MA104 Cell line presents characteristics suitable for enterovirus A71 isolation and proliferation

Enterovirus A71 (EV‐A71), one of the most important causative agents of hand, foot and mouth disease (HFMD) in children, can lead to severe clinical outcomes, even death. However, the infection spectrum of EV‐A71 in different cell lines remains unknown. Therefore, in this study, the biological characteristics of EV‐A71 Subgroup C4 in different cell lines were investigated. To this end, the infectivity of EV‐A71Jinan1002 isolated from children with severe HFMD was assessed in 18 different host cell lines. It was found that the MA104 cell line displayed biological characteristics suitable for EV‐A71 Subgroup C4 strain isolation and proliferation; indeed, it was found that a broad spectrum of cell lines can be infected by EV‐A71Jinan1002. Among the screened cells, four cell lines (HEK293, RD, MA104 and Marc145) produced high 50% tissue culture infective dose (TCID₅₀) values calculated in viral proliferations (ranged from 10^7.6 to 10^7.8); the TCID₅₀ being negatively associated with the time to appearance of CPE. Proliferation curves demonstrated that EV‐A71Jinan1002 amplifies more efficiently in MA104, Hep‐2 and RD cells. Remarkably, the virus isolation rate was much higher in MA104 cells than in RD cells. Thus this study, to our knowledge, is for the first to explore the infection spectrum of EV‐A71 subgroup C4 in such a large number of different cell lines. Our data provide useful reference data for facilitating further study of EV‐A71.     

Development of a sandwich ELISA for the quantification of enterovirus 71

Since 2008, enterovirus 71 (EV71) has been responsible for high-mortality seasonal epidemics of hand, foot and mouth disease in China. Currently many groups in the world are in the process of developing EV71 vaccines to combat this deadly disease. We have developed three EV71-specific monoclonal antibodies, and in this study we report the establishment of a fast and cost-effective sandwich ELISA kit for measurement of virus concentration in EV71 vaccines using a pair of mouse anti-EV71 monoclonal antibodies. The system is specific for EV71 virus, with no cross-reactivity to coxsackievirus A16, H1N1, rabies, and hepatitis A. Using a reference EV71 vaccine standard, the sensitivity of the assay kit was determined to be 0.82 U/ml, with a linear range between 3.75 and 120 U/ml.     

应用生物反应器培养Vero细胞制备EV71病毒初探

目的应用生物反应器培养Vero细胞制备EV71病毒。方法以3 L生物反应器采用4 g/L、8 g/L Cytodex-1微载体培养比较Vero细胞比生长率,并以4 g/L微载体培养EV71病毒。结果 4 g/L微载体培养Vero细胞3～4 d微载体细胞密度达2.3×106/mL,按0.001的感染复数(MOI)接种EV71病毒,病毒收获液的滴度最高达7.90 lgPFU/mL,较静置培养平均高出0.92 lgPFU/mL。结论初步建立了3 L生物反应器微载体培养Vero细胞制备EV71病毒的工艺,为进一步放大生产规模奠定了基础。     

肠道病毒71型感染诱导细胞程序性死亡形态学及分子生物学特征的初步研究

肠道病毒 71型(enterovirus type 71,EV71)感染常可引起婴幼儿手足口病(hand,foot and mouth disease,HFMD),还可引起中枢神经系统并发症等重症,甚至死亡。研究认为,EV71诱发重症的原因主要与病毒感染诱导细胞程序性死亡(programmed cell death,PCD)及诱导细胞产生大量炎症因子有关。病毒感染可通过激活不同的信号通路触发细胞程序性死亡,主要包括含半胱氨酸的天冬氨酸蛋白水解酶(cysteinyl aspartate specific proteinase,caspase)依赖的细胞凋亡、细胞焦亡,以及非caspase依赖的细胞坏死性凋亡。本研究旨在探讨EV71感染诱导细胞程序性死亡的形态学和分子生物学特征,利用显微镜和免疫荧光技术检测EV71感染后细胞形态变化,JC-1染色检测感染后细胞线粒体膜电位变化,流式细胞术及Annexin V-FITC/PI双染法、乳酸脱氢酶释放量法检测感染细胞的细胞膜损伤程度,结合蛋白免疫印迹法检测病毒感染后细胞中多聚ADP核糖聚合酶[poly(ADP-ribose) polymerase,PARP]、caspase-9、caspase-3等凋亡因子,以及细胞焦亡关键效应蛋白Gasdermin D、坏死性凋亡效应蛋白MLKL的磷酸化情况。结果显示,EV71感染后细胞主要呈现凋亡特征,并伴随少量细胞坏死。与细胞凋亡相关的PARP被剪切,caspase-9和caspase-3等相关因子被激活。经泛caspase抑制剂处理后,细胞程序性死亡被抑制,但仍有部分细胞坏死。结果提示,EV71感染以细胞凋亡为主,也可能存在非caspase依赖的细胞程序性死亡。     

