Single-Cell Virology: On-Chip Investigation of Viral Infection Dynamics

Abstract

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; (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.

More than efficacy revealed by single-cell analysis of antiviral therapeutics

“…The era of single-cell virology has begun (7,(32)(33)(34)(35). Infection outcomes in cells are determined, in part, by stochastic processes (36).…”

“…As observed previously, viruses with the highest yields (maximum) and fastest rates of replication (slope) were sensitive to 2′-C-Me-A ( fig. S6B) (7). An efficient polymerase is likely required to replicate fast and to the highest levels.…”

“…To address this problem, we created a multilayer device as previously described ( Fig. 1A) (7) but added a trapping structure to the channel layer of the device ( Fig. 1B and fig.…”

“…In a previous study, we evaluated infections that survived treatment when present at a concentration that reduces the number of infections by 50% [median inhibitory concentration (IC 50 )]. To our surprise, we observed selective ablation of cells infected with virus variants capable of the fastest rates of replication and the highest yields of replicated RNA (7). This experiment suffered from the inability to evaluate concentrations of drug higher than the value of the IC 50 .…”

“…As a part of a study evaluating PV infection dynamics on the singlecell level, we observed that a chain-terminating antiviral ribonucleotide selectively eliminates the most-fit members of the viral population (7). This class of antiviral agent has always been touted as having a high barrier to resistance (8).…”