviruses and the EV71 prevention in China.
文摘Objective:To explore which pattern recognition receptors(PRRs)play a key role in the development of hand,foot,and mouth disease(HFMD)by analyzing PRR-associated genes.Methods:We conducted a comparative analysis of PRR-associated gene expression in human peripheral blood mononuclear cells(PBMCs)infected with enterovirus 71(EV-A71)which were derived from patients with HFMD of different severities and at different stages.A total of 30 PRR-associated genes were identified as significantly upregulated both over time and across different EV-A71 isolates.Subsequently,ELISA was employed to quantify the expression of the six most prominent genes among these 30 identified genes,specifically,BST2,IRF7,IFI16,TRIM21,MX1,and DDX58.Results:Compared with those at the recovery stage,the expression levels of BST2(P=0.027),IFI16(P=0.016),MX1(P=0.046)and DDX58(P=0.008)in the acute stage of infection were significantly upregulated,while no significant difference in the expression levels of IRF7(P=0.495)and TRIM21(P=0.071)was found between different stages of the disease.The expression levels of BST2,IRF7,IFI16 and MX1 were significantly higher in children infected with single pathogen than those infected with mixed pathogens,and BST2,IRF7,IFI16 and MX1 expression levels were significantly lower in coxsackie B virus(COXB)positive patients than the negative patients.Expression levels of one or more of BST2,IRF7,IFI16,TRIM21,MX1 and DDX58 genes were correlated with PCT levels,various white blood cell counts,and serum antibody levels that reflect disease course of HFMD.Aspartate aminotransferase was correlated with BST2,MX1 and DDX58 expression levels.Conclusions:PRR-associated genes likely initiate the immune response in patients at the acute stage of HFMD.
基金supported by the Research Projects of Hainan Province Health Planning Industry(grant numbers:2012ZD-03)
文摘Objective:To investigate the relationship between the levels of plasma adrenaline and norepinephrine and gene polymorphism of β1 adrenergic receptor G1165 C in children with enterovirus 71(EV71) infection in hand foot and mouth disease(HFMD). Methods:The polymerase chain reaction(PCR) was used to detect the expression of gene polymorphism of β1 adrenergic receptor G1165 C in vitro. The levels of plasma adrenaline and norepinephrine were measured by enzyme-linked immunosorbent assay(ELISA). Results:The plasma norepinephrine level of severe group was significantly higher than the mild group in children with EV71 infection in HFMD(P<0.05); however,the levels of plasma adrenalinein in two groups had no statistical differences(P>0.05); There was no significant difference in the distribution of β1 adrenergic receptor G1165 C genotype and allele between EV71 infection group and healthy control group(P> 0.05). Further analysis of EV71 infection group by dividing it into mild and severe groups showed that there was no significant difference in the distribution of genotype and allele between these two groups as well(P> 0.05). There was no significant difference in the levels of epinephrine and norepinephrine in different genotypes of EV71 infection group(P> 0.05),and in the levels of plasma epinephrine and norepinephrine in the mild and severe groups(P> 0.05). Conclusions:As the disease gets worse,the plasma norepinephrine level has a rising trend in children with EV71 infection in HFMD,which is an important indicator to evaluate the progress of the disease. However,the gene polymorphism of eptor G1165 C have no significant correlation,not only with the susceptibility and severit β1 adrenergic recy of EV71 infection in hand,foot and mouth disease,but also with the levels of catecholamine.
基金supported by the National Science-technology Support Plan Projects 'The development of EV71 genetic engineering vaccine'[2008BAI69B02]
文摘Objective To eliminate the side effects of aluminum adjuvant and His-tag,we constructed chimeric VLPs displaying the epitope of EV71(SP70) without His-tagged.Then evaluating whether the VLPs could efficiently evoke not only humoral but also cellular immune responses against EV71 without adjuvant.Methods The fusion protein was constructed by inserting SP70 into the MIR of truncated HBc Ag sequence,expressed in E.Coli,and purified through ion exchange chromatography and density gradient centrifugation.Mice were immunized with the VLPs and sera were collected afterwards.The specific antibody titers,Ig G subtypes and neutralizing efficacy were detected by ELISA,neutralization assay,and EV71 lethal challenge.IFN-γ and IL-4 secreted by splenocytes were tested by ELISPOT assay.Results HBc-SP70 proteins can self-assemble into empty VLPs.After immunization with HBc-SP70 VLPs,the detectable anti-EV71 antibodies were effective in neutralizing EV71 and protected newborn mice from EV71 lethal challenge.There was no significant difference for the immune efficacy whether the aluminum adjuvant was added or not.The specific Ig G subtypes were mainly IgG1 and IgG2 b and splenocytes from the mice immunized produced high levels of IFN-γ and IL-4.Conclusion The fusion proteins without His-tagged was expressed and purified as soluble chimeric HBc-SP70 VLPs without renaturation.In the absence of adjuvant,they were efficient to elicit high levels of Th1/Th2 mixed immune response as well as assisted by aluminum adjuvant.Furthermore,the chimeric VLPs have potential to prevent HBV and EV71 infection simultaneously.