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.

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.

EV71型重症手足口病患儿的临床特征及危险因素的相关性分析

目的探讨人肠道病毒71型(EV71)重症手足口病(HFMD)患儿的临床特征及其感染发生的危险因素,为预防、临床诊治提供参考依据。方法回顾性分析2013年1月-2015年12月67例重症手足口病患儿的病例资料,用描述性方法进行流行病学分析;同时对患儿的一般资料、症状、体征、辅助检查进行回顾性分析,收集患儿发生感染的部位、细菌种类、药敏实验结果,分别采用实时荧光定量聚合酶链反应(q RT-PCR)方法对人肠道病毒普通型(EV)、EV71、柯萨奇病毒A16型(A16)进行检测。结果 67例EV71型重症HFMD患儿以单一感染最多,占50.7%(34例);EV71型的混合感染占49.3%(33例)。继发感染最常见部位是下呼吸道(26.5%)。临床分离病原体46株,其中革兰阳性菌11株(23.9%),革兰阴性菌35株(76.1%)。对病原体进行药敏实验,头孢曲松、亚胺培南、头孢哌酮/舒巴坦普遍对G-性菌敏感,头孢唑林钠、哌拉西林、万古霉素普遍对G+性菌敏感。EV71型重症HFMD以3岁内的男性患儿多见,5岁以上儿童发病率较低,春夏季为发病高峰期,患儿临床症状以高热、皮疹、嗜睡、易惊、肢体抖动、病理征阳性为主,常伴有胸片、头颅CT异常,实验检查结果显示心肌酶谱、肝功能、白细胞、血糖升高、体液免疫及降钙素原等异常;3岁以下的男性患儿、农村住宿条件差、不良卫生习惯、对该疾病不了解未采取预防措施的监护人的散居儿童为该疾病的高发人群,EV71为其感染的常见病原体。结论四川部分地区重症HFMD患儿主要由EV71及其混合型人肠道病毒感染引起,了解EV71型HFMD重症临床特征,掌握本地区EV71型重症HFMD的高危因素对于HFMD防治具有重要意义。

检测EV71和EV-U的TaqMan-LNA探针多重荧光RT-PCR的建立

目的建立一种针对手足口病病原体的快速准确检测方法。方法利用TaqMan-LNA探针建立多重荧光定量RT-PCR方法,同时检测肠道病毒71型(EV71)和肠道病毒通用型(EV-U),用含有目的序列RNA的标准品制备标准曲线,对病毒进行准确定量,同时对该方法的特异性、灵敏度、重复性进行评估。结果用TaqMan-LNA探针多重荧光RT-PCR法检测CoxA16、CoxB2、EV71病毒和其他人类病毒RNA,证实其特异性为100%;对EV71和EV-U的检测灵敏度均达到102拷贝/μl;将EV71和EV-U的RNA标准品进行重复性实验,其批内、批间变异系数分别为0.97%～2.02%和0.89%～2.13%。结论本方法灵敏度较高,特异性强,重复性好,适用于手足口病的临床检测。

2016年上海市普陀区手足口病病原谱及EV71和Cox A16病毒分离株VP1基因特征分析

目的了解上海市普陀区手足口病病原学特点,分析肠道病毒71型(EV71)和柯萨奇病毒A组16型(Cox A16) VP1基因的特征,为手足口病的防治提供科学依据。方法收集2016年上海市普陀区手足口病患者的咽拭子标本,采用实时荧光定量PCR法(Real-time PCR)进行核酸检测,并用RD细胞对核酸检测结果为EV71和Cox A16的标本进行病毒分离,收获的阳性分离株利用逆转录-聚合酶链反应(RT-PCR)法扩增病毒VP1区核苷酸序列,测序后分析其同源性并构建系统进化树。结果 2016年上海市普陀区共检测234份手足口病标本,阳性标本138份,阳性率为58.97%(138/234)。其中EV71阳性率为11.54%(27/234)、Cox A16阳性率为18.80%(44/234)、Cox A6阳性率为27.78%(65/234)和其他肠道病毒阳性率0.85%(2/234)。病毒分离培养阳性株包括10株EV71和20株Cox A16,10株EV71分离株均为C4a型,VP1区核苷酸和氨基酸序列同源性分别为93.6%～99.9%和98.7%～100.0%;20株Cox A16分离株均为B1b亚型,VP1区核苷酸和氨基酸序列同源性分别为93.4%～100.0%和98.2%～100.0%。结论 2016年上海市普陀区手足口病呈现Cox A6、EV71和Cox A16共同流行,而EV71流行株属于C4a亚型,Cox A16流行株属于B1b亚型,均与国内大部分地方的流行株基因亚型基本一致。

