柯萨奇病毒A组16型的3C蛋白抑制RIG-Ⅰ介导的IFN-β的诱导

目的探讨柯萨奇病毒A组16型对RIG-Ⅰ介导的信号通路的影响。方法将带有IFN-β启动子的萤火虫荧光素酶报告质粒pIFN-β-luc及内对照报告载体pRL-CMV转染细胞,之后用CA16感染细胞,24 h后检测报告基因活性。将CA16感染细胞,在感染6 h、12 h、24 h以及48 h后用Real-time PCR方法测定细胞中IFN-β的mRNA的表达水平。将pIFN-β-luc和pRL-CMV以及表达RIG-I的质粒和表达CA16的3C蛋白质粒共转染细胞,24 h后检测报告基因活性。将带有绿色荧光蛋白GFP的IRF3质粒和表达RIG-I基因N端质粒以及表达CA16的3C蛋白共转染细胞,24 h后用荧光显微镜观察。将表达RIG-I的质粒和表达CA16的3C蛋白质粒共转染细胞,24 h后用免疫共沉淀方法验证蛋白质间相互作用。结果 CA16感染细胞不能引起IFN-β表达的上调,CA16的3C蛋白抑制了RIG-I诱导的IFN-β的启动子活性以及IRF3的核迁移,CA16的3C蛋白与RIG-Ⅰ存在相互作用。结论 CA16的3C蛋白通过与RIG-Ⅰ相互作用从而抑制RIG-Ⅰ介导的IFN-β的诱导。     

病毒感染后表达下调的RNF167抑制小鼠腹腔巨噬细胞产生IFN-β

目的探讨RNF167对病毒感染的小鼠腹腔巨噬细胞产生IFN-β的调控作用。方法 RNA病毒VSV和DNA病毒HSV-1感染小鼠腹腔巨噬细胞后,Western blot检测RNF167的表达水平。利用RNA干扰减少小鼠原代腹腔巨噬细胞中RNF167的表达后,病毒感染小鼠腹腔巨噬细胞,实时荧光定量PCR法检测IFN-β的mRNA水平,ELIAS法检测细胞上清中IFN-β的浓度。Western blot法检测IFN-β的转录因子IRF3的磷酸化(p-IRF3)水平。结果VSV感染巨噬细胞4和8 h后,RNF167表达水平显著下降(P0.01),而HSV-1感染后RNF167的表达没有显著变化。si-RNF167降低小鼠腹腔巨噬细胞RNF167表达水平后,与对照细胞相比,感染RNA病毒后细胞中IFN-β的mRNA水平及上清中IFN-β水平显著下降(P0.01),p-IRF3水平也显著下降。而感染HSV-1病毒后上述指标均无差异。结论 RNF167能够特异性地调控RNA病毒感染的巨噬细胞中IFN-β的表达。     

呼吸道合胞病毒感染人肺上皮细胞活化Toll样受体3介导的抗病毒作用研究

目的 探讨呼吸道合胞病毒( RSV)感染人肺上皮A549细胞后,Toll样受体3(TLR3)的水平变化及其产生的Ⅰ型干扰素的抗病毒作用.方法 RSV感染体外培养的人肺上皮A549细胞,并给予TLR3特异性抗体处理,分别感染4、8、12、16、24h后收集各组细胞.未感染病毒的细胞作为对照组.RT-PCR法检测TLR3、IFN-α、IFN-β,RSV F蛋白的mRNA表达水平变化.结果 RSV感染A549细胞后,TLR3、IFN-α、IFN-β,RSV F蛋白的mRNA表达量均升高且有时间依赖性,TLR3 mRNA在24h表达量是基础表达量的5倍,IFN-α、IFN-β mRNA在24 h表达量是基础表达量的4倍多,RSVF蛋白的mRNA表达量近1.7倍.TLR3抗体预先处理以抑制TLR3受体,再行RSV感染,IFN-α和IFN-β mRNA表达量虽然升高,但较感染组均有所下降,mRNA表达在12 h后显著降低,且IFN-ββ的mRNA表达量下调更明显.但RSV F基因的mRNA表达在12 h、24 h升高有显著性差异.结论 RSV感染A549细胞后可上调TLR3表达,其活化细胞介导产生的Ⅰ型干扰素起抗病毒作用,在一定程度上可抑制病毒的增殖水平.     

