肠道病毒71型的全长cDNA感染性克隆的构建与鉴定

目的构建一株肠道病毒71型的全长cDNA克隆并验证感染性。方法用长片段高保真RT-PCR方法扩增肠道病毒71型87-2008 Xi’an株的cDNA,装入pBR322载体。将该克隆线性化,体外转录后转染RD细胞后,通过观察CPE及RT-PCR验证其感染性。随后利用空斑法测定拯救病毒的一步生长曲线。结果成功构建了肠道病毒71型87-2008 Xi’an株的全长cDNA克隆;体外转录及转染RD细胞60 h后可观察到明显的CPE现象;RT-PCR在转染RD细胞中检测到病毒的存在;成功利用空斑法测定了拯救病毒的一步生长曲线,最大滴度达到2×107pfu/mL。结论成功构建了肠道病毒71型87-2008 Xi’an株的全长cDNA克隆并验证了其感染性,同时测定了拯救病毒的一步生长曲线,与野生型病毒相近。     

Generation of murine monoclonal antibodies which cross-neutralize human enterovirus genogroup B isolates

A live enterovirus 71 (EV71) isolate designated, EV71/E59, with genotype B4 produced in Vero cells and purified over a sucrose gradient was used as the immunogen to generate EV71-specific murine monoclonal antibodies. Four hybridoma clones derived from the fusion of splenocytes of EV71/E59-preimmunized BALB/c (H-2^d) mice and the NS-1 myeloma cells that exhibit stable growth were selected for detailed characterization. The proof that the hybridomas produced are indeed true independent clones was based on the obervations that they expressed different complementarity-determining regions (CDRs) in their κ light chain genes. Purified ascitic fluids produced by the individual clones reacted against the viral capsid protein, VP1, in Western blot; and recognized distinct sites of a common epitope localized at the C-terminal half of VP1. Each of the monoclonal antibodies exhibited potent neutralizing activities against the immunizing virus strain, as well as two other isolates namely, N0781-TW-01, and N2838, of subgenogroups B4 and B5, respectively, that were found commonly in recent outbreaks in Taiwan. It was also observed the monoclonal antibodies acted cooperatively in neutralizing the EV71/E59 virus.     

河北省手足口病病例的病原构成及EV71病毒基因特征分析

目的了解河北省不同地区、不同严重程度手足口病病例的病原构成情况及EV71病毒的基因特征。方法采集河北省不同地区的HFMD患者粪便、疱疹液、咽拭子标本进行核酸检测和病毒分离,同时结合所收集的HFMD患病例的居住地、疾病严重程度信息加以分析。选取18株EV71阳性分离株进行VP1编码区基因扩增和核苷酸序列测定和分析,与其它38株各基因型和基因亚型的EV71代表株构建系统发生树。结果2009年河北省HFMD临床诊断病例的EV阳性率为65．13％,其中以EV71为主,占阳性病例的58．0％（752／1296）。秦皇岛、邯郸、保定、邢台地区手足口病例以EV71感染为主,而衡水、沧州等地区则以CA16为主。轻型病例中EV71阳性率为37．74％,重症病例中EV71阳性率为80．64％,死亡病例检测13例,均为EV71阳性。18株EV71分离株的VP114核苷酸同源性为94．9％～99．8％,与C4亚型代表株的VP1区核苷酸同源性最高,为91．9％～99．6％。进化树结果显示,河北省EV71分离株与c4亚型代表株处于同一分支,并在C4a进化分支的不同簇中。结论2009年引起河北省手足口病流行的病原体主要为EV71和CA16。秦皇岛、邯郸、保定、邢台地区手足口病例以EV7I感染为主,而衡水、廊坊、沧州等地区则以CA16为主。EV71是重症病例的主要致病病原体。河北省EV71分离株为c4亚型C4a进化分支。     

