深圳市手足口病重症感染性克隆的构建和分析

目的构建肠道病毒71型（EV71）的感染性克隆,为EV71致病机理的研究和药物的开发建立技术平台。方法选取临床手足口重症儿童粪便样品,经荧光定量PCR检测为阳性,RT-PCR方法扩增出EV71全基因组,通过TA克隆的方法连接到TOPO-XL-PCR载体中,应用T7聚合酶系统将线性化的EV71DNA序列体外转录成RNA,转染人横纹肌肉瘤细胞系RD-A细胞,病毒传代并观察病变,通过间接免疫荧光实验进一步鉴定,获得的感染性克隆进行全基因组测序和序列比对分析。结果 RT-PCR可以获得EV71全长约7.5kb的DNA片段,体外转录并转染后3～5d可观察到典型的肠道病毒致细胞病变,免疫荧光可看到特异标记,序列比对为C4a亚型的EV71病毒。结论构建出具有感染性的EV71全长cDNA克隆,在分子生物学水平上有利于深入研究EV71的致病机制和毒力基因、患者抗体的中和特性、疫苗和药物的效力评估与开发等。     

利巴韦林对肠道病毒体外抑制作用的研究

目的研究抗病毒药物利巴韦林体外抗肠道病毒（EV）的效果。方法采用细胞病变效应（CPE）法和MTT分析法,观察利巴韦林对肠道病毒（EV71、CAV16、CBV3、ECH011、EV84）的抑制作用。结果利巴韦林对Vero细胞的半数毒性浓度（TC50）为2．09mg／mL,0．2mg／mL浓度的利巴韦林对5种肠道病毒均有抑制作用,对CAV16、EV71、ECHO11、EV84、CBV3的抑制率分别为13％、27％、36％、23％、58％。对EV84、EV71病毒,利巴韦林浓度为0．1mg／mL时其抑制率为16．5％、29．5％;对CBV3病毒,利巴韦林抗病毒作用与其剂量呈正相关,半数有效浓度（IC50）为0．125mg／mL,治疗指数（TI）为16．72。结论利巴韦林在体外对肠道病毒具有抑制作用,对不同的肠道病毒其抑制效率不同,对CVB3的抑制率较高。     

自噬抑制剂3-MA抑制EV71病毒颗粒的产生与释放

目的 通过研究肠道病毒71型（Enterovirus 71,EV71）感染引起细胞的自噬及抑制自噬对病毒滴度的影响,为进一步明确EV71的致病机制提供基础.方法 利用Western Blot检测EV71感染后RD-A细胞内源性LC3的型别转换和P62的降解来指示细胞发生自噬的水平,通过测定染毒细胞培养上清中EV71病毒CCID50来观察抑制自噬后细胞释放EV71感染性病毒颗粒的变化.结果 EV71的感染促进了细胞LC3的型别转换和F62的降解,诱导了细胞发生自噬;3-MA抑制细胞自噬后,EV71染毒细胞产生的感染性EV71病毒颗粒数量减少.结论 EV71可以诱导细胞发生自噬,细胞自噬可能促进EV71感染性病毒颗粒的产生和释放.     

蚯蚓体腔液体外抗呼吸道合胞病毒的实验研究

目的研究蚯蚓体腔液（Earthworm coelomic fluid,ECF）体外抗呼吸道合胞病毒（RSV）的作用。方法以Hep-2细胞为宿主细胞,利巴韦林作为阳性对照药物,通过观察细胞病变效应（CPE）和MTT染色法来研究蚯蚓体腔液体外抗RSV的作用。结果ECF和利巴韦林对Hep-2细胞的CC50分别为3．11mg／ml和1．35mg／ml。在直接杀灭作用实验中,IC50为184．1μg／ml,SI值为16．87;在抑制病毒复制实验中,IC50为1555．8μg／ml,SI值为1．99;在阻断病毒侵入实验中,没有观察到抗病毒的作用。结论蚯蚓体腔液具有直接杀灭和抑制RSV复制的作用,其中直接杀灭作用的抗病毒效果较明显,但不具有阻断RSV入侵的作用。     

