全内反射荧光显微技术实时观察人偏肺病毒感染

Visualizing infection of individual human metapneumovirus by internal reflection fluorescence microscopy

目的利用显微成像技术拍摄并分析人偏肺病毒(human metapneumovirus,hMPV)的入胞过程,以探讨hMPV的感染免疫方式。方法使用全内反射荧光技术(total internal reflection fluorescence,TIRF)连续拍摄带有绿色荧光蛋白的病毒影像,并逐帧分析病毒的感染过程。结果感染开始后邻近培养介质的细胞膜上hMPV数量缓慢增多并在15～20 min时大量进入细胞。黏附于细胞上的病毒大部分做小振幅的无规律运动,偶见快速移动。感染12 h后hMPV在细胞膜阴影处、两细胞交界面大量聚集。结论本研究探讨了使用TIRF实时观察病毒入胞的可行性,结果表明TIRF技术能够连续观察病毒的运动规律和分布状况,为进一步揭示hMPV的感染免疫机制以及疫苗研制奠定了实验基础。

Human metapneumovirus （hMPV） is there is no morphology report about the infection. In infection, total internal reflection fluorescence （TIRr） a leading cause of respiratory infection in children. However, this study, with an aim of investigating early events of hMPV microscopy was used to observe GFP-hMPV contiguously and analyze virus characterization in every frame. The results showed that the number of hMPV particles was increased slowly at the beginning of infection and that viruses entered cells at 15-20 min post infection. When hMPV cohered to cell membrane, the movements of most individual viruses were slow and irregular and other viruses moved rapidly and bidirectionally. At 12 hour post infection, images showed that GFP-hMPV was accumulated along the specialized cell membrane sites and adherent surfaces of cells. The results suggested that TIRF provides a direct and effective way to recognize distribution and motion pattern of viruses on plasma membrane. This methodology will offer a solid foundation for further studies on pathogenesis of hMPV and development of attenuated vaccines.