肠道病毒EV71及CA16在不同细胞系中细胞病变作用的研究

目的研究CA16和EV71在4种细胞系中细胞病变作用的差异.方法随机选择滴度相似的EV71轻症、EV71重症和CA16轻症毒株各1株,分别感染RD、Vero、U251和SY5Y细胞,比较3株病毒在细胞病变的形态及程度、感染后的细胞存活率、病毒在细胞中的复制水平和病毒所致凋亡蛋白相关mRNA的表达水平等方面的差别.结果3株病毒的感染在同一细胞系中表现相似.镜下从形态学上无法对三株病毒加以区别;感染后病毒载量均表现为从高到低依次为Vero、RD、SY5Y细胞到U251细胞的顺序;病毒感染后细胞存活率的顺序与细胞内复制水平与相反,两者之间存在类似负相关的趋势.3株病毒的感染在3种细胞系中均有凋亡蛋白caspase-8、9和3相关mRNA的表达,提示可能3种细胞系中凋亡启动的途径相同.结论细胞水平的感染,提示这两种病毒感染所致临床表现的差异性并非单独由病毒所决定.     

寨卡病毒调控子宫成纤维细胞先天免疫的机制

目的探讨寨卡病毒(Zika virus,ZIKV)逃避子宫成纤维细胞先天免疫的分子机制。方法使用ZIKV感染子宫成纤维细胞后,检测YWHAE-RIG-I-MAVS-IFN-β通路关键分子的表达情况。通过实时荧光定量PCR检测YWHAE mRNA的表达情况。通过高通量测序检测ZIKV感染子宫成纤维细胞后的miRNA表达谱。通过荧光素酶报告实验确定调控YWHAE的miRNA。结果 ZIKV感染子宫成纤维细胞后,IFN-β的分泌和IFN-β的启动子活性水平下降(P0.05),YWHAE、p-IRF3S396的表达水平下降,MAVS与RIG-I、RIG-I与YWHAE的相互作用水平下降。但是,YWHAE启动子活性没有显著改变(P0.05),而YWHAE mRNA水平下降(P0.05)。通过高通量测序发现ZIKV感染后子宫成纤维细胞miRNA的表达发生改变。其中hsamiR-15a-5p、hsa-miR-6780b-3p、hsa-miR-6845-3p、hsa-miR-16-5p、hsa-miR-590-3p、hsa-miR-575、hsa-miR-4674、hsamiR-371a-5p、hsa-miR-4642、hsa-miR-4676-5p的表达差异在5倍以上。子宫成纤维细胞中过表达上述差异miRNA后,miR-545-3p能够降低YWHAE mRNA的表达水平(P0.05)。敲低miR-545-3p后,YWHAE m RNA和蛋白的表达水平上升(P0.05)。此外,miR-545-3p靶向YWHAE的3端非编码区(P0.05)。ZIKV感染子宫成纤维细胞的同时过表达miR-545-3p后,YWHAE mRNA水平显著下降(P0.05)、IFN-β分泌水平显著下降(P0.05)、ZIKV复制水平均显著上升(P0.05);但是,ZIKV感染子宫成纤维细胞的同时敲低miR-545-3p后,YWHAE m RNA水平显著上升(P0.05)、IFN-β分泌水平显著上升(P0.05)、ZIKV复制水平均显著下降(P0.05)。结论 ZIKV通过miR-545-3p靶向YWHAE促进了自身的复制。     

模式识别受体TLR3、RIG-I和MDA5在慢性乙肝患者外周血的表达水平

目的:观察慢性乙型肝炎(CHB)患者外周血中单个核细胞胞膜和胞质模式识别受体mRNA的表达表达水平。方法:54例慢性乙型肝炎患者为实验组,40例健康体检者为对照组。采集实验组和对照组的新鲜空腹抗凝血,分离单个核细胞,提取单个核细胞中RNA,并反转录为c DNA,应用实时定量PCR技术检测Toll样受体3(TLR3)、维甲酸诱导基因-I(RIG-I)、黑色素瘤分化相关分子5(MDA5)、I型干扰素(IFN-α、IFN-β)、转录因子3(IRF-3)mRNA表达水平。结果:与正常对照组比较,实验组的TLR3、RIG-I、MDA5、IFN-α、IFN-β、IRF-3 mRNA表达水平均明显降低,具有显著性差异(P0.05)。高病毒载量组中的各分子表达水平与低病毒载量组和健康对照组比较降低更明显,且TLR3、RIG-I、MDA5、IFN-α、IFN-β、IRF-3mRNA水平分别与HBV-DNA的含量呈负相关(r=-0.697、-0.738、-0.867、-0.618、-0.415、-0.573)。结论:细胞胞膜(TLR3)和胞质(RIG-1、MDA5)模式识别受体、IFN-α、IFN-β、IRF-3 mRNA水平在慢性乙型肝炎患者的外周血单个核细胞中的表达降低,可能与HBV感染的慢性化状态有关。     