EV71致重症手足口病引起心肌损伤的病理机制

目的观察由新肠道病毒(EV71)引起的重症手足口病(HFMD)患儿心肌损伤的病理变化。方法对7例由EV71引起的HFMD死亡患儿进行解剖,取心肌组织进行病理学检查,探讨其损伤及损伤机制。结果 HFMD患儿心外膜内有灶性炎细胞浸润;心肌纤维增粗、水肿,心肌间质内见散在的炎细胞浸润等病变。结论 EV71引起的HFMD可致心肌组织受损,且以炎症病变为主。

EV71与Cox A16病原体RT-PCR检测在预防手足口病传播中的意义

目的探讨EV71和Cox A16病毒的RT-PCR检测在早期诊断与预防手足口病传播中的应用价值。方法选择2013年11月至2014年10月间疑似手足口病患儿120例纳入疑似组,同期排除手足口病诊断患儿100例纳入对照组,均行粪便RTPCR检测,比较两组EV、EV71、Cox A16的检出率。结果疑似组的EV、EV71、Cox A16和EV71联合Cox A16检出率分别为100.0%、37.5%、31.7%和11.7%,均显著高于对照组的15.0%、0.0%、0.0%和0.0%,两组间差异均具有统计学意义(P<0.01)。结论 EV71和Cox A16的RT-PCR检测对于早期评价手足口病发病作用明显,有利于早期发现和隔离患儿,预防疫情传播扩散。

EV71-2A突变质粒的构建及体内外功能初探

目的构建pc DNA3.1-EV71-2A的突变质粒(EV71-2Amut),并对其功能进行检测,为进一步研究肠道病毒71型(Enterovirus type 71,EV71)2A蛋白酶的活性位点及其生物学功能奠定实验基础。方法利用聚合酶链式反应(PCR)定点诱变技术,定点突变编码EV71-2A第21His、39Asp和110Cys氨基酸的位点,构建EV71-2Amut,并测序鉴定。将所构建突变质粒与巨细胞病毒(Cytomegalovirus,CMV)启动子介导的真核细胞荧光素酶报告基因(pRL-CMV)共转染人横纹肌肉瘤细胞(RD细胞),通过观察转染组的EV71-2Apro和EV71-2Amut对pRL-CMV荧光强度的影响,判断突变质粒的细胞内酶活性。将EV71-2Apro或EV71-2Amut质粒转染BALB/c小鼠股四头肌,用RT-PCR检测局部相应质粒的mRNA水平,并观察局部肌肉组织的病理变化。结果构建的突变质粒EV71-2Amut经测序及比对,结果与预计突变位点一致;在细胞学水平检测到的pRL-CMV荧光强度,EV71-2Amut组比EV71-2Apro组显著增高(P<0.05);小鼠股四头肌注射局部均检出EV71-2Apro和EV71-2Amut的mRNA表达,且与EV71-2Apro组相比,EV71-2Amut注射组的肌肉组织的肌肉凝固性坏死、炎症反应病理损伤较轻。结论成功构建了突变的EV71-2Amut质粒,为研究EV71-2A蛋白酶的生物学功能奠定了实验基础。

循证护理对EV71感染重症手足口病患儿临床症状及预后的影响

目的探讨循证护理对肠道病毒71型(EV71)感染重症手足口病患儿的护理效果。方法方便选取2018年4月—2019年6月期间在该院接受治疗的84例EV71感染重症手足口病患儿,采用随机数字表法将入选患儿分为观察组和对照组,各42例。对照组使用常规护理模式进行干预,观察组使用循证护理模式进行干预,对比分析两组患儿干预后临床症状的改善时间、并发症发生情况。结果观察组患儿发热持续时间、口腔疱疹愈合时间、口腔疼痛改善时间、神经系统受累时间、手足皮疹愈合时间及总患病时间分别为(3.49±1.35)、(4.22±1.59)、(2.03±0.69)、(4.12±1.39)、(4.12±1.64)、(9.71±3.12)d,均明显短于对照组患儿的(5.02±2.39)、(6.13±2.31)、(3.98±1.35)、(6.38±2.57)、(6.84±1.91)、(14.53±4.02)d,差异有统计学意义(t=3.612、4.414、8.335、5.013、7.002、6.139,P=0.001、0.000、0.000、0.000、0.000、0.000);观察组患儿并发症发生率为5.56%,显著低于对照组患儿的28.57%,差异有统计学意义(χ^2=4.941,P=0.026)。结论对EV71感染重症手足口病患儿使用循证护理模式进行干预,可缩短其发热持续时间、口腔疱疹愈合时间,降低并发症的发生率,护理效果较好。

