Morales Marisol, Zhang Fushun, Xiang Yan
The University of Texas Health San Antonio, Department of Microbiology, Immunology, & Molecular Genetics, San Antonio, TX, USA.
Methods Mol Biol. 2025;2860:219-227. doi: 10.1007/978-1-0716-4160-6_14.
To investigate the impact of a host factor on viral replication, conventional techniques involve analyzing viral replication within a homogeneous cell population engineered to uniformly express the host factor. In this chapter, we present a method to explore the intricate dynamics between host factors and vaccinia virus (VACV) infection using flow cytometry, which enables the rapid, multi-parametric analysis of individual cells in solution. Cultured cells are transfected to express a host factor fused with a fluorescent protein, followed by infection with a VACV encoding a different fluorescent reporter. Subsequently, cells are washed, fixed, and filtered for flow cytometry analysis. The data are then analyzed with flow cytometry analysis software to evaluate the influence of the host factor on various infection parameters, including the stage of viral infection and viral replication level. This method allows for the precise investigation of proviral or antiviral factor expression in relation to viral replication within a heterogeneous population of cells.
为了研究宿主因子对病毒复制的影响，传统技术包括在经过基因工程改造以均匀表达宿主因子的同质细胞群体中分析病毒复制。在本章中，我们介绍一种使用流式细胞术探索宿主因子与痘苗病毒（VACV）感染之间复杂动态关系的方法，该方法能够对溶液中的单个细胞进行快速、多参数分析。培养细胞被转染以表达与荧光蛋白融合的宿主因子，随后用编码不同荧光报告基因的痘苗病毒进行感染。随后，对细胞进行洗涤、固定并过滤以进行流式细胞术分析。然后使用流式细胞术分析软件对数据进行分析，以评估宿主因子对各种感染参数的影响，包括病毒感染阶段和病毒复制水平。该方法能够在异质细胞群体中精确研究与病毒复制相关的前病毒或抗病毒因子表达。
