Hand foot and mouth disease is a febrile sickness complex characterized by cutaneous eruption (exanthem) on the palms and soles with simultaneous occurrence of muco-cutanous vesiculo-ulcerative lesions (enanthem) affe...Hand foot and mouth disease is a febrile sickness complex characterized by cutaneous eruption (exanthem) on the palms and soles with simultaneous occurrence of muco-cutanous vesiculo-ulcerative lesions (enanthem) affecting the mouth. The illness is caused by a number of enteroviruses with coxsackievirus A16 and enterovirus 71 as the main causative agents. Human enterovirus 71 (EV71) belongs to the species Human enterovirus A under the genus Enterovirus within the family Picornaviridae. EV71 has been associated with an array of clinical diseases including hand foot and mouth disease (HFMD), aseptic meningitis, encephalitis and poliomyelitis-like acute flaccid paralysis. A large outbreak of HFMD due to highly neurovirulent EV71 emerged in Malaysia in 1997, and caused 41 deaths amongst young children. In late 2000, a recurrence of an outbreak of HFMD occurred in Malaysia with 8 fatalities in peninsular Malaysia. Outbreak of HFMD due to EV71 recurred in 2003 with an unknown number of cases and mortalities. A similar outbreak of HFMD with 2 recorded deaths in young children occurred in peninsular Malaysia in late 2005 and this was followed by a larger outbreak in Sarawak (Malaysian Borneo) with 6 reported fatalities in the early part of 2006. The current on-going outbreak of HFMD started in peninsular Malaysia in epidemiological week 12 of 2010. As with other HFMD outbreaks in Malaysia, both EV71 and CA16 were the main aetiological viruses isolated. In similarity with the HFMD outbreak in 2005, the isolation of CA16 preceded the appearance of EV71. Based on the VP1 gene nucleotide sequences, 4 sub-genogroups of EV71 (C1, C2, B3 and B4) co-circulated and caused the outbreak of hand, foot and mouth disease in peninsular Malaysia in 1997. Two sub-genogroups (C1 and B4) were noted to cause the outbreak in 2000 in both peninsular Malaysia and Sarawak. EV71 of sub-genogroup B5 with smaller contribution from sub-genogroup C1 caused the outbreak in 2003. In the 2005 outbreak, besides the EV71 strains of sub-genogroup C1, EV71 strains belonging to sub-genogroup B5 were isolated but formed a cluster which was distinct from the EV71 strains from the sub-genogroup B5 isolated in 2003. The four EV71 strains isolated from clinical specimens of patients with hand, foot and mouth disease in the Sarawak outbreak in early 2006 also belonged to sub-genogroup B5. Phylogenetic analysis of the VP1 gene suggests that the EV71 strains causing the outbreak in Sarawak could have originated from peninsular Malaysia. Epidemiological and molecular data since 1997 show the recurrence of HFMD due to EV71 in Malaysia every 2 to 4 years. In each of the past outbreaks, more than one sub-genogroup of the virus co-circulate.展开更多
Enterovirus 71 （EV71） is a common cause of Hand, foot, and mouth disease （HFMD） and may also cause severe neurological diseases, such as encephalitis and poliomyelitis-like paralysis. To examine the genetic divers...Enterovirus 71 （EV71） is a common cause of Hand, foot, and mouth disease （HFMD） and may also cause severe neurological diseases, such as encephalitis and poliomyelitis-like paralysis. To examine the genetic diversity of EV71, we determined and analyzed the complete VP1 sequences （891 nueleotides） from nine EV71 strains isolated in Fuyang, China. We found that nine EV71 strains isolated were over 98% homologous at the nucleotide level and 93%-100% homologous tO members of the C4 subgenogroup. At the amino acid level, these Fuyang strains were 99% -100% homologous to one another, 97%-100% homologous to members of the C4 subgenogroup, and the histidine（H） at amino acid position 22 was conserved among the Fuyang strains. The results indicate that Fuyang isolates belong to genotype C4, and an H at position 22 appears to be a marker for the Fuyang strains.展开更多
In this study,we have investigated the antiviral activity of GuiQi polysaccharides (GQP) upon enterovirus 71 (EV71) in vitro.An assay using methyl thiazolyl tetrazolium (MTT),and analyses of cytopathic effects (CPE)we...In this study,we have investigated the antiviral activity of GuiQi polysaccharides (GQP) upon enterovirus 71 (EV71) in vitro.An assay using methyl thiazolyl tetrazolium (MTT),and analyses of cytopathic effects (CPE)were used to examine the antiviral activity of GQP upon Vero cells infected with EV71.The results revealed that GQP at concentrations below 31.2μg/mL exhibited significant antiviral effects upon EV71 when applied under three different experimental protocols.GQP was most strongly active in preventing the adsorption of EV71 to target cells and in this respect it was significantly more effective than ribavirin.In addition,it was clear that GQP could inhibit viral replication when added to cells 2 h after infection,but if added at the point of infection its effect was weak.GQP is considered to be less toxic than ribavirin,and may warrant further evaluation as a possible agent in the treatment of hand,foot and mouth disease (HFMD).展开更多
