Single-cell longitudinal analysis of SARS-CoV-2 infection in human airway epithelium

Abstract

SARS-CoV-2, the causative agent of COVID-19, has resulted in more than 3,000,000 infections and 200,000 deaths. There are currently no approved drugs or vaccines for the treatment or prevention of COVID-19. Enhanced understanding of SARS-CoV-2 infection and pathogenesis is critical for the development of therapeutics. To reveal insight into viral replication, cell tropism, and host-viral interactions of SARS-CoV-2 we performed single-cell RNA sequencing of experimentally infected human bronchial epithelial cells (HBECs) in air-liquid interface cultures over a time-course. This revealed novel polyadenylated viral transcripts and highlighted ciliated cells as the major target of infection, which we confirmed by electron microscopy. Over the course of infection, cell tropism of SARS-CoV-2 expands to other epithelial cell types including basal and club cells. Infection induces cell intrinsic expression of type I and type III IFNs and IL6 but not IL1. This results in expression of interferon stimulated genes in both infected and bystander cells. Here, we have conducted an in-depth analysis of SARS-CoV-2 infection in HBECs and provide a detailed characterization of genes, cell types, and cell state changes associated with the infection.CoVs are enveloped viruses with positive-sense, single-stranded RNA genomes ranging from 26-30 kb [3]. Six human CoVs have been previously identified: HCoV-NL63 and HCoV-229E, which belong to the Alphacoronavirus genus; and HCoV-OC43, HCoV-HKU1, SARS-CoV, and Middle East Respiratory Syndrome CoV (MERS-CoV), which belong to the Betacoronavirus genus [4]. In the past two decades, CoVs have become a major public health concern due to potential zoonotic transmission, as revealed by the emergence of SARS-CoV in 2002, which infected 8, 000 people worldwide with a mortality rate of 10-15%, and MERS-CoV in 2012 and 2019, which infected 2, 500 people with a mortality rate of 35%, and now SARS-CoV-2 (WHO).Tissue and cell tropism are key determinants of viral pathogenesis. SARS-CoV-2 entry into cells depends on the binding of the viral spike (S) protein to its cognate receptor angiotensin-converting enzyme II (ACE2) on the cell surface [2]. ACE2 is also the receptor for SARS-CoV and HCoV-NL63, yet these viruses induce profoundly different morbidity and mortality suggesting unknown determinants of coronavirus pathogenesis [5,6]. Additionally, proteolytic priming of the S protein by host proteases is also critical for viral entry [7]. The cellular serine protease Type II transmembrane (TMPRSS2) is used by SARS-CoV-2 for S protein priming [8,7,9,10]. This is also used by SARS-CoV alongside the endosomal cysteine proteases cathepsin B and L [11,12]. Another host protease, furin, has been suggested to mediate SARS-CoV-2 pathogenesis; however, the precise role of host proteases in SARS-CoV-2 entry remains to be determined [13,10].SARS-CoV and MERS-CoV caused fatal pneumonia associated with rapid virus replication, elevation of proinflammatory cytokines, and immune cell infiltration [14]. These characteri...

Transcriptional response modules characterise IL-1β and IL-6 activity in COVID-19

“…The protracted clinical course, inverse relationship between viral load and symptom progression, and the association between inflammation and worse clinical outcomes support a hypothesis whereby severe COVID-19 disease is predominantly driven by an exaggerated inflammatory response (1,2). Both IL-1 and IL-6 may play a role in this process (1,(4)(5)(6)(7)(8), and in this study we utilised transcriptional modules derived from cytokine stimulated cells to demonstrate that their expression, but not that of their cognate cytokine genes, provided a quantitative readout for cytokine bioactivity in vivo, both in the context of COVID-19 and other chronic inflammatory conditions.…”

“…Severe Coronavirus Disease 2019 (COVID-19) typically occurs over a week from symptom onset, when viral titres have diminished, suggesting an unregulated host inflammatory response may be driving the pathogenesis of severe disease (1)(2)(3). Elevated IL-1 and IL-6 responses have each been associated with disease severity (1,(4)(5)(6)(7)(8). In addition, the hyperinflammatory state in COVID-19 is reported to resemble some aspects of haemophagocytic lymphohistiocytosis (HLH), a condition that may benefit from therapeutic IL-1 blockade (9).…”

Genome-wide CRISPR screen reveals host genes that regulate SARS-CoV-2 infection

“…An XGBoost model was trained to predict infected cells based on 24,714 genes from SARS-CoV-2 infected HBEC scRNA-seq, which was composed of 4,745 infected and 50,297 bystander cells (39,77). The trained model reported an accuracy of 88%, a sensitivity of 62% and a specificity of 91%.…”

“…We recently performed single-cell RNA sequencing of experimentally infected primary human bronchial epithelial cells. Eight epithelial cell populations were observed and ciliated cells, basal cells, and club cells were the primary target cells of infection (39). In the single-cell transcriptomic dataset, ACE2, CTSL, and TMPRSS2 expressions were of limited value in predicting whether a cell was infected.…”

“…In the single-cell transcriptomic dataset, ACE2, CTSL, and TMPRSS2 expressions were of limited value in predicting whether a cell was infected. To evaluate whether SARS-CoV-2-interacting genes identified in the CRISPR screen were predictive of infection, we assessed the predictive value of the top 50 resistance and sensitization genes for the ability to predict SARS-CoV-2 infection of primary human bronchial epithelial cells (39). We found that these 100 genes obtained from the CRISPR screen are significantly predictive (Wilcoxon rank sum, see Methods) of viral infection, suggesting that these genes are involved in variability of infection at the single cell level.…”