体外培养的嵌合体丙型肝炎病毒感染Huh7．5细胞的透射电镜观察

Observation of chimeric hepatitis C virus in infected Huh7.5 cell through transmission electron microscopy

目的通过透射电子显微镜技术观察体外培养的嵌合体丙型肝炎病毒（HCV）感染Huh7．5细胞后胞内病毒颗粒的形态学特征及细胞内部超微结构的变化。方法将含有全长HCV嵌合基因组的质粒pFL-J6／JFH体外转录为HCVRNA，电穿孔转染至Huh7．5细胞，实时定量聚合酶链反应（qRT-PCR）测定培养上清中病毒数量；间接免疫荧光检测病毒蛋白的表达；收取转染后细胞培养上清感染原始Huh7．5细胞，制作超薄细胞切片，透射电子显微镜技术观察被感染细胞中病毒颗粒的形态学特征及细胞超微结构的变化。结果qRT-PCR显示不同时间点收取的转染后细胞培养上清中含有高水平的病毒量；间接免疫荧光显示病毒NSSA非结构蛋白高表达；透射电子显微镜观察到被感染的Huh7．5细胞内含有大量有包膜或无包膜的病毒样颗粒，细胞质内部分膜性细胞器增生，出现黄病毒科病毒感染后特征性结构及某种未知结构等。结论体外培养的嵌合体HCV具有HCV颗粒的形态学特征，并能够有效感染人源性肝细胞Huh7．5。

Objective To observe the morphological characteristics of HCV particles and intracellular uhrastructure changes in HuhT. 5 cells which was infected with chimeric HCV via transmission electron microscopy. Methods Plasmid J6/JFH encoding the full length HCV chimeric genome was transcribed to HCV RNA in vitro and the RNA was transfected into Huh7.5 cells by electroporation. Quantitative real-time PCR（qRT-PCR） was used to assay HCV copies of the supernatant of transfected cells. Indirect immunofluorescence was used to detect the expression of HCV proteins. The cell culture supernatant were used to infect naive Huh7.5 cells, transmission electron microscopy was used to observe morphological characteristics of vires particles and intracellular uhrastructure changes in infected Huh7.5 cells. Results qRT-PCR showed high level virus copies in supernatant of transfected cells collected in different times, indirect immuno-fluorescence proved high expression of HCV NSSA proteins in the transfected cells. Large numbers of enveloped or unenveloped virus-like particles （VLPs） were observed in infected Huh7.5 cells via transmission electron microscopy. We also found hyperplasia of some membrane-enclosed organelles in the cytoplasm. Several features characterizing flavivirus infected ceils and a cytoplasmic inclusion of unknown origin were observed. Conclusion The chimeric HCV from in vitro cell culture system is proved to be intact virus particles which can efficiently infect Huh7.5 cells.