MEK1/2抑制剂U0126抑制肠道病毒71型的复制

为了揭示肠道病毒71型(enterovirus71,EV71)的复制与宿主细胞Raf/MEK/ERK信号通路(简称ERK通路)的相互关系,本研究应用临床诊断为手足口病的患儿疱疹液,通过易感细胞分离培养、RT-PCR及序列测定,以及Western印迹技术等方法,成功分离到EV71临床株.进一步用该分离株感染易感细胞,通过观察宿主细胞p-ERK1/2蛋白磷酸化水平、病毒特异性衣壳蛋白VP1水平、病毒半数组织培养感染量(50%tissue culture infectious dose,TCID50),以及感染细胞的CPE等指标,以期揭示ERK通路在EV71复制的作用.结果表明,EV71的复制可引起细胞ERK通路的活化;而用MEK1/2特异性的抑制剂U0126预先抑制ERK通路的活化,可显著地降低受染细胞上清液中的病毒的感染滴度(以TCID50表示)、受染细胞中EV71VP1蛋白水平、受染细胞中EV71核酸水平,以及受染细胞的细胞病变效应(cytopathic effect,CPE).提示ERK信号通路的活化对EV71的复制具有重要的作用.本研究为进一步阐明EV71在宿主细胞内的复制机制、寻找新型抗病毒靶标等研究奠定了良好的基础.     

EV71诱导人神经细胞SH-SY5Y自噬的分子机制

【背景】EV71感染所致的重症手足口病易导致神经系统并发症,使患儿预后较差,甚至死亡。【目的】从EV71可诱导神经细胞自噬这一现象出发,探索该病毒诱导神经细胞自噬的miRNA机制,探讨EV71损伤神经细胞可能的分子机制。【方法】通过RT-PCR及Westernblot技术,在感染EV71病毒的人神经母细胞瘤细胞SH-SY5Y中检测细胞自噬变化;通过芯片分析细胞感染前后差异表达的miRNA分子,再使用miRNA mimics调节工具明确与EV71诱导神经细胞自噬有关的miRNA分子。【结果】EV71可诱导SH-SY5Y细胞自噬增加,下调细胞内miRNA29b(miR29b)分子的表达水平;当上调细胞内miR29b的表达后,EV71诱导细胞自噬增加的现象可被逆转,病毒复制水平下降。【结论】EV71诱导神经细胞自噬是通过下调miR29b分子的表达水平实现;miR29b不仅与自噬相关,它与EV71病毒复制也存在密切关系。因此,该研究不仅有助于阐明EV71导致神经系统损伤的具体分子机制,还为miR29b成为治疗EV71感染可能的新药物靶点奠定了理论基础。     

Recent Progress on Functional Genomics Research of Enterovirus 71

Enterovirus 71(EV71) is one of the main pathogens that causes hand-foot-and-mouth disease(HFMD). HFMD caused by EV71 infection is mostly self-limited; however, some infections can cause severe neurological diseases, such as aseptic meningitis, brain stem encephalitis, and even death. There are still no effective clinical drugs used for the prevention and treatment of HFMD. Studying EV71 protein function is essential for elucidating the EV71 replication process and developing anti-EV71 drugs and vaccines. In this review, we summarized the recent progress in the studies of EV71 noncoding regions(50 UTR and 30 UTR) and all structural and nonstructural proteins, especially the key motifs involving in viral infection, replication, and immune regulation. This review will promote our understanding of EV71 virus replication and pathogenesis, and will facilitate the development of novel drugs or vaccines to treat EV71.     