中国大陆EV71病毒分离株的分子流行病学研究

目的了解1987-2010年中国大陆肠道病毒71型（EV71）分离株的分子流行病学特点及其种系进化、基因分型和遗传变异性。方法从GenBank/NCBI上获得中国大陆来源的具有完整VP1或近似完整VP1基因的核苷酸序列信息的413株EV71毒株进行分析,采用MEGA5.0软件,构建系统进化树,计算相同或不同基因型及基因亚型毒株的核苷酸与氨基酸的相似性。结果 1987-2010年,中国大陆20个省、市或地区均分离到具有完整VP1序列的毒株,且2008年以来数量陡增;中国大陆流行的主要是C型,只有2008年安徽和2009年湖北发现了A型;各基因型在43、58、142、164、167、184、240、249、292等氨基酸位点发生了特异性变异;从健康人体内分离的HQ129932毒株与其他序列比较,氨基酸无特异性变异。结论 C型株可能具有更强的传染力;氨基酸位点变异对于EV71病毒进化有重要意义;VP1基因与疾病的严重程度无明显关联;应加强C4a亚型疫苗候选疫苗株对其他基因型毒株的交叉保护作用研究。     

EV71疫苗的研究进展

肠道病毒71型(Enterovirus 71,EV71)的感染主要引起儿童的手足口病(hand-foot-and-mouth disease,HFMD),以手、脚和口腔粘膜疤疹或破溃成溃疡为主要临床特征,严重者可引起无菌性脑膜炎、脊髓灰质炎样瘫痪、致命性脑炎等中枢神经系统并发症[1].自从EV71病毒于1969年首次从美国加利福尼亚州患有中枢神经系统疾病的婴儿粪便标本中分离出来后[2],它在欧洲和亚洲国家已引起多次高死亡率的流行暴发[3-7].     

人肠道病毒71型感染免疫抑制恒河猴的分析

目的: 通过建立人肠道病毒71型(EV71)感染在免疫抑制恒河猴的动物模型,进一步分析人EV71在免疫抑制动物体内病毒增殖、病理变化以及病毒抗原在不同组织的表达情况。方法: 按照每天15 mg/kg的剂量给8只恒河猴口服环孢菌素A连续7 d后造成其免疫抑制状态,再经呼吸道和消化道感染EV71病毒FY23株,对其进行14 d的临床观察后处死,采集各器官组织进行病毒载量检测,同时对不同的组织进行病理学分析和免疫组化分析。结果: 感染EV71病毒后,免疫抑制组的临床症状、病毒载量、病理分析和免疫组化结果都明显比非免疫抑制组严重,从病毒对机体造成的影响来看呼吸系统感染较消化系统感染明显严重。结论: 本实验分析了人EV71在免疫抑制动物体内病毒增殖的情况,为建立用于EV71疫苗评价的恒河猴动物模型提供了依据,并对未来的EV71疫苗的使用人群范围做出了新的限定。     

RNA interference against enterovirus 71 infection

Enterovirus 71 (EV71) is a highly infectious major causative agent of hand, foot, and mouth disease (HFMD) which could lead to severe neurological complications. There is currently no effective therapy against EV71. In this study, RNA interference (RNAi) is employed as a therapeutic approach for specific viral inhibition. Various regions of the EV71 genome were targeted for inhibition by chemically synthesized siRNAs. Transfection of rhabdomyosarcoma (RD) cells with siRNA targeting the 3'UTR, 2C, 3C, or 3D region significantly alleviated cytopathic effects of EV71. The inhibitory effect was dosage-dependent with a corresponding decrease in viral RNA, viral proteins, and plaque formations by EV71. Viral inhibition of siRNA transfected RD cells was still evident after 48 h. In addition, no significant adverse off-target silencing effects were observed. These results demonstrated the potential and feasibility for the use of siRNA as an antiviral therapy for EV71 infections.     

Comparative study of enterovirus 71 infection of human cell lines

The cell tropism of enterovirus 71 (Enteroviridae) in neuronal, glial and laryngeal cells. The 4643 strain, an enterovirus 71 isolate from a patient in Taiwan, was used to infect three human cell lines representing neuronal cells (SK-N-SH, neuroblastoma), glial cells (U373MG, glioblastoma), and laryngeal cells (HEp-2, larynx epidermoid carcinoma). Immunofluorescent staining and transmission electron microscopy (TEM) were used to detect mature enterovirus 71 4643 virions in these cell lines. The three cell lines were also compared for presence of virus-mediated cytopathic effect (CPE), synthesis of infected cell-specific proteins, viral (-) RNA, and virus replication rate. Virus particles were detected by TEM, and viral replication increased over time, indicating the existence and release of mature viruses from all three infected cell lines. The most severe CPE and the highest viral replication rate were observed in the SK-N-SH cells. Further screening of the infected cell lines by microarray analysis revealed that the neuron growth factor receptor (NGFR) gene was uniquely upregulated in infected SK-N-SH cells, implying that the receptor encoded by this gene may be involved in cell tropism. The data show that neurons are vulnerable to enterovirus 71 4643 infection and are consistent with the clinical observation that enterovirus 71 4643 targets mainly neuronal cells but is also found in many organs in conjunction with an inflammatory reaction.     

