The use of single-cell RNA-seq to study heterogeneity at varying levels of virus-host interactions.

The outcome of viral infection depends on the diversity of the infecting viral population and the heterogeneity of the cell population that is infected. Until almost a decade ago, the study of these dynamic processes during viral infection was challenging and limited to certain targeted measurements. Presently, with the use of single-cell sequencing technology, the complex interface defined by the interactions of cells with infecting virus can now be studied across the breadth of the transcriptome in thousands of individual cells simultaneously. In this review, we will describe the use of single-cell RNA sequencing (scRNA-seq) to study the heterogeneity of viral infections, ranging from individual virions to the immune response between infected individuals. In addition, we highlight certain key experimental limitations and methodological decisions that are critical to analyzing scRNA-seq data at each scale

Single-cell RNA sequencing methods discussed in this review.

Single-cell RNA sequencing methods discussed in this review.</p

Table describing the caveats and experimental modifications that can aid in scRNA-seq to assess heterogeneity at different levels.

Table describing the caveats and experimental modifications that can aid in scRNA-seq to assess heterogeneity at different levels.</p

Example computational tools useful in the analysis scRNA-seq datasets during the study of virus–host interactions.

Example computational tools useful in the analysis scRNA-seq datasets during the study of virus–host interactions.</p

Broad examples of heterogeneity probed by scRNA-seq in the context of viral infection.

Specific instances of each provided in the text of the appropriate section.</p

Single-cell sequencing workflow for droplet-based approaches exemplified by the 10x Genomics Chromium platform.

Infected cells from cell culture, tissues/organoids, or infected individuals are dissociated into a single-cell suspension. The cell suspension is loaded onto a microfluidic chip, and cells are partitioned into nanoliter-scale Gel Beads-in-emulsion (GEMs) droplets containing barcoded gel beads and reagents for reverse transcription (RT). Following cell lysis, the beads capture the mRNA molecules. Reverse transcription (RT) by template switching using a template switching oligonucleotide (TS) generates cDNA tagged with a 10x barcode (BC) to identify the cell and a unique molecular identifier (UMI) to label the mRNA transcript. The pooled cDNA is amplified in bulk, fragmented by enzymatic fragmentation, and sequencing adapters (P5 and P7) including a sample index, are added to the fragments by PCR to generate sequencing libraries. The sequencing libraries are sequenced, and the data are analyzed by alignment and demultiplexing, following which the data are interpreted.</p

Heterogeneity of ROS levels in antibiotic-exposed mycobacterial subpopulations confers differential susceptibility

Phenotypically heterogeneous but genetically identical mycobacterial subpopulations exist in in vitro cultures, in vitro-infected macrophages, infected animal models and tuberculosis patients. In this regard, we recently reported the presence of two subpopulations of cells, which are phenotypically different in length and buoyant density, in mycobacterial cultures. These are the low-buoyant-density short-sized cells (SCs), which constitute similar to 10-20% of the population, and the high-buoyantdensity normal/long-sized cells (NCs), which form similar to 80-90% of the population. The SCs were found to be significantly more susceptible to rifampicin (RIF), isoniazid (INH), H2O2 and acidified nitrite than the NCs. Here we report that the RIF-/INH-/H2O2 -exposed SCs showed significantly higher levels of oxidative stress and therefore higher susceptibility than the equivalent number of exposed NCs. Significantly higher levels of hydroxyl radical and superoxide were found in the antibiotic-exposed SCs than in the equivalently exposed NCs. Different proportions of the subpopulation of SCs were found to have different levels of reactive oxygen species (ROS). The hydroxyl radical quencher, thiourea, and the superoxide dismutase mimic, TEMPOL, significantly reduced hydroxyl radical and superoxide levels, respectively, in the antibiotic-exposed SCs and NCs and thereby decreased their differential susceptibility to antibiotics. Thus, the present study shows that the heterogeneity of the reactive oxygen species (ROS) levels in these mycobacterial subpopulations confers differential susceptibility to antibiotics. We have discussed the possible mechanisms that can generate differential ROS levels in the antibiotic-exposed SCs and NCs. The present study advances our current understanding of the molecular mechanisms underlying antibiotic tolerance in mycobacteria

Maximal interferon induction by influenza lacking NS1 is infrequent owing to requirements for replication and export.

Influenza A virus exhibits high rates of replicative failure due to a variety of genetic defects. Most influenza virions cannot, when acting as individual particles, complete the entire viral life cycle. Nevertheless influenza is incredibly successful in the suppression of innate immune detection and the production of interferons, remaining undetected in >99% of cells in tissue-culture models of infection. Notably, the same variation that leads to replication failure can, by chance, inactivate the major innate immune antagonist in influenza A virus, NS1. What explains the observed rarity of interferon production in spite of the frequent loss of this, critical, antagonist? By studying how genetic and phenotypic variation in a viral population lacking NS1 correlates with interferon production, we have built a model of the "worst-case" failure from an improved understanding of the steps at which NS1 acts in the viral life cycle to prevent the triggering of an innate immune response. In doing so, we find that NS1 prevents the detection of de novo innate immune ligands, defective viral genomes, and viral export from the nucleus, although only generation of de novo ligands appears absolutely required for enhanced detection of virus in the absence of NS1. Due to this, the highest frequency of interferon production we observe (97% of infected cells) requires a high level of replication in the presence of defective viral genomes with NS1 bearing an inactivating mutation that does not impact its partner encoded on the same segment, NEP. This is incredibly unlikely to occur given the standard variation found within a viral population, and would generally require direct, artificial, intervention to achieve at an appreciable rate. Thus from our study, we procure at least a partial explanation for the seeming contradiction between high rates of replicative failure and the rarity of the interferon response to influenza infection

Defective populations show a depletion in full-length polymerase segments.

Indicated viral populations were subjected to influenza-specific cDNA synthesis and analyzed by a set of primers that either sit external to deletions (All) or internal (Full-length). The Full-length signal was normalized to the All signal for each segment, smaller numbers represent a depletion of full-length segment relative to the total population. Asterices indicate a significant depletion between high and low defective populations, two-tailed t test with Benjamini-Hochberg multiple testing correction at an FDR of 0.05, n = 4 two technical and two biological replicates. (TIF)</p