Department of Medical Biology, Medical School, University of Pécs, Szigeti 12., H-7624 Pécs, Hungary.
Enzyme Microb Technol. 2011 Aug 10;49(3):255-9. doi: 10.1016/j.enzmictec.2011.06.005. Epub 2011 Jun 12.
The present study demonstrates that the fluorescent general membrane dyes PKH67 and PKH26 are suitable to label Newcastle disease virus, an enveloped virus belonging to the family of paramyxoviridae. Adsorption of the labeled virus particles was tracked, visualized and quantitated using confocal laser scanning microscopy. The specificity of PKH-labeling was determined by colocalization analysis of the PKH signal with NDV-specific immunolabeling, and by using mock-infected controls and infection with detergent-pretreated labeled virus particles. The infectivity of the NDV particles was not affected by the labeling procedure as indicated by the results of a cytotoxicity ATP assay, an apoptosis assay and detection of virus-specific RNA and protein by qPCR and Western blotting, respectively, in cells infected with PKH-labeled and unlabeled virus particles. This technique can be used as an inexpensive, sensitive and rapid alternative method in the analysis of adsorption and internalization of enveloped viruses by the infected cells.
本研究表明，荧光通用膜染料 PKH67 和 PKH26 适合标记新城疫病毒，该病毒属于副粘病毒科的包膜病毒。使用共聚焦激光扫描显微镜跟踪、可视化和定量标记病毒颗粒的吸附。PKH 标记的特异性通过 PKH 信号与 NDV 特异性免疫标记的共定位分析以及使用mock 感染对照和用去污剂预处理的标记病毒颗粒感染来确定。细胞毒性 ATP 测定、细胞凋亡测定以及通过 qPCR 和 Western blot 分别检测感染细胞中病毒特异性 RNA 和蛋白质的结果表明，标记程序不影响 NDV 颗粒的感染性。该技术可以作为一种廉价、敏感和快速的替代方法，用于分析感染细胞中包膜病毒的吸附和内化。