Human enterovirus 71 viruses have been long circulating throughout the world. In this study, we performed a positive selection analysis of the VP1 genes of capsid proteins from Enterovirus 71 viruses. Our results show...Human enterovirus 71 viruses have been long circulating throughout the world. In this study, we performed a positive selection analysis of the VP1 genes of capsid proteins from Enterovirus 71 viruses. Our results showed that although most sites were under negative or neutral evolution, four positions of the VP1 genes were under positive selection pressure. This might account for the spread and frequent outbreaks of the viruses and the enhanced neurovirulence. In particular, position 98 might be involved in neutralizing antibodies, modulating the virus-receptor interaction and enhancing the virulence of the viruses. Moreover, both positions 145 and 241 might correlate to determine the receptor specificity. However, these positions did not display much difference in amino acid polymorphism. In addition, no position in the VP1 genes of viruses isolated from China was under positive selection.展开更多
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
AIM:To characterise the viral kinetics of enterovirus 71 (EV71).METHODS:In this study,human rhabdomyosarcoma (RD) cells were infected with EV71 at different multiplicity of infection (MOI).After infection,the cytopath...AIM:To characterise the viral kinetics of enterovirus 71 (EV71).METHODS:In this study,human rhabdomyosarcoma (RD) cells were infected with EV71 at different multiplicity of infection (MOI).After infection,the cytopathic effect (CPE) was monitored and recorded using a phase contrast microscope associated with a CCD camera at different time points post viral infection (0,6,12,24 h post infection).Cell growth and viability were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in both EV71 infected and mock infected cells at each time point.EV71 replication kinet-ics in RD cells was determined by measuring the total intracellular viral RNA with real-time reverse-transcription polymerase chain reaction (qRT-PCR).Also,the intracellular and extracellular virion RNA was isolated and quantified at different time points to analyze the viral package and secretion.The expression of viral protein was determined by analyze the levels of viral structure protein VP1 with Western blotting.RESULTS:EV71 infection induced a significant CPE as early as 6 h post infection (p.i.) in both RD cells infected with high ratio of virus (MOI 10) and low ratio of virus (MOI 1).In EV71 infected cells,the cell growth was inhibited and the number of viable cells was rapidly decreased in the later phase of infection.EV71 virions were uncoated immediately after entry.The intracellular viral RNA began to increase at as early as 3 h p.i.and the exponential increase was found between 3 h to 6 h p.i.in both infected groups.For viral structure protein synthesis,results from western-blot showed that intracellular viral protein VP1 could not be detected until 6 h p.i.in the cells infected at either MOI 1 or MOI 10;and reached the peak at 9 h p.i.in the cells infected with EV71 at both MOI 1 and MOI 10.Simultaneously,the viral package and secretion were also actively processed as the virus underwent rapid replication.The viral package kinetics was comparable for both MOI 1 and MOI 10 infected groups.It was observed that at 3 h p.i,the intracellular virions obviously decreased,thereafter,the intracellular virions began to increase and enter into the exponential phase until 12 h p.i.The total amounts of intracellular virons were decreased from 12 to 24 h p.i.Consistent with this result,the increase of virus secretion occurred during 6 to 12 h p.i.CONCLUSION:The viral kinetics of EV71 were established by analyzing viral replication,package and secretion in RD cells.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
