减蛋综合征病毒进入宿主细胞途径的研究

发布时间：2018-12-07 07:07

【摘要】：鸡产蛋下降综合症是由减蛋综合征病毒(Egg drop syndrome virus,EDSV)引起的以产蛋鸡的产蛋量降低、蛋体畸形、蛋品品质低劣等症状的传染性疾病。目前,关于EDSV相关的研究文献相对较少,大多集中于疾病诊断,血清抗体水平监测以及对该病毒基因组的相关分析。因此,关于EDSV感染特性以及病毒进入细胞的途径还不是很清楚。本试验目的在于探究EDSV经入宿主细胞的途径,以及病毒对鸭胚成纤维(Duck embryonic fibroblast,DEF)细胞和鸡胚肝(chick embryo liver,CEL)细胞的感染特性,从而为研究该病毒对宿主细胞的感染机制提供相关的理论依据及技术支持。1.根据GenBank上已发表的EDSV全基因序列(Y09598.1),设计合成了一对扩增Penton基因的特异性引物,通过基因扩增,载体构建及标准阳性质粒的梯度稀释,构建了一条用于实时荧光定量PCR方法检测EDSV的标准曲线。结果表明,该方法具有敏感、重复性好等优点,可直接用于对EDSV病毒粒子的定量检测。用EDSV感染原代DEF和CEL细胞,同时在感染后不同时间点用实时荧光定量PCR方法检测病毒的增殖情况。结果显示,EDSV能够引起DEF和CEL细胞发生细胞病变,在感染后72 h,细胞病变最为明显,且细胞上清液中病毒的滴度最高,病毒HA效价分别为212和211。此外,病毒增殖曲线显示DEF细胞的病毒滴度在整个检测时间点上都要比CEL细胞的病毒滴度高。2.用EDSV感染DEF细胞后0 min,10 min,20 min,30 min,2 h,and 72 h,分别制备透射电子显微镜超薄切片并用4%乙酸双氧铀染色。结果表明,在感染后10 min,EDSV就能触发DEF细胞膜内陷,在20 min时,即能够形成完整的包裹病毒的Qg吞囊泡,随后包裹病毒粒子的囊泡向细胞内部运输。在感染72 h时,细胞内聚集大量的病毒粒子,且细胞发生破裂。3.用chlorpromazine(CPZ,特异性抑制clathrin介导内吞作用)、sucrose(高浓度的蔗糖能够抑制clathrin介导内吞作用)、NH4Cl和MβCD(caveolin介导内吞作用和脂质筏介导的内吞作用)处理DEF细胞后感染EDSV,用实时荧光定量PCR检测EDSV含量变化。结果显示,CPZ和sucrose处理组能够显著降低病毒含量,并呈现剂量依赖性,而MβCD处理组无显著性差异。同时,NH4Cl也能够显著降低病毒含量。4.为观察抑制剂对EDSV进入的影响,用CPZ(50μmol/L)和sucrose(100 mmol/L)在37°C条件下处理DEF细胞1 h,感染EDSV,未感染的DEF细胞作为阴性对照,感染EDSV的未处理的DEF细胞作为阳性对照。以制备的鼠抗EDSV高免血清作为一抗进行间接免疫荧光染色。结果表明,与对照相比,CPZ和sucrose处理的DEF细胞,荧光聚集在细胞周围,成点状分布,而阳性对照则呈弥散状分布于细胞内部。所有结果表明EDSV能够通过内吞作用进入细胞,且进入细胞的途径是clathrin介导的内吞作用,并且具有PH依赖性。
[Abstract]:Egg drop syndrome (EPS) is a kind of infectious disease caused by egg drop syndrome virus (Egg drop syndrome virus,EDSV), which is characterized by egg production reduction, egg body malformation and poor egg quality. At present, there are relatively few studies on EDSV, mostly focused on disease diagnosis, serum antibody level monitoring and the analysis of the genome of the virus. Therefore, the characteristics of EDSV infection and the way the virus enters cells are not well understood. The purpose of this study was to investigate the pathway of EDSV into host cells and the infection characteristics of the virus on duck embryo fibroblast (Duck embryonic fibroblast,DEF cells and chicken embryo liver (chick embryo liver,CEL cells. Therefore, it provides relevant theoretical basis and technical support for studying the infection mechanism of the virus on host cells. 1. According to the published EDSV gene sequence of GenBank (Y09598.1), a pair of specific primers for amplification of Penton gene were designed and synthesized. The primers were constructed by gene amplification, vector construction and gradient dilution of standard positive plasmid. A standard curve for real-time fluorescence quantitative PCR detection of EDSV was constructed. The results show that the method is sensitive and reproducible, and can be directly used for quantitative detection of EDSV virus particles. The primary DEF and CEL cells were infected with EDSV, and the virus proliferation was detected by real-time fluorescence quantitative PCR at different time points after infection. The results showed that EDSV could induce cytopathic changes in DEF and CEL cells. The cytopathic effect was the most obvious at 72 h after infection, and the titer of virus in the supernatant was the highest. The titer of virus HA was 212 and 211respectively. In addition, the virus proliferation curve showed that the virus titer of DEF cells was higher than that of CEL cells. Transmission electron microscope (TEM) ultrathin sections were prepared at 0 min,10 min,20 min,30 min,2 and 72 h after DEF cells were infected with EDSV. The results showed that at 10 min,EDSV after infection, the DEF cell membrane trapping could be triggered. At 20 min, the complete Qg endocytosis vesicles could be formed, and then the vesicles encapsulated the virus particles were transported into the cells. After 72 h of infection, a large number of virus particles were accumulated in the cells and the cells ruptured. ), sucrose (high concentration sucrose can inhibit clathrin mediated endocytosis) NH4Cl and M 尾 CD (caveolin mediated endocytosis and lipid raft mediated endocytosis) treatment of DEF cells infected with EDSV, Real-time fluorescence quantitative PCR was used to detect the change of EDSV content. The results showed that CPZ and sucrose could significantly decrease the virus content in a dose-dependent manner, but there was no significant difference in M 尾 CD treatment. At the same time, NH4Cl can also significantly reduce the virus content. In order to observe the effect of inhibitor on EDSV entry, DEF cells were treated with CPZ (50 渭 mol/L) and sucrose (100 mmol/L) at 37 掳C for 1 hour, and DEF cells infected with EDSV, were used as negative control. Untreated DEF cells infected with EDSV served as positive control. The prepared mouse anti-EDSV high-immune serum was used as the first antibody for indirect immunofluorescence staining. The results showed that the fluorescence of DEF cells treated with CPZ and sucrose gathered around the cells and distributed in dots, while the positive control cells distributed diffusely in the cells. All the results showed that EDSV could enter cells through endocytosis, and the pathway to enter cells was clathrin mediated endocytosis, which was PH dependent.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.65

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