荧光标记新城疫病毒在检测病毒入侵过程中的应用

Application of fluorescent labeled virus in the detection of the entry process of Newcastle disease virus

本研究利用荧光染料标记的新城疫病毒(NDV)检测病毒的吸附、融合和进入,旨在为研究NDV入侵细胞的机制提供一种工具。首先通过DiOC标记NDV(DiOC-NDV)检测病毒的吸附和进入,随后利用R18标记NDV(R18-NDV)检测病毒的融合,最后利用R18和DiOC同时标记NDV(DiOC/R18-NDV)进一步检测NDV在内体中的融合。病毒吸附的检测结果显示,代表NDV的绿色荧光定位于细胞表面。病毒进入的检测结果显示,60~120 min不能被台盼蓝淬灭以及被台盼蓝淬灭的绿色荧光强度均无明显变化(P>0.05);病毒融合的检测结果显示,30~120 min时均能在细胞表面和细胞质中观察到红色荧光,且60~120 min时细胞的总荧光强度无显著差异(P>0.05)。以上结果表明,NDV吸附细胞后,在细胞表面与内体中发生膜融合及进入,且该过程在60 min内完成。综上所述,荧光标记的NDV能直观、可靠地展现病毒的吸附、进入和融合过程,荧光标记NDV可作为一种研究NDV入侵细胞机制的工具。

In this study,in order to provide a tool for the study of the entry mechanism of Newcastle disease virus(NDV),virus labeled with fluorescent dye was used to detect the adsorption,fusion and internalization of NDV.Firstly,the adsorption and internalization of NDV was analyzed by DiOC-labeled NDV(DiOC-NDV).Subsequently,R18-labeled NDV(R18-NDV)was used to analyze the fusion of virus.Furthermore,R18 together with DiOC(DiOC/R18-NDV)were used to label the virus in order to further detect the fusion of NDV in the endosome compartments.The results of virus adsorption assay showed that green fluorescence representing NDV was located on the cell surface after adsorption.Subsequently,the internalization of NDV was analyzed by using DiOC-NDV,and the results of virus internalization assay revealed that there was no significant change in the intensity of green fluorescence during 60 to 120 min which resistant to trypan blue treatment and which was quenched by trypan blue(P>0.05).Furthermore,R18-labeled NDV was used to analyze fusion of virus.The results of virus fusion assay demonstrated that red fluorescence could be observed on the cell surface and cytoplasm during 30 to 120 min,and there was no significant difference in total fluorescence intensity during 60 to 120 min(P>0.05).The results ofvirus fusion occurred in the endosome showed that the overlap of red and green fluorescence signals wasobserved in the cytoplasm,indicating that the membrane fusion of NDV occurred in the endosome.Theabove results indicated that the fusion and entry process occurred on the cell surface and in the endosomewas completed within 60 min after the adsorption of NDV.Collectively,fluorescence dye labeled-NDV could intuitively and reliably exhibited the process of virus adsorption,internalizationand fusion.Therefore,NDV labeled with fluorescence dye which could be used as a tool to study the entrymechanism of NDV.