Gp78 regulates PMP22 and causes ER stress and autophagy in EV71-VP1-overexpressing mouse Schwann cells

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.

红景天苷体外抗EV71病毒的作用

应用MTT法研究红景天苷对Vero细胞的毒性以及对肠道病毒71型(Enterovirus 71,EV71)的抑制作用,通过吸附试验和穿入试验观察红景天苷是否对EV71的吸附和穿入具有抑制作用。结果表明,红景天苷的细胞毒性低,半数细胞毒性浓度(CC50)为4.25 mg/mL。药物在病毒感染前加入(药物预处理)、药物和病毒同时加入及药物在病毒感染后加入都能够抑制病毒,以药物预处理细胞抗病毒作用最好;红景天苷能够抑制病毒的吸附和穿入作用,对吸附作用抑制效果更强。表明红景天苷能够在体外抑制病毒对细胞的感染。

Expression,purification and characterization of enterovirus-71 virus-like particles

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.

Hand Foot and Mouth Disease Due to Enterovirus 71 in Malaysia

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.

Genetic Analysis of the VP1 Region of Human Enterovirus 71 Strains Isolated in Fuyang,China,During 2008

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.

A Comparison of the Biological Characteristics of EV71 C4 Subtypes from Different Epidemic Strains

The comparative analysis of the biological characterization and the genetic background study of EV71 circulating strains is commonly recognized as basic work necessary for development of an effective EV71 vaccine. In this study, we sequenced five EV71 circulating strains, isolated from Fuyang, Hefei, Kunming and Shenzhen city of China and named them FY-23, FY-22, H44, K9 and S1 respectively. The sequence alignment demonstrated their genotypes be C4. The genetic distance of the VP1 gene from these isolates suggested that they were highly co-related with genetic identity similar to other previously reported EV71 strains in China. Additionally, these strains were identified to display some obvious proliferation dynamics and plaque morphology when propagated in Vero cells. However, a distinctive difference in pathogenic ability in neonatal mice was found. Some differences in cross neutralization test & immunogenic analysis were also found. All these results are related to the biological characterization of circulating EV71 strains in China and aid in the development of an EV71 vaccine in the future.

Positive Selection Analysis of VP1 Genes of Worldwide Human Enterovirus 71 Viruses

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.

Antiviral Activity of GuiQi Polysaccharides against Enterovirus 71 in vitro

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).

Comparative Genomic Analysis of Enterovirus 71 Revealed Six New Potential Neurovirulence-associated Sites

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）.

Viral kinetics of Enterovirus 71 in human habdomyosarcoma cells

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.

Human IgG Fc promotes expression, secretion and immunogenicity of enterovirus 71 VP1 protein

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.

Clinical characteristics and treatment of severe encephalitis associated with neurogenic pulmonary edema caused by enterovirus 71 in China

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.

Relationship between catecholamine level and gene polymorphism of β1 adrenergic receptor G1165C in children with EV71 infection in hand foot and mouth disease

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.

Contribution of 3CD Region to the Virulence of Enterovirus 71

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.

Efficient Humoral and Cellular Immune Responses Induced by a Chimeric Virus-like Particle Displaying the Epitope of EV71 without Adjuvant

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.

Safe and Objective Assay of Enterovirus 71 Neutralizing Antibodies via Pseudovirus

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.

A Novel Pharmacophore Model Derived from a Class of Capsid Protein Enterovirus 71 Inhibitors

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.

Expression and clinical significance of pattern recognition receptor-associated genes in hand, foot and mouth disease

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.

Rational design of a DNA-launched live attenuated vaccine against human enterovirus 71

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.

Investigation on Factors Associated with Severe Hand, Foot and Mouth Disease

Objective To analyze the clinical and laboratory features of patients with mild and severe HFMD to identify early predictive or diagnostic markers for severe cases. Methods Samples of feces, nasopharyngeal-swab specimens, peripheral blood, serum and cerebral spinal fluid were collected. Postmortem pathological examination was conducted on 2 dead patients with complication due to neurogenic pulmonary edema. Reverse transcription-polymerase chain-reaction(RT-PCR), culture and isolation of enterovirus 71(EV71) were performed to detect EV71 infection. Both univariate and multivariate logistic analysis were used to identify factors associated with severe cases. Results EV71 was mainly responsible for HFMD. In this study, 5 isolated EV71 strains belonged to C4 gene subtype. Compared with mild patients, EV71-RNA detection rate was higher and Cox A16 detection rate was lower among severe patients(P < 0.01). Inflammatory cell infiltration in the lung, cardiac and liver tissues were mild by postmortem pathological examination. It was found that body temperature, vomitting, limb tremor, neutrophil, blood glucose and EV71 infection were significantly related to the severe cases by univariate logistic analysis. However, after multivariate logistic regression analysis, only vomiting(OR 16.1, CI 2.3-110.5, P < 0.01) and limb tremor(OR 117.6, CI 13.8-1004.5, P < 0.01) were significantly and independently correlated with the severe cases.Conclusions EV71 was mainly responsible for HFMD, particularly for severe cases. Vomiting and limb tremor were predictive markers for severe cases.