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( A ) Schematic representation of primary <t>human</t> <t>bronchial</t> epithelial cell (BEpC) differentiation into a pseudostratified airway epithelium by culturing at air-liquid interface (ALI) on transwell plates. ( B ) Differentiated <t>BEpCs</t> from donor 1 were infected with 6000 PFU of each SARS-CoV-2 isolate from the apical side. At several times post-infection, apical washes were harvested and virus titers determined by plaque assay. Data represent means and standard deviations from 3 independent experiments, with each experiment performed using 1 individual transwell. The dotted line crossing the y-axis at 10 2 PFU/mL indicates the assay limit of detection (LoD). ( C ) Area under the curve (AUC) values for the virus replication data shown in (B), normalized to inoculum titer of the respective isolate. Data represent means and standard deviations from the 3 independent experiments. A one-way ANOVA was performed on log2-transformed data to test for significant differences between the individual isolates ( p <0.0001). To test for statistical significance against BavPat1, an unpaired t-test was used on log2-transformed data (* p <0.05; *** p <0.001). ( D ) Indirect immunofluorescent staining of SARS-CoV-2 infected BEpCs. Differentiated BEpCs were infected from the apical side with 6000 PFU of the indicated SARS-CoV-2 isolate. At 72 h post-infection, cells were fixed, permeabilized and stained for the presence of infected cells (dsRNA; magenta) and ciliated cells (β-tubulin; cyan), goblet cells (Muc5AC; cyan), club cells (uteroglobin; cyan), or basal cells (P63; yellow). Nuclei were stained with DAPI (blue). Z-stacks were transformed into maximum projection images. Scale bar represents 9.6µm. Arrows indicate co-localization. Data show a subset of representative images from 2 independent experiments performed in different BEpC donors (see also Supplementary Figures S5-10).
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( A ) Schematic representation of primary <t>human</t> <t>bronchial</t> epithelial cell (BEpC) differentiation into a pseudostratified airway epithelium by culturing at air-liquid interface (ALI) on transwell plates. ( B ) Differentiated <t>BEpCs</t> from donor 1 were infected with 6000 PFU of each SARS-CoV-2 isolate from the apical side. At several times post-infection, apical washes were harvested and virus titers determined by plaque assay. Data represent means and standard deviations from 3 independent experiments, with each experiment performed using 1 individual transwell. The dotted line crossing the y-axis at 10 2 PFU/mL indicates the assay limit of detection (LoD). ( C ) Area under the curve (AUC) values for the virus replication data shown in (B), normalized to inoculum titer of the respective isolate. Data represent means and standard deviations from the 3 independent experiments. A one-way ANOVA was performed on log2-transformed data to test for significant differences between the individual isolates ( p <0.0001). To test for statistical significance against BavPat1, an unpaired t-test was used on log2-transformed data (* p <0.05; *** p <0.001). ( D ) Indirect immunofluorescent staining of SARS-CoV-2 infected BEpCs. Differentiated BEpCs were infected from the apical side with 6000 PFU of the indicated SARS-CoV-2 isolate. At 72 h post-infection, cells were fixed, permeabilized and stained for the presence of infected cells (dsRNA; magenta) and ciliated cells (β-tubulin; cyan), goblet cells (Muc5AC; cyan), club cells (uteroglobin; cyan), or basal cells (P63; yellow). Nuclei were stained with DAPI (blue). Z-stacks were transformed into maximum projection images. Scale bar represents 9.6µm. Arrows indicate co-localization. Data show a subset of representative images from 2 independent experiments performed in different BEpC donors (see also Supplementary Figures S5-10).
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( A ) Schematic representation of primary human bronchial epithelial cell (BEpC) differentiation into a pseudostratified airway epithelium by culturing at air-liquid interface (ALI) on transwell plates. ( B ) Differentiated BEpCs from donor 1 were infected with 6000 PFU of each SARS-CoV-2 isolate from the apical side. At several times post-infection, apical washes were harvested and virus titers determined by plaque assay. Data represent means and standard deviations from 3 independent experiments, with each experiment performed using 1 individual transwell. The dotted line crossing the y-axis at 10 2 PFU/mL indicates the assay limit of detection (LoD). ( C ) Area under the curve (AUC) values for the virus replication data shown in (B), normalized to inoculum titer of the respective isolate. Data represent means and standard deviations from the 3 independent experiments. A one-way ANOVA was performed on log2-transformed data to test for significant differences between the individual isolates ( p <0.0001). To test for statistical significance against BavPat1, an unpaired t-test was used on log2-transformed data (* p <0.05; *** p <0.001). ( D ) Indirect immunofluorescent staining of SARS-CoV-2 infected BEpCs. Differentiated BEpCs were infected from the apical side with 6000 PFU of the indicated SARS-CoV-2 isolate. At 72 h post-infection, cells were fixed, permeabilized and stained for the presence of infected cells (dsRNA; magenta) and ciliated cells (β-tubulin; cyan), goblet cells (Muc5AC; cyan), club cells (uteroglobin; cyan), or basal cells (P63; yellow). Nuclei were stained with DAPI (blue). Z-stacks were transformed into maximum projection images. Scale bar represents 9.6µm. Arrows indicate co-localization. Data show a subset of representative images from 2 independent experiments performed in different BEpC donors (see also Supplementary Figures S5-10).

Journal: bioRxiv

Article Title: SARS-CoV-2 Variants Reveal Features Critical for Replication in Primary Human Cells

doi: 10.1101/2020.10.22.350207

Figure Lengend Snippet: ( A ) Schematic representation of primary human bronchial epithelial cell (BEpC) differentiation into a pseudostratified airway epithelium by culturing at air-liquid interface (ALI) on transwell plates. ( B ) Differentiated BEpCs from donor 1 were infected with 6000 PFU of each SARS-CoV-2 isolate from the apical side. At several times post-infection, apical washes were harvested and virus titers determined by plaque assay. Data represent means and standard deviations from 3 independent experiments, with each experiment performed using 1 individual transwell. The dotted line crossing the y-axis at 10 2 PFU/mL indicates the assay limit of detection (LoD). ( C ) Area under the curve (AUC) values for the virus replication data shown in (B), normalized to inoculum titer of the respective isolate. Data represent means and standard deviations from the 3 independent experiments. A one-way ANOVA was performed on log2-transformed data to test for significant differences between the individual isolates ( p <0.0001). To test for statistical significance against BavPat1, an unpaired t-test was used on log2-transformed data (* p <0.05; *** p <0.001). ( D ) Indirect immunofluorescent staining of SARS-CoV-2 infected BEpCs. Differentiated BEpCs were infected from the apical side with 6000 PFU of the indicated SARS-CoV-2 isolate. At 72 h post-infection, cells were fixed, permeabilized and stained for the presence of infected cells (dsRNA; magenta) and ciliated cells (β-tubulin; cyan), goblet cells (Muc5AC; cyan), club cells (uteroglobin; cyan), or basal cells (P63; yellow). Nuclei were stained with DAPI (blue). Z-stacks were transformed into maximum projection images. Scale bar represents 9.6µm. Arrows indicate co-localization. Data show a subset of representative images from 2 independent experiments performed in different BEpC donors (see also Supplementary Figures S5-10).

Article Snippet: Primary human bronchial epithelial cells (BEpCs) from a 73 year-old female donor (donor 1) were purchased from Promocell (#C-12640).

Techniques: Infection, Plaque Assay, Transformation Assay, Staining