三白草水提液抗肠道病毒71型作用初探

目的：初步探讨三白草水提液抗肠道病毒71型（EV71）的活性及其机制。方法：采用EV71感染非洲绿猴肾上皮（Vero细胞）模型,以不同浓度的三白草水提液提前预处理细胞,结合MTT方法检测细胞活力,采用TCID50的方法检测子代病毒的释放,评价三白草水提物抗EV71病毒能力。应用Realtime PCR的方法检查三白草水提物对炎症因子产生的作用。以免疫印迹法检测三白草水提物对NF-κB信号通路的影响。利巴韦林为阳性对照。结果：三白草水提物0.1、0.03、0.015 mg/ml的浓度下能够显著抑制EV71感染诱导的细胞死亡,并且这种抑制作用具有剂量依赖性。与病毒组相比,三白草水提液在0.1、0.03以及0.015 mg/ml的浓度下分别将病毒滴度降低了1.60 logs、1.85 logs和3.45 logs。利巴韦林将病毒滴度降低了3.54 logs。Realtime PCR的结果显示,三白草水提液能够明显降低EV71诱导的IL-1β、IL-6以及IL-8 mRNA的表达。提取核蛋白,免疫印迹检测结果显示,三白草水提物能够阻断EV71诱导的NF-κB 核转移。结论：三白草水提液在体外具有抗EV71病毒复制的活性,并且可能通过抑制NF-κB 核转移抑制炎症因子的产生。     

自噬抑制剂3-MA抑制EV71病毒颗粒的产生与释放

目的 通过研究肠道病毒71型(Enterovirus 71,EV71)感染引起细胞的自噬及抑制自噬对病毒滴度的影响,为进一步明确EV71的致病机制提供基础.方法 利用Western Blot检测EV71感染后RD-A细胞内源性LC3的型别转换和P62的降解来指示细胞发生自噬的水平,通过测定染毒细胞培养上清中EV71病毒CCID50来观察抑制自噬后细胞释放EV71感染性病毒颗粒的变化.结果 EV71的感染促进了细胞LC3的型别转换和F62的降解,诱导了细胞发生自噬;3-MA抑制细胞自噬后,EV71染毒细胞产生的感染性EV71病毒颗粒数量减少.结论 EV71可以诱导细胞发生自噬,细胞自噬可能促进EV71感染性病毒颗粒的产生和释放.

Abstract：

Objective To determine whether or not enterovirus 71 ( enteroviurs 71, EV71) may induce autophagy and affect the production and release of EV71 after the treatment of autophagy inhibitor. Methods Western blots were performed to examine the conversion of LC3- Ⅰ to LC3-Ⅱ and the degradation of P62 after the RD-A cells were infected with EV71. CCID50 was determined by checking the virus titer in the supernatant of cells that treated with autophagy inhibitor 3-MA. Results EV71 infection enhances the type conversion of LC3 and degradation of P62. The infectious virus particles were decreased after the treatment of 3-MA. Conclusion EV71 infection could induce cell autophagy and the autophagy might contribute to the production and release of infectious EV71 particles.     

清开灵注射液对登革病毒Ⅱ型的抗病毒作用研究

目的 检测清开灵注射液在细胞模型上抗登革病毒Ⅱ型(DENV-Ⅱ型)作用.方法 本实验以白纹伊蚊C6/36细胞为宿主细胞,阿昔洛韦(ACV)为阳性对照药物,通过观察细胞病变效应(CPE)和改良MTT法检测细胞存活率来测定药物的细胞毒性、药物对DENV-Ⅱ的直接灭活作用、以及药物抗DENV-Ⅱ对细胞的吸附和对DENV-Ⅱ在细胞内复制的抑制作用.结果 该药在体外对DENV-Ⅱ无直接灭活作用,也不能阻止其对细胞的吸附,但对病毒在细胞内的增殖有明显的抑制作用,且呈一定的剂量效应依赖性.结论 该药在体外有一定的抗DENV-Ⅱ感染作用.     

药物抗CBV3及ECHO11的体外实验研究

目的 寻找抗肠道病毒（EV）感染的安全有效的药物。方法 采用细胞形态观察、MTT法经色检测利巴韦林、双黄连、大蒜素的细胞毒性,并以观察CPE、MTT法比色及蚀斑抑制实验判断其抗CBV3和ECHO11的活性和进行三者间及后二者病毒吸附前后的药效比较。结果 ①利巴韦林TC50为2mg/ml,在1mg/ml-1.5mg/ml时有抑制CBV3和ECHO11的活性。②双黄连TC50为5mg/ml,在0.5mg/ml时即有抑制CBV3和ECHO11的活性,且与药物浓度呈正相关。③大蒜素TC50为12.5μg/ml,在2.5μg/ml-7.5μg/ml时有抑制CBV3和ECHO11的活性。④1.5mg/ml利巴韦林对CBV3及ECHO11的蚀斑抑制率分别为43.2%和37.2%,2.5mg/ml双黄连为81.1%和88.0%,5μg/ml大蒜素为66.2%和77.4%。⑤双黄连在病毒吸附前用药蚀斑抑制率高于吸附手用药（P＜0.05）;大蒜素的差异则不显著（P＞0.05）。结论 三种药物均有体外抗CBV3、ECHO11的作用,但双黄连、大蒜素优于利巴韦林。三种药物中以双黄连毒性最小,抑制病毒活性最高。双黄连在病毒吸附前用药抗病毒活性优于病毒吸附后,有一定预防作用。     

