SARS-CoV-2 variants reveal features critical for replication in primary human cells
Fig 2
Characterization of SARS-CoV-2 isolates in primary human BEpCs.
(A) Schematic representation of primary human BEpC differentiation into a pseudostratified airway epithelium by culturing at ALI on transwell plates. (B) Differentiated BEpCs from donor 1 were infected with 6,000 PFU of each SARS-CoV-2 isolate from the apical side. At several times postinfection, apical washes were harvested, and virus titers were determined by plaque assay. Data represent means and standard deviations from 3 in dependent experiments, with each experiment performed using 1 individual transwell. The dotted line crossing the y-axis at 102 PFU/mL indicates the assay LoD. (C) 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 1-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 6,000 PFU of the indicated SARS-CoV-2 isolate. At 72 h postinfection, 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 9.6 μm. Arrows indicate co-localization. Data show a subset of representative images from 2 independent experiments performed in different BEpC donors (see also S5–S10 Figs). For underlying data, see S1 Data. ALI, air–liquid interface; AUC, area under the curve; BEpC, bronchial epithelial cell; LoD, limit of detection; PFU, plaque-forming unit; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2.