肠道病毒71型感染人横纹肌肉瘤细胞不同时期凋亡相关基因差异表达研究

目的 研究肠道病毒71型(EV71)感染人横纹肌肉瘤(RD)细胞后不同时期凋亡相关基因差异表达及信号通路转导机制.方法 锥虫蓝染色观察EV71感染RD细胞的存活率,Annexin V-FITC/PI双染色法检测病毒感染后凋亡细胞形态学及凋亡发生率,PCR芯片分析病毒感染RD细胞8h和20h后88个凋亡相关基因的表达情况.结果 EV71(MOI=5)感染RD细胞8h后存活率显著下降.流式细胞仪检测显示病毒感染20 h后早期凋亡及晚期凋亡细胞分别上升至18.0％和19.1％.PCR芯片检测88个凋亡相关基因,EV71感染8h后,除IFN-β1上调5.22倍外,ACIN1、Akt、Apaf1、caspase、CIDEB等47个基因发生明显下调.病毒感染20 h后,FasL、CD40L、TNF、caspase-10、caspase-3等28个基因发生2倍以上上调.结论 EV71感染早期RD细胞凋亡相关基因表达下调与病毒凋亡抑制效应相关,而感染晚期病毒可激活Fas/FasL、TNF/TNFR死亡受体信号通路诱导细胞凋亡.同时宿主细胞也通过调节CD40/CD40L、NF-κB/RelA、PI3 K/Akt等信号通路途径抑制凋亡作用.     

EV71感染患儿Toll样受体表达初探

目的 探讨肠道病毒71型( EV71)感染患儿免疫活性细胞模式识别受体(pattern recognition receptor,PRR)及细胞因子水平的变化。方法 EV71感染患儿71例,其中轻症EV71感染组20例,重症EV71感染组25例,危重症EV71感染组26例,同年龄正常对照组20例。采用real -time PCR检测外周血单个核细胞维甲酸蛋白Ⅰ(retinoic acidinducible gene Ⅰ,RIG-Ⅰ)、黑色素瘤分化相关基因5（melanoma differentitation-associated gene 5,MDA5）及细胞因子(IL-12、IFN-α)表达;流式细胞术检测外周血单核/巨噬细胞( monocyte/macrophages,MC)、髓样树突状细胞(myloid dentritic cells,mDC)及浆样树突状细胞（plasmacytoid dentritic cells,pDC）Toll样受体(TLRs)表达率;ELISA检测细胞因子IL-12及IFN-α的变化。结果 (1)轻症患儿仅TLR7升高,重症EV71感染患儿外周血MC、mDC、pDCTLR7表达明显升高(P＜0.05),MC、mDC高表达TLR2、3、4,危重症患儿呈下降趋势。(2)EV71感染患儿胞内模式识别受体RIG- Ⅰ/MDA5 mRNA表达明显增加;(3)轻症患儿DC相关细胞因子有上升趋势,重症患儿DC相关细胞因子IL-12、IFN-α明显增高(P＜0.05),轻症及危重症患儿明显降低(P＜0.05)。结论 TLR7可能是免疫活性细胞识别EV71的主要模式识别受体;RIG-I/MDA5也可能参与识别EV71;合并细菌感染或细胞破坏释放的内源性配体导致TLR2或TLR4活化,诱导炎症反应。     

DUT通过靶向RIG-I正调控RLR介导的抗病毒信号通路

目的探究DUT(deoxyuridine triphosphate nucleotidohydrolase, dUTPase)对RIG-I(retinoic acid-inducible gene I)介导的RLR(RIG-I-like receptors)抗病毒先天免疫的研究。方法采用免疫共沉淀的方法检测DUT与RIG-I之间的相互作用以及DUT对RIG-I介导的泛素化修饰的影响;应用非变性聚丙烯酰胺凝胶电泳实验验证DUT对RIG-I或仙台病毒(Sendai virus,SeV)诱导的IRF3二聚化的作用;利用双荧光素酶报告基因实验检测RIG-I或SeV介导的DUT对干扰素启动子IFN-β的激活效果;通过实时荧光定量PCR实验检测DUT对SeV诱导的IFN-β转录的影响。结果 DUT与RIG-I相互作用,过表达DUT促进RIG-I或SeV诱导的IRF3的二聚化水平以及IFN-β启动子的活性。此外,过表达DUT也加强了SeV诱导的IFN-β启动子的转录水平,研究结果还表明DUT促进RIG-I泛素化修饰及RIG-I K63泛素化修饰,并且通过与RIG-I之间的相互作用增强RNF135、MEX3C、TRIM4介导的RIG-I K63泛素化修饰。结论 DUT是RIG-I介导的针对RNA病毒天然免疫应答的正调节因子。     

