Abstract
Cell-to-cell variability of infection has long been known, yet it has remained one of the least understood phenomena in infection research. It impacts on disease onset and development, yet only recently underlying mechanisms have been studied in clonal cell cultures by single-virion immunofluorescence microscopy and flow cytometry. In this review, we showcase how single-cell RNA sequencing (scRNA-seq), single-molecule RNA-fluorescence in situ hybridization (FISH), and copper(I)-catalyzed azide-alkyne cycloaddition (click) with alkynyl-tagged viral genomes dissect infection variability in human and mouse cells. We show how the combined use of scRNA-FISH and click-chemistry reveals highly variable onsets of adenoviral gene expression, and how single live cell plaques reveal lytic and nonlytic adenovirus transmissions. The review highlights how scRNA-seq profiling and scRNA-FISH of coxsackie, influenza, dengue, zika, and herpes simplex virus infections uncover transcriptional variability, and how the host interferon response tunes influenza and sendai virus infections. We introduce the concept of “cell state” in infection variability, and conclude with advances by single-cell simultaneous measurements of chromatin accessibility and mRNA counts at high-throughput. Such technology will further dissect the sequence of events in virus infection and pathology, and better characterize the genetic and genomic stability of viruses, cell autonomous innate immune responses, and mechanisms of tissue injury.
Highlights
Virus infections are multi-step processes comprising entry, genome trafficking, activation of viral gene expression, replication of the genome, particle assembly, and release of progeny
Recent advances in single-cell analyses, such as single-cell RNA sequencing, single-cell, single-molecule RNA fluorescence in situ hybridization, high-throughput fluorescence in situ DNA hybridization, and single-cell mass cytometry, have unambiguously demonstrated that biology is highly heterogeneous at the single-cell level [7,8]
The number of GFP puncta per cell increased over time, which was indicative of viral RNA replication
Summary
Virus infections are multi-step processes comprising entry, genome trafficking, activation of viral gene expression, replication of the genome, particle assembly, and release of progeny. Recent advances in single-cell analyses, such as single-cell RNA sequencing (scRNAseq), single-cell, single-molecule RNA fluorescence in situ hybridization (scRNA-FISH), high-throughput fluorescence in situ DNA hybridization, and single-cell mass cytometry, have unambiguously demonstrated that biology is highly heterogeneous at the single-cell level [7,8] This heterogeneity applies to different cell types in a tissue but is observed with clonal tissue culture cells (reviewed in [9,10]). Virus infections of single cells proceed with different kinetics and have different outcomes This has been visualized by a second-generation plaque assay, Plaque 2.0, in live cell mode using fluorescent viruses, such as human adenovirus (HAdV) (see Figure 2, and [11,12,13,14]). For coronavirus single-cell infection analyses, the reader may consider recent reviews [20,21,22,23]
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