The effect of enterovirus 71 immunization on neuropathogenesis and protein expression profiles in the thalamus of infected rhesus neonates

Enterovirus 71 (EV71) is a major pathogen that causes hand-foot-mouth disease (HFMD). Our previous studies have demonstrated that the complete process of pathogenesis, which may include tissue damage induced by host inflammatory responses and direct tissue damage caused by viral infection, can be observed in the central nervous system (CNS) of animals infected in the laboratory with EV71. Based on these observations, the neuropathogenesis and protein expression profiles in the thalamic tissues of EV71-infected animals were further analyzed in the present study. Changes in protein expression profiles following immunization with the inactivated EV71 vaccine followed by virus challenge were observed and evaluated, and their physiological roles in viral pathogenesis are discussed. Taken together, the results of these experiments provide evidence regarding the neuropathogenesis and molecular mechanisms associated with EV71 infection and identify several protein indicators of pathogenic changes during viral infection.     

Therapeutic and prevention strategies against human enterovirus 71 infection

Human enterovirus 71 (HEV71) is the cause of hand, foot and mouth disease and associated neurological complications in children under five years of age. There has been an increase in HEV71 epidemic activity throughout the Asia-Pacific region in the past decade, and it is predicted to replace poliovirus as the extant neurotropic enterovirus of highest global public health significance. To date there is no effective antiviral treatment and no vaccine is available to prevent HEV71 infection. The increase in prevalence, virulence and geographic spread of HEV71 infection over the past decade provides increasing incentive for the development of new therapeutic and prevention strategies against this emerging viral infection. The current review focuses on the potential, advantages and disadvantages of these strategies. Since the explosion of outbreaks leading to large epidemics in China, research in natural therapeutic products has identified several groups of compounds with anti-HEV71 activities. Concurrently, the search for effective synthetic antivirals has produced promising results. Other therapeutic strategies including immunotherapy and the use of oligonucleotides have also been explored. A sound prevention strategy is crucial in order to control the spread of HEV71. To this end the ultimate goal is the rapid development, regulatory approval and widespread implementation of a safe and effective vaccine. The various forms of HEV71 vaccine designs are highlighted in this review. Given the rapid progress of research in this area, eradication of the virus is likely to be achieved.     

肠病毒71型分离株的鉴定及免疫原性研究

目的 在对肠病毒71型(enterovirus 71,EV71)分离株进行鉴定的基础上,对其诱导小鼠产生的免疫应答水平进行研究,拟为进一步的EV71候选疫苗研究奠定基础.方法 超速离心纯化病毒后,采用磷钨酸负染法通过电镜观察病毒形态、大小;采用特异性EV71单抗通过间接免疫荧光法(IFA)检测病毒的特异性;合成EV71特异性引物,通过RT-PCR对病毒进行分子生物学鉴定;病毒的VP1基因序列测定后,与其他EV71序列进行比较,绘制种系发育树,确定病毒基因型;通过腹腔注射途径免疫小鼠,免疫后分离血清通过微量细胞病变抑制法测定血清中和抗体效价,ELISA法检测血清抗体水平和抗体亚型水平.结果 电镜下可观察到典型的圆形病毒颗粒,直径为20～30 nm,呈典型的肠病毒形态;085和087株病毒感染细胞中均能观察到黄绿色荧光,提示该两株病毒可与EV71单抗特异性结合;RT-PCR可以从病毒感染细胞中扩增出226 bp大小的特异性产物带,而采用CA16特异性引物则不能扩增出相应大小的产物带;基于VP1基因序列的种系发育分析显示,087和085株均为C4基因型;085和087株EV71免疫小鼠后可诱导产生能中和包括其本身在内的多个病毒株的中和抗体,针对同一株中和病毒,实验组间差异无统计学意义;针对不同中和病毒株,各实验组中和本株、523-07T株的能力明显高于FY-02T株(P<0.05);ELISA法检测结果显示,接种灭活前后的EV71病毒085和087株后均能诱导小鼠产生抗EV71特异性IgG;IgG亚型为IgG1/IgG2a混合型,灭活病毒免疫组小鼠血清EV71特异性IgG、IgG1、IgG2a水平明显高于活病毒免疫组(P<0.05).两株病毒之间差异无统计学意义.结论 本研究分离到的085和087株病毒均为EV71 C4基因亚型,并具有一定的免疫原性.     