肠道病毒71型抵抗Ⅰ型干扰素诱导的抗病毒作用

目的 研究肠道病毒71型(EV71)抵抗Ⅰ型干扰素(interferon,IFN)诱导的抗病毒作用.方法 将1000 U/ml的Ⅰ型干扰素(α,β)加入HeLa细胞后,去除上清中的干扰素,感染带有GFP的重组单纯疱疹病毒(HSV-1)和EV71,观察GFP的表达和PCR检测HSV-1核酸,判断Ⅰ型干扰素对HSV-1的作用.通过RT-PCR方法检测EV71 2A基因的表达从而判断EV71病毒的复制能力.结果 Ⅰ型干扰素(α,β)诱导HeLa细胞产生抗病毒蛋白而有效地抑制重组HSV-1 GFP的表达和核酸扩增.而EV71 2A的RT-PCR结果证实EV71可在Ⅰ型干扰素(α,β)作用后的HeLa细胞中有效增殖.结论 Ⅰ型干扰素(a,β)诱导产生抗病毒蛋白;EV71可在Ⅰ型干扰素(α,β)诱导产生抗病毒蛋白的HeLa细胞中有效增殖.     

Enterovirus 71 blocks selectively type I interferon production through the 3C viral protein in mice

Type I interferons (IFNs) represent an essential innate defense mechanism for controlling enterovirus 71 (EV 71) infection. Mice inoculated with EV 71 produced a significantly lower amount of type I IFNs than those inoculated with poly (I:C), adenovirus type V, or coxsackievirus B3 (CB3). EV 71 infection, however, mounted a proinflammatory response with a significant increase in the levels of serum and brain interleukin (IL)‐6, monocyte chemoattractant protein‐1, tumor necrosis factor, and IFN‐γ. EV 71 infection abolished both poly (I:C)‐ and CB3‐induced type I IFN production of mice. Such effect was not extended to other enteroviruses including coxsackievirus A24, B2, B3, and echovirus 9, as mice infected with these viruses retained type I IFN responsiveness upon poly (I:C) challenge. In addition, EV 71‐infected RAW264.7 cells produced significantly lower amount of type I IFNs than non‐infected cells upon poly (I:C) stimulation. The inhibitory effect of EV 71 on type I IFN production was attributed to the viral protein 3C, which was confirmed using over‐expression systems in both mice and RAW264.7 cells. The 3C over‐expression, however, did not interfere with poly (I:C)‐induced proinflammatory cytokine production. These findings indicate that EV 71 can hamper the host innate defense by blocking selectively type I IFN synthesis through the 3C viral protein. J. Med. Virol. 84:1779–1789, 2012. © 2012 Wiley Periodicals, Inc.     

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.     

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.     

Cerebrospinal fluid cytokines in enterovirus 71 brain stem encephalitis and echovirus meningitis infections of varying severity

Taiwan has experienced several outbreaks of enterovirus 71 (EV71) infections since 1998. This study examined the quantitative relationship between specific cytokines in the cerebrospinal fluid (CSF) and the severity of EV71 brain stem encephalitis (BE), and investigated whether the CSF cytokine response differed from that to uncomplicated echovirus meningitis (EM). The study included 57 children with EV71 BE, of whom 24 had isolated BE, 24 had autonomic nervous system (ANS) dysregulation, and nine had pulmonary oedema (PE), and 15 children with EM. All were confirmed by virus culture. Mean CSF glucose, total protein and lactate levels were increased significantly in association with the severity of EV71 BE. The mean CSF concentration of interleukin (IL)-1beta in children with EV71 PE was significantly higher than in those with isolated BE. IL-6 and interferon (IFN)-gamma levels were significantly higher for EV71 PE and ANS dysregulation than for isolated BE. In contrast, EM was associated with high levels of IL-1beta and low levels of IFN-gamma. Cytokines in the central nervous system, as well as in blood, appear to be involved in the pathogenesis of EV71 BE.     

