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

Abstract

Since its emergence in December 2019, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has spread globally and become a major public health burden. Despite its close phylogenetic relationship to SARS-CoV, SARS-CoV-2 exhibits increased human-to-human transmission dynamics, likely due to efficient early replication in the upper respiratory epithelium of infected individuals. Since different temperatures encountered in the human upper and lower respiratory tract (33°C and 37°C, respectively) have been shown to affect the replication kinetics of several respiratory viruses, as well as host innate immune response dynamics, we investigated the impact of temperature on SARS-CoV-2 and SARS-CoV infection using the primary human airway epithelial cell culture model. SARS-CoV-2, in contrast to SARS-CoV, replicated to higher titers when infections were performed at 33°C rather than 37°C. Although both viruses were highly sensitive to type I and type III interferon pretreatment, a detailed time-resolved transcriptome analysis revealed temperature-dependent interferon and pro-inflammatory responses induced by SARS-CoV-2 that were inversely proportional to its replication efficiency at 33°C or 37°C. These data provide crucial insight on pivotal virus-host interaction dynamics and are in line with characteristic clinical features of SARS-CoV-2 and SARS-CoV, as well as their respective transmission efficiencies.

Fig 1. SARS-CoV and SARS-CoV-2 replication kinetics in hAEC cultures.

Well-differentiated hAEC cultures were infected with SARS-CoV (a) and SARS-CoV-2 (b) using 30,000 PFU or remained uninfected (mock) and were incubated at 37°C or 33°C. Inoculated virus was removed at 1 hpi and the apical side was washed. Cultures were further incubated at the indicated temperature. At the indicated time post infection, apical virus release was assessed by plaque titration (a, b). Data represent the mean ± 95% CI of hAEC cultures from 7 different human donors. Individual points represent the average of 2 technical replicates. Values at 0 hpi indicate the titer of the inoculum used to infect the hAEC cultures, and values at 1 hpi indicate the remaining titer after the third wash. The p-values were computed by using two-sided paired sample t tests. At 96 hpi, hAEC cultures were fixed and processed for immunofluorescence analysis using antibodies against SARS-CoV Nucleocapsid protein (green), β-tubulin (cilia, red), ZO-1 (tight junctions, white), and DAPI (blue) (c). Representative z-projections of one donor are shown. Scale bar, 20 μm. Infected cells were quantified after segmentation of individual cells based on the ZO-1 staining and measuring the mean intensity of the nucleocapsid protein staining (d). Data represent the mean ± 95% CI of multiple images acquired from hAEC cultures derived from 4 different human donors. On average, more than 10⁴ cells per donor and per condition were analyzed. The data underlying this figure are found in S1 Data. hAEC, human airway epithelial cell; hpi, hours post infection; PFU, plaque-forming unit; SARS-CoV, Severe Acute Respiratory Syndrome Coronavirus; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2.

Fig 2. SARS-CoV and SARS-CoV replication upon IFN-I and IFN-III pretreatment.

hAEC cultures were treated from the basolateral side with recombinant universal type I IFN (50 IU or 5 IU) or recombinant IFN-λ3 (50 ng or 5 ng) for 18 hours. Before infection, medium was removed and replaced with IFN-free medium, and hAEC cultures were infected with SARS-CoV and SARS-CoV-2 using 30,000 PFU and were incubated at 37°C or 33°C. Inoculated virus was removed at 1 hpi, and the apical side was washed. Cultures were further incubated at the indicated temperature. At the indicated time, apical virus release was assessed by plaque titration (a, b). Data represent the mean ± 95% CI of hAEC cultures from 3 different human donors. Individual points represent 1 (b) or the average of 2 technical replicates (a). Values at 0 hpi indicate the titer of the inoculum used to infect the hAEC cultures, and values at 1 hpi indicate the remaining titer after the third wash. The p-values were computed by using two-sided paired sample t tests. The data underlying this figure are found in S1 Data. At 72 hpi, hAEC cultures pretreated with 50 IU type I IFN or 50 ng type III IFN were fixed and processed for immunofluorescence analysis using antibodies against SARS-CoV Nucleocapsid protein (green), β-tubulin (cilia, red), ZO-1 (tight junctions, white), and DAPI (blue) (c). Representative z-projections of 1 donor are shown. Scale bar, 20 μm. hAEC, human airway epithelial cell; hpi, hours post infection; IFN, interferon; PFU, plaque-forming unit; SARS-CoV, Severe Acute Respiratory Syndrome Coronavirus; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2.

Fig 3. Temperature-dependent host transcriptional response in SARS-CoV and SARS-CoV-2 infected hAEC cultures.

Venn diagram showing the overlap among DE genes identified in SARS-CoV (purple) and SARS-CoV-2 (orange) infected hAEC cultures at either 33°C or 37°C (a). Enrichment map illustrating the pathway enrichment analysis results for the DE gene clusters identified in SARS-CoV and SARS-CoV-2 infected hAEC cultures at either 33°C or 37°C. Circle size represents the number of genes associated with a given pathway (b). Hierarchical cluster analysis of DE genes identified in SARS-CoV and SARS-CoV-2 infected hAEC cultures at either 33°C or 37°C compared to uninfected hAEC cultures (Mock). Expression levels for individual DE genes are shown in rows as the log₂ mean normalized counts for all 7 human donors stratified by condition, temperature, and hpi (columns; representative colors shown in legends). The 40 of 45 DE genes unique to SARS-CoV-2 at both 33°C and 37°C are shown on the right (y-axis) (c). Dotplot illustrating pathway enrichment analysis performed on the 16 distinct DE gene profiles. Significantly enriched pathways for SARS-CoV and SARS-CoV-2 are shown for both 33°C and 37°C incubation temperatures at 72 and 96 hpi. Dots were adjusted in size and color to illustrate the gene ratio and adjusted p-value (<0.05) for a given pathway, respectively (d). Boxplot graphs showing the distribution of the Z-score abundance from DE genes associated with cluster 1, 2, and 3 at different hpi for Mock (green), SARS-CoV (purple), and SARS-CoV-2 (orange) for both 33°C and 37°C. The overall mean Z-score abundance over time is for each condition indicated with a solid line (e). Enrichment map illustrating the pathway enrichment analysis on the temporal DE genes identified in SARS-CoV and SARS-CoV-2 infected and uninfected (Mock) hAEC cultures at either 33°C or 37°C. Circle size represents the number of genes associated with a given pathway (f). DE, differentially expressed; hAEC, human airway epithelial cell; hpi, hours post infection; SARS-CoV, Severe Acute Respiratory Syndrome Coronavirus; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2.

Fig 4. Innate immune response in SARS-CoV and SARS-CoV-2 infected hAEC cultures.

Gene-concept network plot illustrating the individual relationships between temporal DE genes from cluster 1 (IFN-signaling) and the top 5 significantly enriched biological processes. Enriched pathway hubs (colored by biological process) were adjusted in size to reflect the number of genes associated with each respective pathway, whereas for individual genes, their relationship is colored by the respective biological process (a). Bar graph illustrating the mean normalized expression levels over time for IFNL1, IFNL2, IFNL3, and IFNB1, at the respective temperatures for uninfected (Mock) hAEC cultures (top two panels) and for SARS-CoV (middle two panels) and SARS-CoV-2 (bottom two panels) infected hAEC cultures. Bars were adjusted in color to illustrate the respective SD among donors (b). The data underlying this figure are found in S1 Data. DE, differentially expressed; hAEC, human airway epithelial cell; IFN, interferon; SARS-CoV, Severe Acute Respiratory Syndrome Coronavirus; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2; SD, standard deviation.