Extreme heterogeneity of influenza virus infection in single cells

Abstract

Viral infection can dramatically alter a cell’s transcriptome. However, these changes have mostly been studied by bulk measurements on many cells. Here we use single-cell mRNA sequencing to examine the transcriptional consequences of influenza virus infection. We find extremely wide cell-to-cell variation in the productivity of viral transcription – viral transcripts comprise less than a percent of total mRNA in many infected cells, but a few cells derive over half their mRNA from virus. Some infected cells fail to express at least one viral gene, but this gene absence only partially explains variation in viral transcriptional load. Despite variation in viral load, the relative abundances of viral mRNAs are fairly consistent across infected cells. Activation of innate immune pathways is rare, but some cellular genes co-vary in abundance with the amount of viral mRNA. Overall, our results highlight the complexity of viral infection at the level of single cells.

Dissection of Influenza Infection In Vivo by Single-Cell RNA Sequencing

“…We found that epithelial cells manifest a wide range of viralload states spanning two orders of magnitude, with 33%, 23%, and 44% of infected epithelial cells characterized respectively by low, medium, and high viral-load states (Figures 2D and S4C). Our data therefore confirm the previously reported epithelial heterogeneity in vitro (Russell et al, 2018) and, in addition, extends this result to include the case of in vivo infection. Interestingly, we found a clear distinction between epithelial cells and the rest of the cell types, with the observation that infected cells, in all of the non-epithelial cell types, mainly displayed low heterogeneity of viral-load states: the vast majority (85%-100% of the infected cells in the different cell types) maintained low viral-load states, whereas high viral-load states were maintained by only up to 1.1% of those cells ( Figure 2D and Table S3).…”

“…Recent studies have demonstrated a wide range of intracellular virus-related states, including heterogeneity in viral transcription and virus titers. For example, in vitro infection of epithelial cells results in wide heterogeneity of transcriptional viral loads (Russell et al, 2018), and during in vitro infection, low viral loads are prevalent in Madin-Darby canine kidney epithelial (MDCK) cells (Brooke et al, 2013). Here, focusing on in vivo infection, we found that epithelial cells indeed display high viral-load heterogeneity, whereas the remaining cell types maintain low viral-load states ( Figure 2D).…”

“…We next focused on the composition of viral loads in the subpopulation of infected cells. A recent report described a wide range of viral loads during influenza infection of epithelial cells in vitro (Russell et al, 2018). We wanted to find out whether this epithelial cell property also applies during in vivo infection, and if so, we further aimed to assess the relevance of this finding to additional cell types.…”

“…'Bystanders' were defined as cells derived from an influenza-treated mouse in which no viral segments were detected. These definitions were previously used in a combined single-cell mapping of host and viral transcriptome (Russell et al, 2018). To determine the quality of this categorization we used precision and recall (sensitivity) measures, as well as the percentage of false negatives (Supplemental Experimental Procedures).…”

“…For example, while epithelial cells are known to be the main targets of the influenza virus, several studies have documented that other cell types, such as endothelial cells, natural killer (NK) cells, macrophages, and dendritic cells (DCs), are also susceptible to the influenza virus, with potential implications of intracellular infection for their functionality (Manicassamy et al, 2010;McFadden et al, 2009). Complexity may also be related to a wide range of viral transcriptional states within the infected cells (Russell et al, 2018;Xin et al, 2018;Zanini et al, 2018), as well as to the heterogeneity of host-response states (Avraham et al, 2015). Another complication may be attributable to the dual role of the metabolic machinery in supporting the host while also limiting the energetic demands of the viral life cycle (Jovanovic et al, 2015;Kissig et al, 2017).…”