雄黄纳米微粒在细胞水平上抑制腺病毒复制的初步研究

目的 建立腺病毒3型( HAdV-3)感染Hep-2细胞模型,观察中药雄黄对腺病毒3型( HAdV-3)感染Hep-2细胞病变的抑制作用.方法 用高能球磨机研磨双蒸水水飞处理制备雄黄纳米微粒,应用砷钼蓝染色法测定雄黄纳米微粒浓度并在Nano Series粒度测定仪上测定其粒度.以MTT法计算药物的半数中毒剂量(TC50).通过三种不同给药方式即预防给药、治疗给药及直接灭活给药方式进行体外实验,以利巴韦林为阳性对照药,观察雄黄纳米微粒对HAdV-3感染Hep-2细胞病变所起的作用,并对药物的量效关系进行分析.结果 雄黄纳米微粒TC50值为0.649 μg/ml.预防、治疗及直接灭活给药方式均可减轻HAdV-3感染Hep-2细胞的CPE程度,其抗HAdV-3的半数有效浓度( IC50)分别为0.255 μg/ml、0.142 μg/ml、0.117 μg/ml,治疗指数(TI)分别为2.55、4.57和5.55,雄黄纳米微粒对HAdV-3感染Hep-2细胞CPE的抑制作用存在着明显的量效关系.结论 雄黄纳米微粒在体外有抑制HAdV-3病毒复制和直接灭活病毒的作用,并有一定保护Hep-2细胞预防HAdV-3病毒感染的作用.     

雄黄纳米微粒在细胞水平上抑制腺病毒复制的初步研究

目的 建立腺病毒3型（ HAdV-3）感染Hep-2细胞模型,观察中药雄黄对腺病毒3型（ HAdV-3）感染Hep-2细胞病变的抑制作用.方法 用高能球磨机研磨双蒸水水飞处理制备雄黄纳米微粒,应用砷钼蓝染色法测定雄黄纳米微粒浓度并在Nano Series粒度测定仪上测定其粒度.以MTT法计算药物的半数中毒剂量（TC50）.通过三种不同给药方式即预防给药、治疗给药及直接灭活给药方式进行体外实验,以利巴韦林为阳性对照药,观察雄黄纳米微粒对HAdV-3感染Hep-2细胞病变所起的作用,并对药物的量效关系进行分析.结果 雄黄纳米微粒TC50值为0.649 μg/ml.预防、治疗及直接灭活给药方式均可减轻HAdV-3感染Hep-2细胞的CPE程度,其抗HAdV-3的半数有效浓度（ IC50）分别为0.255 μg/ml、0.142 μg/ml、0.117 μg/ml,治疗指数（TI）分别为2.55、4.57和5.55,雄黄纳米微粒对HAdV-3感染Hep-2细胞CPE的抑制作用存在着明显的量效关系.结论 雄黄纳米微粒在体外有抑制HAdV-3病毒复制和直接灭活病毒的作用,并有一定保护Hep-2细胞预防HAdV-3病毒感染的作用.     

Antiviral effect of nicotinamide on enterovirus‐infected human islets in vitro: Effect on virus replication and chemokine secretion

Type 1 diabetes is a chronic disease characterized by the selective destruction of insulin‐producing cells in the pancreas. Enterovirus (EV) is the prime candidate to initiate this destruction and several inflammatory chemokines are induced by EV infection. Nicotinamide has been shown to protect isolated human islets, and to modulate chemokine expression. The aim of this study was to evaluate the effect of nicotinamide on EV replication and EV‐induced chemokine secretion and cytolysis of human islets. Two EV strains were used to infect human islets in vitro, one lytic (Adrian) isolated from a child at onset of type 1 diabetes, and one non‐lytic (VD2921). Secretion of the chemokines IP‐10 and MCP‐1, viral replication, and virus‐induced cytopathic effect (CPE), were measured at different time points post‐infection. Addition of nicotinamide to the culture medium reduced viral replication and virus‐induced islet destruction/CPE, significantly. Both EV strains increased secretion of IP‐10 and MCP‐1, when measured days 2–3, and days 5–7 post infection, compared to mock‐infected control islets. IP‐10 was not produced by uninfected isolated islets, whereas a basal secretion of MCP‐1 was detected. Interestingly, addition of nicotinamide blocked completely (Adrian), or reduced significantly (VD2921), the virus‐induced secretion of IP‐10. Secretion of MCP‐1 was also reduced in the presence of nicotinamide, from infected and uninfected islets. The reported antiviral effects of nicotinamide could have implications for the treatment/prevention of virus‐ and immune‐mediated disease. Also, this study highlights a possible mechanism of virus‐induced type 1 diabetes through the induction of MCP‐1 and IP‐10 in pancreatic islets. J. Med. Virol. 81:1082–1087, 2009. © 2009 Wiley‐Liss, Inc.     