Association of EV71 3C polymorphisms with clinical severity

Objectives

Enterovirus 71 (EV71) may cause neurological and fatal cases. EV71 3C plays an important role on viral replication and possess proteolysis activity. To delineate pathogenesis of EV71 virulence, we studied EV71 3C genetics, protease activity and correlated the results with clinical severity.

肠道病毒71型诱导Bax依赖的细胞凋亡

目的研究肠道病毒71型（EV71）诱导细胞凋亡的分子机制。方法采用CCK8比色法检测EVT1感染对于人横纹肌肉瘤细胞RD增殖的影响,通过Hoechst33342染色和Caspase3活性测定检测细胞凋亡特征,用WesternBlot方法检测凋亡相关蛋白Caspase3和8的激活。同时通过免疫共沉淀检测EV71感染后细胞内Bax的活化。结果EV71感染RD细胞可以明显抑制细胞增殖,引起Caspase3、8和PARP蛋白的激活,同时引起Bax蛋白表达增加并发生构象变化。结论EV71通过Bax构象变化诱导Caspase依赖的细胞凋亡。     

EV71型手足口病乳鼠动物模型的建立及免疫、内分泌及病理特征研究

目的建立EV71型手足口病乳鼠动物模型并进行免疫、致病特性研究。方法将临床分离的EV71病毒株经蚀斑纯化,3T3细胞适应,乳鼠驯化,最终获得1株能致死7日龄乳鼠的EV71毒株,命名为BJ09/07(GenBank Accession NO.JQ319054,EV71-BJ)。EV71-BJ感染7日龄ICR乳鼠后,观测临床疾病得分、体质量变化、死亡率并测定病毒载量、免疫分子、内分泌水平、组织病理损伤等病毒、免疫、内分泌、病理指标。结果 EV71-BJ毒株感染7日龄ICR乳鼠,其病毒毒力为150 LD50/ml,感染后不同时期肌肉病毒载量均高于脑中,至第4天达到高峰,后不断下降。至感染后第6天发病达高峰时做病理检测,相对于脑组织,肌肉中有更严重的淋巴细胞浸润,引起更严重的炎性分子升高。肌肉研磨液和血清中MCP-1、IFN-γ、IL-6和TNF-α显著升高,而肾上腺素和皮质醇未见明显变化。结论初步建立了EV71型手足口病乳鼠动物模型,为药物筛选、疫苗研发及免疫机理研究奠定了基础。     

VP1-Binding Protein Glucose-regulated protein 78 as an important mediator for Enterovirus 71 infecting Human Brain Microvascular Endothelial Cells

Purpose: Enterovirus 71 (EV71) is one of the main pathogens causing hand, foot and mouth disease, which could even induce severe brain damage in some patients. As the underlying mechanism of the invasion and replication process still remains largely unknown, we investigated the role of candidate proteins expressed during EV71 invasion in human brain microvascular endothelial cells (HBMECs) to delineate the pathophysiological mechanism of EV-71 infection. Materials and Methods: Ninety-one candidate EV71-associated proteins which could bind the major capsid protein (viral protein 1 [VP1]) of EV71 on the HBMEC were identified by applying an analysis of glutathione-S-transferase pull-down coupling with liquid chromatography-electrospray ionisation-tandem mass spectrometry (LC-ESI-MS/MS). Seventy-eight kDa glucose-regulated protein 78 (GRP78) binding to the VP1 protein was further validated by co-immunoprecipitation, immunofluorescence and western blot analysis. To explore the role of GRP78 in EV71 infection, GRP78 was knocked down and overexpressed in HBMEC and was verified by TCID50 assay. Results: LC-ESI-MS/MS-identified 91 proteins were subjected to gene ontology analysis, and on molecular and biological function analysis revealed GRP78 act as an important binding protein in mediating EV71 infection. In addition, immunofluorescence demonstrated the co-localisation of GRP78 and VP1 in cytoplasm of the infected HBMEC. The TCID50 assay showed that knockdown of GRP78 could attenuate the replication capacity of EV71 in HBMEC, and the overexpression could increase the virus titre in HBEMC at 24 h post-infection suggesting that GRP78 was associated with the replication capacity of EV71 in HBMEC. Conclusion: These findings provided evidence that GRP78 plays an important role during the progression of EV71 infection as a mediator in HBMEC.