Department of Engineering Science and Mechanics, The Pennsylvania State University, University Park, PA 16802, USA.
Molecular Cellular and Integrative Biosciences Graduate Program, The Huck Institutes of the Life Sciences, The Pennsylvania State University, University Park, PA 16802, USA.
Cell Rep. 2017 Nov 7;21(6):1692-1704. doi: 10.1016/j.celrep.2017.10.051.
We have developed a high-throughput, microfluidics-based platform to perform kinetic analysis of viral infections in individual cells. We have analyzed thousands of individual poliovirus infections while varying experimental parameters, including multiplicity of infection, cell cycle, viral genotype, and presence of a drug. We make several unexpected observations masked by population-based experiments: (1) viral and cellular factors contribute uniquely and independently to viral infection kinetics; (2) cellular factors cause wide variation in replication start times; and (3) infections frequently begin later and replication occurs faster than predicted by population measurements. We show that mutational load impairs interaction of the viral population with the host, delaying replication start times and explaining the attenuated phenotype of a mutator virus. We show that an antiviral drug can selectively extinguish the most-fit members of the viral population. Single-cell virology facilitates discovery and characterization of virulence determinants and elucidation of mechanisms of drug action eluded by population methods.
我们开发了一种高通量、基于微流控的平台，可对单个细胞中的病毒感染进行动力学分析。我们在改变实验参数的情况下分析了数千个单独的脊髓灰质炎病毒感染，包括感染复数、细胞周期、病毒基因型和药物存在。通过基于群体的实验，我们观察到了几个出人意料的现象：（1）病毒和细胞因素对病毒感染动力学的贡献是独特且独立的；（2）细胞因素导致复制起始时间的广泛变化；（3）感染通常比群体测量预测的开始得更晚，复制得更快。我们表明，突变负荷会损害病毒群体与宿主的相互作用，从而延迟复制起始时间，并解释了突变病毒的减毒表型。我们表明，一种抗病毒药物可以选择性地消灭病毒群体中最适应的成员。单细胞病毒学有助于发现和鉴定毒力决定因素，并阐明群体方法无法阐明的药物作用机制。