In vitro and in vivo evaluation of ribavirin and pleconaril antiviral activity against enterovirus 71 infection

Enterovirus 71(EV71) causes recurring outbreaks of hand, foot and mouth disease and encephalitis leading to complications or death in young children. More effective antiviral drugs are needed to prevent or reduce EV71-related disease and complications. However, there are no standard models currently in use to evaluate activity against EV71 infection both in vitro and in vivo. In this study, the activity of ribavirin and pleconaril against EV71 infection was evaluated in two models. An in vitro EV71 infection model was developed in RD cells, and an in vivo EV71 infection model was applied. Ribavirin and pleconaril effectively increased the viability of infected cells. Pleconaril reduced the morbidity and mortality of one-day-old infected mice, but ribavirin did not protect the infected mice. In all, the results demonstrated that infected cells and infected mice can be used to evaluate antiviral activity of ribavirin and pleconaril against EV71 infection in vitro and in vivo.     

microRNA-124-3p靶向调控STAT3抑制神经细胞中肠道病毒71型的复制

目的构建microRNA-124-3p表达上调和下调的SK-N-SH细胞,探讨microRNA-124-3p对肠道病毒71型(EV71)在SK-N-SH中的复制机制。方法构建过表达miRNA的miRNA mimic和敲低miRNA的miRNA inhibitor。检测miRNA-124-3p,EV71核酸,细胞病变效应(CPE),病毒滴度TCID₅₀,Ratio值,VP1、STAT3、P-STAT3、CC-ND2、MMP2蛋白表达。结果与miR-124-3p mimic NC组比,miR-124-3p mimic组miR-124-3p上升,LgTCID₅₀降低,EV71核酸的复制受抑,转染野生型miRSTAT3-WT质粒细胞活性被上调的miRNA-124-3p抑制,STAT3、P-STAT3、CC-ND2和MMP2降低(均P<0.05),与miR-124-3p inhibitorNC组比,miR-124-3p inhibitor组miR-124-3p降低,CPE增高,STAT3、P-STAT3、CC-ND2和MMP2升高(均P<0.05)。结论EV71感染SK-N-SH神经细胞,复制受抑,与microRNA-124-3p靶向抑制STAT通路分子STAT3、P-STAT3、CCND2和MMP2有关。     

Human endothelial cell activation and apoptosis induced by enterovirus 71 infection

Enterovirus 71 (EV71), a neurotropic virus, its infection is transmitted mainly by the oral-fecal route. However, it is unclear how EV71 is disseminated/spread from initial replication sites to the central nervous system. Since endothelial cells form the interface between blood and tissues throughout the body, it is likely that EV71 can infect and then exit endothelial cells to establish infection. In this study, human endothelial cells were examined for susceptibility to EV71 infection using human microvascular endothelial cell line (HMEC-1 cell). Immunofluorescence assay confirmed EV71 infection of HMEC-1. Viable viruses were cultured from both the culture supernatant and the cell lysate. Live but not UV-inactivated EV71 induced HMEC-1 to secrete IL-6, macrophage migration inhibition factor, and macrophage chemo-attractant protein 1, and to express toll-like receptor 4. In addition, EV71 decreased the viability and increased the apoptosis of HMEC-1 cells after 36-48 hr of infection. These results demonstrate that EV71 is able to infect, activate, and induce apoptosis of endothelial cells, which may play a role in the pathogenesis of EV71 infection.     

肠道病毒71型（EV71）AH3株感染性克隆的构建与鉴定

目的 构建肠道病毒71型（Enterovirus71,EV71） AH3株的全基因序列感染性克隆.方法 通过分段扩增和酶切连接将EV71全基因组cDNA片段逐步克隆入PWSK29载体,构建含全病毒基因组序列的重组质粒.转染Vero细胞后,获得拯救病毒.经RT-PCR、酶切、测序、间接免疫荧光（IFA）等方法进行鉴定.结果 酶切鉴定结果与预期一致,序列分析显示为EV71基因;其转染Vero细胞后,可观察到典型的细胞病变;所获得的拯救子代病毒滴度（TCID50）为107.5;IFA检测可见感染细胞出现绿色荧光.结论 成功构建EV71全基因序列感染性克隆,为病毒致病机理及减毒疫苗的研究奠定基础.