BACKGROUND： Hand-foot-mouth disease has become a major public health issue in children in China. In the present prospective study we investigated the clinical characteristics and emergency management of children with...BACKGROUND： Hand-foot-mouth disease has become a major public health issue in children in China. In the present prospective study we investigated the clinical characteristics and emergency management of children with severe encephalitis associated with NPE caused by enterovirus 71.METHODS： The study was conducted in 2 pediatric intensive care units （PICUs） over a 2-month period. Clinical records were reviewed of critically ill children with severe encephalitis associated with NPE caused by EV71 who were admitted to PICUs during the period of May to June 2008 in Fuyang.RESULTS： We reviewed the complete records of 36 children, of whom 23 （63.9%） were male and 13 （36.1%） female. Their age ranged from 4 to 48 months, with an average of 15.8 months. All children except one were under 3 years of age. The overall mortality in these children was 19.4%. The average duration of critical life threatening signs and symptoms was 2.1 days （12 hours-5 days）. Nervous system diseases included brainstem encephalitis in 27 children （75%）, brainstem encephalitis associated with myelitis in 6 children （16.7%）, and general encephalitis in 3 chidren （8.3%）, respectively. In 12 patients of NPE （33.3%） pink or bloody bubble sputum and asymmetric pulmonary edema or hemorrhage was the primary manifestation but no typical exanthema was observed. Five children died of acute onset of NPE and / or pulmonary hemorrhage with rapid progression of cardiopulmonary failure within hours after admission. Therapeutic management consisted of mechanical ventilation and administration of mannitol, methylprednisolone, intravenous immunoglobulin （IVIG） and vasoactive drugs, associated with the need of fluid volume resuscitation in 9 （25%） of the 36 children.CONCLUSION： In children less than 3 years of age found to be affected by severe EV71 encephalitis associated with NPE, one fifth may die. The major organ systems infected by severe EV71 include the central nervous system, the respiratory system, and the cardiovascular system. Early diagnosis and evaluation, respiratory support, treatment of intracranial hypertension, and mainttenance of function of the cardiovascular system are the most important therapeutic measures.展开更多
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 depositio...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.展开更多
Human Enterovirus 71(EV71)has emerged as one of the predominant causative agents of hand,foot and mouth disease(HFMD)with global impact.Despite the inactivated vaccine being licensed,other vaccine candidates based on ...Human Enterovirus 71(EV71)has emerged as one of the predominant causative agents of hand,foot and mouth disease(HFMD)with global impact.Despite the inactivated vaccine being licensed,other vaccine candidates based on advanced technology platforms are under development.In this report,we rationally designed and constructed two DNA-launched live attenuated vaccine candidates(pDL-EV71)under the control of specific promoters.In vitro and in vivo transfection with pDL-EV71 driven by the CMV promoter successfully yielded fully infectious EV71.More importantly,the administration of pDL-EV71 did not cause clinical symptoms following intracranial or intramuscular inoculation in neonatal and IFNα/βR/mice,demonstrating its safety profile.Moreover,a single-dose or two-dose immunization with pDL-EV71 elicited robust neutralizing antibodies against EV71 as well as an antigen-specific cellular response in mice.A single-dose immunization with 10μg of pDL-EV71 conferred complete protection against lethal EV71 infection in neonates born to immunized maternal mice.Overall,our present results demonstrate that pDL-EV71 is a safe and effective vaccine candidate against EV71 for further development.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
AIM： Enterovirus 71 （EV71） has been implicated as the etiological agent responsible for the recent outbreaks of hand, foot and mouth disease associated with severe neurological diseases in the Asia-Pacific region. ...AIM： Enterovirus 71 （EV71） has been implicated as the etiological agent responsible for the recent outbreaks of hand, foot and mouth disease associated with severe neurological diseases in the Asia-Pacific region. METHODS： The assembly process was hypothesized to occur via an orchestrated proteolytic processing of the P1 precursor by the viral protease 3CD. To test this hypothesis, we constructed 3 recombinant baculoviruses： Bac-P1 expressing P1; Bac-3CD expressing 3CD; and Bac-P1-3CD co-expressing P1 and 3CD. RESULTS： Both single infection by Bac-P1-3CD and coinfection by Bac-P1 and Bac-3CD resulted in correct cleavage of P1 to yield individual proteins VP0, VP1 and VP3, while the former approach yielded higher VLP production. In the cells, the structural proteins selfassembled into clusters of virus-like particles （VLP） resembling the authentic EV71 particle aggregates. After ultracentrifugation purification, the dispersed VLPs were indistinguishable from the authentic virus in size, appearance, composition and surface epitopes, as determined by SDS-PAGE, Western blot, transmission electron microscopy and immunogold labeling. CONCLUSION： Our data, for the first time, suggest that in insect cells EV71 structural proteins adopt a processing and assembly pathway similar to poliovirus assembly. The preservation of particle morphology and composition suggest that the VLP may be a valuable vaccine candidate to prevent EV71 epidemics.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