First detection of enterovirus 71 from an acute flaccid paralysis case with residual paralysis in Iran

In an attempt to determine the types of non-polio enteroviruses (NPEVs) in acute flaccid paralysis (AFP) cases in Iran, we detected enterovirus 71 (EV71) in an AFP case with residual paralysis for the first time. Cell culture detected no enteroviruses, while RT-PCR and subsequent sequencing revealed that the specimen was positive for EV71. EV71 is the causative agent of a variety of diseases from hand, foot and mouth disease to severe neurological complications and is now considered as an important cause of childhood acute flaccid paralysis.     

Genetic characterization of enterovirus 71 isolated from patients with severe disease by comparative analysis of complete genomes

Enterovirus 71 (EV71) which causes mild illness in children is also associated with severe neurological complications. This study analyzed the complete genomes of EV71 strains derived from mild and severe diseases in order to determine whether the differences of EV71 genomes were responsible for different clinical presentations. Compared to complete genomes of EV71 strains derived from mild cases (less virulent strains), nucleotide differences in EV71 strains isolated from severe cases (more virulent strains) were observed primarily in the internal ribosomal entry site (IRES) of the 5'-untranslated region (UTR), which is vital for the cap-independent translation of viral proteins. In the protein-coding region, an E-Q substitution at amino acid position 145 of structural protein VP1 that occurred in more than one of more virulent strains was observed. This site is known to be related functionally to receptor binding and virulence in mice. Overall, strains (Group III) isolated from patients with fatal or severe sequelae outcomes had greater sequence substitutions in the 5'-UTR and/or protein-coding region and exhibited a relatively low-average homology to less virulent strains across the entire genome, indicating the possibility of significant genomic diversity in the most virulent EV71 strains. Further studies of EV71 pathogenesis should examine the significance of genomic diversity and the effects of multiple mutations in a viral population.     

Detecting natural selection in high-altitude human populations

High-altitude natives have distinctive biological characteristics that appear to offset the stress of hypoxia. Evolutionary theory reasons that they reflect genetic adaptations resulting from natural selection on traits with heritable variation. Furthermore, high-altitude natives of the Andean and Tibetan Plateaus differ from one another, perhaps resulting from different evolutionary histories. Three approaches have developed a case for the possibility of population genetic differences: comparing means of classical physiological traits measured in samples of natives and migrants between altitudes, estimating genetic variance using statistical genetics techniques, and comparing features of species with different evolutionary histories. Tibetans have an inferred autosomal dominant major gene for high oxygen saturation that is associated with higher offspring survival, a strong indicator of ongoing natural selection. New approaches use candidate gene and genomic analyses. Conclusive evidence about population genetic differences and associations with phenotypes remains to be discovered.     

深圳市肠道病毒71型血清流行病学初步调查

目的探讨深圳市不同人群肠道病毒71型血清流行病学规律。方法收集1999—2003年深圳地区正常人血清584份，按年龄0-岁、1-岁、2-岁、5-岁、15-岁和〉30岁分成6个组，用ELISA方法对其肠道病毒71型抗体进行检测。结果1999—2003年深圳地区正常人血清中肠道病毒71型抗体阳性率最高的年龄组为5-岁组，阳性率超过50％；15-岁和〉30岁组血清阳性率分别为47．7％和39．8％；2．岁血清阳性率约为30％，其中0-岁和1-岁两个低年龄组的血清阳性率最低。不超过20％。结论5岁以下低年龄儿童是肠道病毒71型的易感人群。     

Patterns of polymorphism and divergence in the VP1 gene of enterovirus 71 circulating in the Asia-Pacific region between 1994 and 2013