抗肠道病毒71型的药物研究进展

肠道病毒71型（Enterovirus 71,EV71）是引起重症手足口病（Hand,Foot and Mouth Disease,HFMD）的主要病原体。重症HFMD进展迅速,可表现为严重的神经系统并发症,甚至危及生命。目前临床上防治EV71感染缺乏特异、高效的药物,其残疾率和死亡率很高。随着研究的深入,已经发现了大量具有抗EV71能力的化合物,人们探索的药物机制和药物靶点各不相同。因此,本文从药物靶向病毒、宿主等角度出发,针对抗EV71感染的天然药物、合成药物及常见中药中活性成分作用机制的最新进展进行综述与讨论。此外,对抗病毒药物筛选技术进行简要概述,以期为抗EV71药物的筛选与研发设计等相关研究提供参考。     

Reverse Genetic Analysis of Adaptive Mutations within the Capsid Proteins of Enterovirus 71 (EV-A71) Strains Necessary for Infection of CHO-K1 Cells

正Dear Editor,Previous studies had described the adaptation of enterovirus 71 (EV-A71) strains that enabled entry and viral replication in Chinese Hamster Ovary (CHO) cell line(Zaini and Mc Minn 2012; Zaini et al. 2012). These adapted     

肠道病毒EV71 IgM抗体参考品的建立

目的建立肠道病毒EV71 IgM抗体检测用参考血清盘。方法通过收集手足口病感染早期患者的咽拭子和血清,对咽拭子病毒进行分离、鉴定、基因分型,通过血清中和试验及酶联免疫吸附试验(捕获法)的验证,确定EV71 IgM抗体阳性样品,组建EV71 IgM抗体参考血清盘,并经3家实验室对该参考血清盘进行协同标定和确认。结果 12名患儿的咽拭子中均分离到EV71病毒,在RD细胞上引起细胞病变。通过套式PCR扩增、序列测定后进化树分析确认均为流行的C4基因亚型。每名患儿的双份血清均可中和EV71病毒,抗体效价为1∶512~1∶2048。通过捕获法进行EV71 IgM抗体检测结果均为阳性。以此12份样品为原料制备EV71 IgM抗体阳性参考品,同时以12份EV71 IgM抗体阴性血清制备阴性参考品,建立了由12份阳性参考品、12份阴性参考品、1份最低检出限参考品和1份精密性参考品组成的肠道病毒EV71 IgM抗体参考血清盘。通过3家实验室协同标定,各实验室间差异均无统计学意义。结论建立了EV71 IgM抗体检测参考血清盘,为相关检测试剂的质量控制和评价提供了参考标准。     

细菌脂多糖对肠道病毒71型感染影响的初步研究

肠道病毒71型(enterovirus 71, EV71)感染常导致婴幼儿手足口病(hand, foot and mouth disease, HFMD),细菌脂多糖(lipopolysaccharide, LPS)在多种肠道病毒感染过程中起重要作用。本研究旨在探讨细菌LPS对EV71感染的影响。将EV71与LPS共孵育,测定病毒被热处理后残留病毒的活力,以及病毒感染过程中病毒基因拷贝数的变化。结果显示,热处理后病毒活力逐渐丧失,而经LPS处理的病毒活力丧失的速度减缓,且残留病毒活力与LPS浓度呈正相关;LPS处理后的病毒在黏附、侵入、胞内复制及释放过程中基因拷贝数较对照组均降低;免疫印迹分析表明LPS与抗VP1单克隆抗体可竞争性结合EV71,且粪便中的EV71可被抗大肠埃希菌抗体识别。上述结果提示,LPS可增强EV71热稳定性,抑制EV71感染过程,且LPS可能与EV71结合。     

Generation of neutralizing monoclonal antibodies against Enterovirus 71 using synthetic peptides

Enterovirus 71 (EV71) has led to recent outbreaks of hand, foot and mouth disease (HFMD) in China, resulting in high mortality. In this study, several monoclonal antibodies were generated by immunizing mice with two synthetic peptides, SP55 and SP70, containing amino acids 163-177 and 208-222 of VP1. The specificities of the anti-EV71 peptide monoclonal antibodies were confirmed by Western blot analysis and immunocytochemistry against EV71 virus. Most importantly, we have identified a monoclonal antibody, clone 22A12, which shows strong neutralizing activity against EV71 in an in vitro neutralization assay. Because there is no vaccine available and treatment is very limited, mouse anti-EV71 monoclonal antibody, clone 22A12, could be a promising candidate to be humanized and used for treatment of EV71 infection.     