文摘Hand foot and mouth disease is a febrile sickness complex characterized by cutaneous eruption (exanthem) on the palms and soles with simultaneous occurrence of muco-cutanous vesiculo-ulcerative lesions (enanthem) affecting the mouth. The illness is caused by a number of enteroviruses with coxsackievirus A16 and enterovirus 71 as the main causative agents. Human enterovirus 71 (EV71) belongs to the species Human enterovirus A under the genus Enterovirus within the family Picornaviridae. EV71 has been associated with an array of clinical diseases including hand foot and mouth disease (HFMD), aseptic meningitis, encephalitis and poliomyelitis-like acute flaccid paralysis. A large outbreak of HFMD due to highly neurovirulent EV71 emerged in Malaysia in 1997, and caused 41 deaths amongst young children. In late 2000, a recurrence of an outbreak of HFMD occurred in Malaysia with 8 fatalities in peninsular Malaysia. Outbreak of HFMD due to EV71 recurred in 2003 with an unknown number of cases and mortalities. A similar outbreak of HFMD with 2 recorded deaths in young children occurred in peninsular Malaysia in late 2005 and this was followed by a larger outbreak in Sarawak (Malaysian Borneo) with 6 reported fatalities in the early part of 2006. The current on-going outbreak of HFMD started in peninsular Malaysia in epidemiological week 12 of 2010. As with other HFMD outbreaks in Malaysia, both EV71 and CA16 were the main aetiological viruses isolated. In similarity with the HFMD outbreak in 2005, the isolation of CA16 preceded the appearance of EV71. Based on the VP1 gene nucleotide sequences, 4 sub-genogroups of EV71 (C1, C2, B3 and B4) co-circulated and caused the outbreak of hand, foot and mouth disease in peninsular Malaysia in 1997. Two sub-genogroups (C1 and B4) were noted to cause the outbreak in 2000 in both peninsular Malaysia and Sarawak. EV71 of sub-genogroup B5 with smaller contribution from sub-genogroup C1 caused the outbreak in 2003. In the 2005 outbreak, besides the EV71 strains of sub-genogroup C1, EV71 strains belonging to sub-genogroup B5 were isolated but formed a cluster which was distinct from the EV71 strains from the sub-genogroup B5 isolated in 2003. The four EV71 strains isolated from clinical specimens of patients with hand, foot and mouth disease in the Sarawak outbreak in early 2006 also belonged to sub-genogroup B5. Phylogenetic analysis of the VP1 gene suggests that the EV71 strains causing the outbreak in Sarawak could have originated from peninsular Malaysia. Epidemiological and molecular data since 1997 show the recurrence of HFMD due to EV71 in Malaysia every 2 to 4 years. In each of the past outbreaks, more than one sub-genogroup of the virus co-circulate.
基金Scientific Research Fund of Institute of Pathogen Biology(2008IPB108)
文摘Enterovirus 71 （EV71） is a common cause of Hand, foot, and mouth disease （HFMD） and may also cause severe neurological diseases, such as encephalitis and poliomyelitis-like paralysis. To examine the genetic diversity of EV71, we determined and analyzed the complete VP1 sequences （891 nueleotides） from nine EV71 strains isolated in Fuyang, China. We found that nine EV71 strains isolated were over 98% homologous at the nucleotide level and 93%-100% homologous tO members of the C4 subgenogroup. At the amino acid level, these Fuyang strains were 99% -100% homologous to one another, 97%-100% homologous to members of the C4 subgenogroup, and the histidine（H） at amino acid position 22 was conserved among the Fuyang strains. The results indicate that Fuyang isolates belong to genotype C4, and an H at position 22 appears to be a marker for the Fuyang strains.
基金supported by research grants from The National Natural Science Foundation of China(NO81260070)The Project of Science and Technology of Lanzhou(NO 2011-1-71)The Doctor Project of Lanzhou University of Technology(NO 0908ZXC127)
文摘In this study,we have investigated the antiviral activity of GuiQi polysaccharides (GQP) upon enterovirus 71 (EV71) in vitro.An assay using methyl thiazolyl tetrazolium (MTT),and analyses of cytopathic effects (CPE)were used to examine the antiviral activity of GQP upon Vero cells infected with EV71.The results revealed that GQP at concentrations below 31.2μg/mL exhibited significant antiviral effects upon EV71 when applied under three different experimental protocols.GQP was most strongly active in preventing the adsorption of EV71 to target cells and in this respect it was significantly more effective than ribavirin.In addition,it was clear that GQP could inhibit viral replication when added to cells 2 h after infection,but if added at the point of infection its effect was weak.GQP is considered to be less toxic than ribavirin,and may warrant further evaluation as a possible agent in the treatment of hand,foot and mouth disease (HFMD).