Enterovirus 71 has been implicated in several outbreaks of hand, foot and mouth disease in the Asia-Pacific region. The present study aimed to achieve comprehensive evolutionary dynamic aspects of EV71 during 1994–2013, based on phylogenetic analyses of the VP1 sequences. The results indicated that 4 genotypes, namely C4, C1, C2 and B4 are the predominant strains, especially in Southeast Asian countries. No common ancestor was shared in different countries. Fourteen sites of substitutions were detected in the VP1 gene sequences; including the most common sites related to neutralization at position V249I [47.1% (189/401)] and A289T [42.6% (171/401)]. However, the sites Q22H and Q22R associated with increased virulence were recognized only in 13.7% (55/401) and 18% (72/401), respectively. None of the above mutations seemed to become fixed because the ratio of Ka/Ks was greater than 1.0. Mutations K43E, A58T, S184T, and T240S could possibly change the spatial structure. Two mutations, G145E and T240S, could obviously affect the hydrophobicity of VP1 and thus alter the EV71 immunoreactivity. In conclusion, the VP1 gene of EV71 strains circulating in the Asia-Pacific region during 1994–2013, showed polymorphisms and divergence with very slow evolution rate, which may be one of the reasons for periodic outbreaks in this area.     

Enterovirus 71‐induced autophagy detected in vitro and in vivo promotes viral replication

Enterovirus 71 (EV71) is an important pathogen causing death in children under 5 years old worldwide. However, the underlying pathogenesis remains unclear. This study reveals that EV71 infection in rhabdomyosarcoma (RD) and neuroblastoma (SK‐N‐SH) cells stimulated the autophagic process, which was demonstrated by an increase of punctate GFP‐microtubule‐associated protein 1 light chain 3 (GFP‐LC3), the level of autophagosome‐bound LC3‐II protein and double‐membrane autophagosome formation. EV71‐induced autophagy benefited EV71 replication, which was confirmed by the autophagic inducer rapamycin and the inhibitor 3‐methyladenine. Signaling pathway investigation revealed that the decreased expression of phosphorylated mTOR and phosphorylated p70S6K is involved in EV71‐induced autophagy in a cell‐specific manner. The expression of phosphorylated extracellular signal‐regulated kinase (Erk) was suppressed consistently in EV71‐infected cells. However it did not participate in the autophagic response of the cell. Other signaling pathway molecules, such as Erk, PI3K/Akt, Bcl‐2, BNIP3, and Beclin‐1 were not affected by infection with EV71. Electron microscopy showed co‐localization of autophagosome‐like vesicles with either EV71‐VP1 or LC3 protein in neurons of the cervical spinal cord in ICR mice infected with EV71. In conclusion, EV71 infection triggered autophagic flux and induced autophagosome formation both in vitro and in vivo. Autophagy induced by EV71 is beneficial for viral replication. Understanding the role of autophagy induced by EV71 in vitro and the formation of autophagosome‐like vesicle in vivo provide new insights into the pathogenesis of EV71 infection. J. Med. Virol. 81:1241–1252, 2009. © 2009 Wiley‐Liss, Inc.     

肠道病毒71型感染人神经母细胞瘤细胞(SH-SY5Y)的miRNA表达谱

目的 研究肠道病毒71型(EV71)感染神经细胞的miRNA表达谱,探讨miRNA在病毒感染神经细胞中的可能作用.方法 建立EV71感染人神经母细胞瘤细胞(SH-SY5Y)模型,收集感染后48 h细胞.以Taqman低密度芯片检测miRNA表达谱,使用实时RT-PCR对芯片结果进行验证并在TargetScan和miRanda网站预测靶基因,采用GO和KEGG分析靶基因功能.结果 成功建立EV71感染SH-SY5Y细胞模型,通过低密度芯片筛选出215种显著升高的miRNA和25种显著下调的miRNA.经过RT-PCR验证,3种miRNA(MiR-10a*、miR-15b*和miR-195)显著下调,7种miRNA(miR-10a、miR-342-5p、miR-483-5p、Let-7b、miR-99a、miR-140-5p和miR-21)显著上调,与芯片结果相符.GO分析显示发展进程和信号调节条目最富集靶基因.KEGG路径分析显示靶基因在肿瘤路径、蛋白水解、Wnt信号传导、黑素形成、粘附连接、MAPK信号通道最富集.结论 EV71感染神经细胞48 h后miRNA表达谱发生改变,10种变化的miRNA靶基因预测在发展进程、信号传导及凋亡中起着重要的作用,可为后期机制研究提供参考.