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。

以主要流行EV71 VP1基因高度保守区为靶点的TaqMan荧光定量PCR检测方法的建立

在我国,肠道病毒71型(enterovirus 71,EV71)C4型是引起手足口病的主要流行基因型。为建立EV71C4型TaqMan荧光定量PCR检测方法,在C4型EV71VP1基因的高保守区,设计合成引物和TaqMan探针,将包含此目的区段的基因片段克隆到pcDNA3.1载体中,通过体外转录获得标准品,并以梯度稀释的标准品为模板建立工作曲线,进而在优化反应条件的基础上建立TaqMan荧光实时定量PCR检测方法。实验中,所设计引物、探针的高度保守性保证了C4型EV71的高效扩增。经反应条件优化,引物和探针的最佳工作浓度分别为300 nmol/L和200 nmol/L,在1×10~301×10~3拷贝数检测范围内具有良好的线性关系(R^2=1),灵敏度可达到10~2 copies/μL。通过对该方法进行检验发现,批间和组间重复实验的变异系数均小于0.5%,且该方法对柯萨奇A16(coxsackievirus A16,CA16)柯萨奇B1(coxsackievirus B1,CB1)人轮状病毒(human rotavirus,HRV)单纯疱疹病毒2型(herpes Simplex virus type 2,HSV-2)均无交叉反应,对6份EV71阳性样本检出率为100%。以上数据表明,文中建立的TaqMan荧光定量PCR方法可为我国主要流行C4型EV71感染的快速诊断及疾病监控提供有效途径。

31例确诊EV71感染所致重型手足口病诊治体会

目的:探讨肠道病毒71型(EV71)感染所致重型手足口病的临床特点和治疗方法。方法:采用描述性流行病学方法对31例确诊EV71感染所致重型手足口病患儿的临床资料进行回顾性分析。结果:男19例,女12例,年龄1岁～6岁8个月,患儿主要表现为持续高热、精神差、易惊、肢体抖动、呕吐;外周血WBC升高为主(67.8%);采用以糖皮质激素加静脉丙种球蛋白为主的综合治疗,31例患儿均治愈。结论:早期识别重型手足口病,采用以激素加丙球为主的综合治疗,能够取得满意的疗效。

雾化吸入重组人干扰素α1b在重症EV71手足口病中的疗效观察

目的探讨雾化吸入重组人干扰素α1b(IFN-α1b)治疗重症肠道病毒71型(EV71)手足口病的临床疗效及安全性。方法 42例重症EV71手足口病患儿,随机分成治疗组和对照组,各21例。对照组给予静脉丙种球蛋白(IVIG)和甘露醇等常规治疗,治疗组在对照组基础上加用IFN-α1b雾化吸入治疗,比较两组的治疗效果。结果治疗组惊跳、眼球运动异常的持续时间与对照组比较,差异无统计学意义(P>0.05);治疗组持续高热、精神萎靡、四肢震颤、肢体无力的持续时间分别为(2.0±0.5)、(3.2±0.4)、(2.6±1.1)、(4.9±2.1)d,均短于对照组的(3.2±1.5)、(5.5±1.7)、(4.2±2.1)、(7.8±3.6)d,差异有统计学意义(P<0.05)。结论雾化吸入IFN-α1b能加速重症EV71手足口病患儿的神经系统表现和其他临床表现的改善,且安全可靠。

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.

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）.

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.

人源抗EV71病毒中和表位SP70的Fab抗体研制

目的构建人源抗EV71病毒Fab噬菌体抗体库,筛选具有中和活性的人源单克隆抗体,为EV71感染引起的手足口病临床诊断及治疗奠定基础。方法从成人志愿者抗凝血中分离外周淋巴细胞,提取总RNA并逆转录为cDNA。以cDNA为模板,用人源IgG Fab段基因引物扩增轻链及重链Fd区基因,分步克隆至pComb3载体,构建Fab噬菌体抗体库。以SP70为抗原对Fab噬菌体抗体库进行富集筛选,将获得的重组噬菌体感染XL1-Blue菌并诱导表达,用SP70对诱导表达的菌上清进行筛选。结果共获得3株轻、重链基因组合序列不同的克隆,且这3株Fab抗体可特异性结合SP70多肽。结论成功构建人源抗EV71病毒Fab噬菌体抗体库,并从中筛选出3株针对EV71病毒结构蛋白VP1上中和线性表位SP70的特异性人源Fab抗体,为研发新的手足口病诊断和治疗工具奠定基础。

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.

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.