文摘Human enterovirus 71 viruses have been long circulating throughout the world. In this study, we performed a positive selection analysis of the VP1 genes of capsid proteins from Enterovirus 71 viruses. Our results showed that although most sites were under negative or neutral evolution, four positions of the VP1 genes were under positive selection pressure. This might account for the spread and frequent outbreaks of the viruses and the enhanced neurovirulence. In particular, position 98 might be involved in neutralizing antibodies, modulating the virus-receptor interaction and enhancing the virulence of the viruses. Moreover, both positions 145 and 241 might correlate to determine the receptor specificity. However, these positions did not display much difference in amino acid polymorphism. In addition, no position in the VP1 genes of viruses isolated from China was under positive selection.
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 Research Grant Council (RGC,CUHK4428/06M)a commissioned grant of the Research Fund for Control of Infectious Diseases (CU-09-02-02)Food and Health Bureau,the Government of Hong Kong Special Administration Region (HKSAR)
文摘AIM:To characterise the viral kinetics of enterovirus 71 (EV71).METHODS:In this study,human rhabdomyosarcoma (RD) cells were infected with EV71 at different multiplicity of infection (MOI).After infection,the cytopathic effect (CPE) was monitored and recorded using a phase contrast microscope associated with a CCD camera at different time points post viral infection (0,6,12,24 h post infection).Cell growth and viability were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in both EV71 infected and mock infected cells at each time point.EV71 replication kinet-ics in RD cells was determined by measuring the total intracellular viral RNA with real-time reverse-transcription polymerase chain reaction (qRT-PCR).Also,the intracellular and extracellular virion RNA was isolated and quantified at different time points to analyze the viral package and secretion.The expression of viral protein was determined by analyze the levels of viral structure protein VP1 with Western blotting.RESULTS:EV71 infection induced a significant CPE as early as 6 h post infection (p.i.) in both RD cells infected with high ratio of virus (MOI 10) and low ratio of virus (MOI 1).In EV71 infected cells,the cell growth was inhibited and the number of viable cells was rapidly decreased in the later phase of infection.EV71 virions were uncoated immediately after entry.The intracellular viral RNA began to increase at as early as 3 h p.i.and the exponential increase was found between 3 h to 6 h p.i.in both infected groups.For viral structure protein synthesis,results from western-blot showed that intracellular viral protein VP1 could not be detected until 6 h p.i.in the cells infected at either MOI 1 or MOI 10;and reached the peak at 9 h p.i.in the cells infected with EV71 at both MOI 1 and MOI 10.Simultaneously,the viral package and secretion were also actively processed as the virus underwent rapid replication.The viral package kinetics was comparable for both MOI 1 and MOI 10 infected groups.It was observed that at 3 h p.i,the intracellular virions obviously decreased,thereafter,the intracellular virions began to increase and enter into the exponential phase until 12 h p.i.The total amounts of intracellular virons were decreased from 12 to 24 h p.i.Consistent with this result,the increase of virus secretion occurred during 6 to 12 h p.i.CONCLUSION:The viral kinetics of EV71 were established by analyzing viral replication,package and secretion in RD cells.
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.