miR-34b调控肠道病毒71型在人横纹肌肉瘤细胞中的复制及机制

【背景】研究发现microRNAs(miRNAs)可以参与调控病毒在宿主细胞内感染和复制的过程。【目的】研究miR-34b对肠道病毒71型(Enterovirus71,EV71)在宿主细胞内的复制及其可能机制。【方法】在人横纹肌肉瘤(Rhabdomyosarcoma,RD)细胞中转染miR-34b mimics和Inhibitor,通过Western blot和Real-time PCR实验检验EV71病毒的复制和表达情况。随后利用双荧光素酶报告系统验证miR-34b与潜在靶点eIF4E的相互作用,并检测miR-34b对RD细胞中eIF4E mRNA表达水平的影响。【结果】miR-34b可以促进病毒在RD细胞中的复制和表达,而miR-34b抑制剂有抑制病毒复制的作用,细胞内miR-34b可以通过作用于靶基因eIF4E调控EV71在宿主细胞中的复制过程。【结论】揭示了miR-34b在EV71病毒复制过程中的调控作用及机制,研究EV71病毒与宿主miRNAs的相互作用机制为进一步阐明EV71病毒感染与复制机理奠定了基础。     

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 31.2 μg/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）.     

C4亚型人肠道病毒71型的小鼠肌肉适应株对小鼠具有致死毒力

目的人肠道病毒71型（EV71）感染婴幼儿可导致手足口病,偶尔伴随有严重的并发症。建立EV71的小鼠模型是深入研究病毒感染的致病机制、进行疫苗和药物评价的基础。方法将EV71的临床分离株在小鼠骨骼肌中进行系列传代,得到了小鼠的肌肉适应株MP10。使用MP10经腹腔注射感染10日龄ICR小鼠,对感染小鼠的症状、病毒复制和病理进行了监测。结果与临床分离株相比,MP10对人横纹肌瘤细胞（rhabdomyosarcoma cell,RD细胞）的感染力提高,并且对小鼠的毒力增强,MP10感染10日龄小鼠可导致其瘫痪和死亡。病毒主要在骨骼肌中复制,并引发大面积的坏死性肌炎,同时神经组织未观察到病变。病理分析发现：感染小鼠体内与呼吸运动相关的肌肉均发生严重的坏死性肌炎,推测此类肌肉的坏死会影响小鼠的呼吸功能,从而成为导致小鼠死亡的因素之一。结论建立了C4亚型EV71毒株感染10日龄ICR小鼠的模型,该模型可作为研究EV71感染的致病机制、以及疫苗和药物评价的工具。     

Enterovirus 71 Virion-Associated Galectin-1 Facilitates Viral Replication and Stability

Enterovirus 71 (EV71) infection causes a myriad of diseases from mild hand-foot-and-mouth disease or herpangina to fatal brain stem encephalitis complicated with pulmonary edema. Several severe EV71 endemics have occurred in Asia-Pacific region, including Taiwan, and have become a serious threat to children’s health. EV71 infection is initiated by the attachment of the virion to the target cell surface. Although this process relies primarily upon interaction between viruses and cell surface receptors, soluble factors may also influence the binding of EV71 to host cells.Galectin-1 has been reported to participate in several virus infections, but is not addressed in EV71. In this study, we found that the serum levels of galectin-1 in EV71-infected children were higher than those in non-infected people. In EV71 infected cells, galectin-1 was found to be associated with the EV71 VP1 and VP3 via carbohydrate residues and subsequently released and bound to another cell surface along with the virus. EV71 propagated from galectin-1 knockdown SK-N-SH cells exhibited lower infectivity in cultured cells and less pathogenicity in mice than the virus propagated from parental cells. In addition, this galectin-1-free EV71 virus was sensitive to high temperature and lost its viability after long-term storage, which could be restored following supplement of recombinant galectin-1. Taken together, our findings uncover a new role of galectin-1 in facilitating EV71 virus infection.