Comprehensive single cell analysis of pandemic influenza A virus infection in the human airways uncovers cell-type specific host transcriptional signatures relevant for disease progression and pathogenesis

Abstract

Respiratory viruses, such as the 2009 pandemic strain of influenza A virus (IAV, H1N1pdm09), target cells found in the human respiratory epithelium. These cells, which form a pseudostratified epithelial layer along the airways, constitute the first line of defence against respiratory pathogens and play a crucial role in the host antiviral response. However, despite their key role in host defence, it remains unknown how distinct cell types in the respiratory epithelium respond to IAV infection and how these responses may contribute to IAV-induced pathogenesis and overall disease outcome. Here, we used single cell RNA-sequencing (scRNA-seq) to dissect the host response to IAV infection in its natural target cells. scRNA-seq was performed on human airway epithelial cell (hAEC) cultures infected with either wild-type pandemic IAV (WT) or with a mutant version of IAV (NS1R38A) that induced a robust innate immune response. We then characterized both the host and viral transcriptomes of more than 19,000 single cells across the 5 major cell types populating the human respiratory epithelium. For all cell types, we observed a wide spectrum of viral burden among single infected cells and a disparate host response between infected and bystander populations. Interestingly, we also identified multiple key differences in the host response to IAV among individual cell types, including high levels of pro-inflammatory cytokines and chemokines in secretory and basal cells and an important role for luminal cells in sensing and restricting incoming virus. Multiple infected cell types were shown to upregulate interferons (IFN), with type III IFNs clearly dominating the antiviral response. Transcriptional changes in genes related to cell differentiation, cell migration, and tissue repair were also identified. Strikingly, we also detected a shift in viral host cell tropism from non-ciliated cells to ciliated cells at later stages of infection and observed major changes in the cellular composition. Microscopic analysis of both WT and NS1R38A virus-infected hAECs at various stages of IAV infection revealed that the transcriptional changes we observed at 18 hpi were likely driving the downstream histopathological alterations in the airway epithelium. To our knowledge, this is the first study to provide a comprehensive analysis of the cell type-specific host antiviral response to a respiratory virus infection in its natural target cells – namely, the human respiratory epithelium.

Disparate temperature-dependent virus – host dynamics for SARS-CoV-2 and SARS-CoV in the human respiratory epithelium

“…Nonetheless, we demonstrate that SARS-CoV-2 and SARS-CoV are sensitive to both type I and III IFN. These data are supported by several studies investigating the outcome of IFN pre-treatment in cultured cell lines 39,47,48 as well as the well-documented dominant antiviral role of type III IFN during virus infection in the respiratory epithelium 31,49,50 .…”

“…Together these results suggest that SARS-CoV infection triggers only very mild IFN induction in hAEC cultures, whereas SARS-CoV-2 infection leads to stronger induction of multiple IFNs that is dominated by type III IFNs and dependent on temperature31 .Taken together, these results clearly demonstrate that SARS-CoV-2 and SARS-CoV induce disparate, virus-specific, and temperature-dependent host responses that inversely correlate with the previously observed dissimilar viral replication efficiencies at temperatures corresponding to the upper and lower respiratory tract. The majority of DEGs are related to the antiviral and pro-inflammatory response, which is much more pronounced in SARS-CoV-2 rather than SARS-CoV virus-infection, and the delayed induction of these DEGs at 33°C coincides with the increased replication of SARS-CoV-2 at temperatures corresponding to the upper respiratory epithelium.…”

“…For the analysis of ACE2, TMPRSS2, IFNAR1, IFNAR2, IFNRL1, and IL10RB mRNA expression we reanalysed the previously obtained single-cell raw sequencing data 31 types found in the respiratory epithelium, as previously described 31 .…”

“…We previously observed, in the context of influenza A/H1N1 virus, that only a small fraction of infected cells produces IFN during infection 31 . Since the majority of upregulated DEGs during SARS-CoV-2 infection were genes induced downstream of the IFN pathway, we next sought to assess the expression of individual type I and III IFN genes over time.…”

LY6E impairs coronavirus fusion and confers immune control of viral disease

“…The RNA-seq data discussed in this publication have been deposited in the Gene Expression Omnibus database, https://www.ncbi.nlm.nih.gov/geo (GSE146074). The data used for scRNA-seq analysis of hAECs have been reported previously 38 . The data used for RNA-seq analysis of LY6E induction in hAECs after viral infection have been reported previously 39 .…”