鸭胚成纤维细胞中鸭瘟强毒的增殖特性研究

Studies on the Proparagation Characteristics of Duck Plague Virulent Virus in Duck Embryo Fibroblasts

通过细胞培养物光学显微观察、细胞超薄切片研究、定量PCR检测技术对鸭胚成纤维细胞中鸭瘟强毒的增殖特性进行了研究。结果表明:鸭瘟强毒接种鸭胚成纤维细胞后42h,可使细胞培养物出现大量明显的蚀斑病变。细胞培养物的超薄切片电镜观察研究表明,病毒核酸在胞核内常集中分布,呈直径35～45nm的圆形颗粒状;核衣壳在胞核和胞浆内都有分布,呈直径90～100nm的圆形颗粒状;成熟病毒位于胞浆空泡内,呈直径150～300nm的圆形,有囊膜和皮层结构;病毒通过囊膜与胞膜融合入侵细胞,在核内生成核酸、装配核衣壳,在胞浆中得到皮层,出芽到胞浆空泡内获得囊膜,通过胞吐作用释放到胞外。定量PCR研究表明:鸭瘟强毒接种细胞后10h开始明显复制,接毒后30h时含量趋于稳定,接毒后22h时开始向胞外释放,50h时达最大值,细胞和上清中病毒含量的增幅均为103左右,病毒主要存在于细胞中,其含量为上清的102～103倍。

The propagation characteristics of virulent duck plague virus （DPV） in duck embryo fibroblast （DEF） were studied by the method of light microscopy observation of DEF cell culture monolayer, electron microscopy observation of infected DEF cell culture, sults demonstrated that on duck embryo fibroblast a real-time PCR detecting virus propagation. The re number of plaques were formed by DPV 42 h postin fection. Electron microscopy of the ultrathin section of infected duck embryo fibroblasts demonstrated that the nucleic acid of DPV was round in shape with diameter of 35-45 nm and was often in a cluster in the nucleus of DEF. The nucleocapsid of DPV was round in shape with diameter of 90-100 nm and could be observed both in nucleus and cytoplasm of DEF. The mature DPV which had the structures of envelop and tegument was spherical in shape with diameter of 150-300 nm and was located in cytoplasmic vacuoles. DPV penetrated the DEF cell membrane by direct fusion between the viral envelop and the plasma membrane. Progeny viral nucleic acid was produced in the nucleus and the assembled nucleocapsids obtained the structure of tegument in the cytoplasm and obtained the structure of envelop by budding into the cytoplasmic vesicles. The mature DPV particles were released out of the cell through exocytosis of the cytoplasmic vesicles. Detection of DPV by real-time PCR demonstrated that virus in DEF began its obvious propagation 10 h postinfection and virus amount tended to increase until 30 h postinfection. DPV began to be released into the supernatant 22 h postinfection and the DPV amount peaked 50 h postinfection, when the virus content in DEF and supernatant both underwent approximately 10^3 fold increase. DPV mainly existed in the DEF and the virus content in DEF was 10^2-10^3 fold than the supernatant.