文摘BACKGROUND： Hand-foot-mouth disease has become a major public health issue in children in China. In the present prospective study we investigated the clinical characteristics and emergency management of children with severe encephalitis associated with NPE caused by enterovirus 71.METHODS： The study was conducted in 2 pediatric intensive care units （PICUs） over a 2-month period. Clinical records were reviewed of critically ill children with severe encephalitis associated with NPE caused by EV71 who were admitted to PICUs during the period of May to June 2008 in Fuyang.RESULTS： We reviewed the complete records of 36 children, of whom 23 （63.9%） were male and 13 （36.1%） female. Their age ranged from 4 to 48 months, with an average of 15.8 months. All children except one were under 3 years of age. The overall mortality in these children was 19.4%. The average duration of critical life threatening signs and symptoms was 2.1 days （12 hours-5 days）. Nervous system diseases included brainstem encephalitis in 27 children （75%）, brainstem encephalitis associated with myelitis in 6 children （16.7%）, and general encephalitis in 3 chidren （8.3%）, respectively. In 12 patients of NPE （33.3%） pink or bloody bubble sputum and asymmetric pulmonary edema or hemorrhage was the primary manifestation but no typical exanthema was observed. Five children died of acute onset of NPE and / or pulmonary hemorrhage with rapid progression of cardiopulmonary failure within hours after admission. Therapeutic management consisted of mechanical ventilation and administration of mannitol, methylprednisolone, intravenous immunoglobulin （IVIG） and vasoactive drugs, associated with the need of fluid volume resuscitation in 9 （25%） of the 36 children.CONCLUSION： In children less than 3 years of age found to be affected by severe EV71 encephalitis associated with NPE, one fifth may die. The major organ systems infected by severe EV71 include the central nervous system, the respiratory system, and the cardiovascular system. Early diagnosis and evaluation, respiratory support, treatment of intracranial hypertension, and mainttenance of function of the cardiovascular system are the most important therapeutic measures.
基金supported by the National Natural Science Foundation of China(31970168)the Key R&D Program of Hubei Province(2021BCD004)+2 种基金the Hubei Central Leading Local Science and Technology Special Project(2022BGE245)the Hubei Science and Technology Major Project[2021ACB004]the Wuhan Knowledge Innovation Special Project(2023020201020303).
文摘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.
基金supported by the National Key Research and Development Program of China(2021YFC2302503)the National Natural Science Foundation of China(82241069)+1 种基金Cheng-Feng Qin was supported by the National Science Fund for Distinguished Young Scholars(81925025)the Innovative Research Group from the NSFC(81621005).
文摘Human Enterovirus 71(EV71)has emerged as one of the predominant causative agents of hand,foot and mouth disease(HFMD)with global impact.Despite the inactivated vaccine being licensed,other vaccine candidates based on advanced technology platforms are under development.In this report,we rationally designed and constructed two DNA-launched live attenuated vaccine candidates(pDL-EV71)under the control of specific promoters.In vitro and in vivo transfection with pDL-EV71 driven by the CMV promoter successfully yielded fully infectious EV71.More importantly,the administration of pDL-EV71 did not cause clinical symptoms following intracranial or intramuscular inoculation in neonatal and IFNα/βR/mice,demonstrating its safety profile.Moreover,a single-dose or two-dose immunization with pDL-EV71 elicited robust neutralizing antibodies against EV71 as well as an antigen-specific cellular response in mice.A single-dose immunization with 10μg of pDL-EV71 conferred complete protection against lethal EV71 infection in neonates born to immunized maternal mice.Overall,our present results demonstrate that pDL-EV71 is a safe and effective vaccine candidate against EV71 for further development.
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.
基金Supported by the National Science Council,No.93-2214-E-007-016 Ministry of Economic Affairs,No.93-EC-17A-17S1-0009
文摘AIM： Enterovirus 71 （EV71） has been implicated as the etiological agent responsible for the recent outbreaks of hand, foot and mouth disease associated with severe neurological diseases in the Asia-Pacific region. METHODS： The assembly process was hypothesized to occur via an orchestrated proteolytic processing of the P1 precursor by the viral protease 3CD. To test this hypothesis, we constructed 3 recombinant baculoviruses： Bac-P1 expressing P1; Bac-3CD expressing 3CD; and Bac-P1-3CD co-expressing P1 and 3CD. RESULTS： Both single infection by Bac-P1-3CD and coinfection by Bac-P1 and Bac-3CD resulted in correct cleavage of P1 to yield individual proteins VP0, VP1 and VP3, while the former approach yielded higher VLP production. In the cells, the structural proteins selfassembled into clusters of virus-like particles （VLP） resembling the authentic EV71 particle aggregates. After ultracentrifugation purification, the dispersed VLPs were indistinguishable from the authentic virus in size, appearance, composition and surface epitopes, as determined by SDS-PAGE, Western blot, transmission electron microscopy and immunogold labeling. CONCLUSION： Our data, for the first time, suggest that in insect cells EV71 structural proteins adopt a processing and assembly pathway similar to poliovirus assembly. The preservation of particle morphology and composition suggest that the VLP may be a valuable vaccine candidate to prevent EV71 epidemics.
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.
