Fig 1.
The growth kinetics of SARS-CoV-2 VOCs in Calu-3 cells.
The cells were infected with each SARS-CoV-2 variant at MOI 0.1 for 2 hours and subsequently washed and incubated further for 12, 24, 48, and 72 hpi. At the indicated time point, the infected cells were then fixed and stained for viral nucleocapsid proteins with anti-SARS-CoV NP mAb. (a) Representative fluorescent images of the infected Calu-3 cells. The SARS-CoV-2 infected cells were detected by a 2.5x lens of BioTek Cytation 7 Cell Imaging Multi-Mode Reader (Agilent Technologies, USA) (SARS-CoV NP- Alexa Fluor™ 488: Green; Hoechst: Blue). Scale bar: 2000 μm. (b) The levels of the infected cells were calculated based on the expression of viral NP at different time points. Statistical analysis was performed by using one-way ANOVA with subsequent Dunnett’s multiple comparisons test. *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. (c) The production of infectious virions at various time points was analyzed from the harvested culture supernatants at the indicated time points by foci-forming assay. All experiments were performed in triplicate.
Fig 2.
Representative fluorescent images of syncytia formation in Calu-3 cells induced by infection with different SARS-CoV-2 variants.
The Calu-3 cells were infected with SARS-CoV-2 at MOI 0.1 including the wild-type strain (a), D614G/B.1.36.16 (b), Alpha/B.1.1.7 (c), Beta/B.1.351 (d), Delta/B.1.617.2 (e), and Omicron/ B.1.1.529.2.10 (BA.2.10) (f). Syncytia formation was monitored based on the expression of anti-SARS-CoV NP mAb at 24 hours post-infection. The images were observed under a 10x lens of BioTek Cytation 7 Cell Imaging Multimode Reader (Agilent Technologies, USA). (SARS-CoV NP- Alexa Fluor™ 488: Green; Hoechst: Blue). Scale bar: 300 μm. (g) The average number of syncytia per 3000 cells in different variants was demonstrated in a bar chart. Syncytia were grouped into two categories, small (5–10 nuclei, white arrow) and large syncytia (>10 nuclei, red arrow). The experiment was performed in triplicate. The statistics were analyzed by using one-way ANOVA with Dunnett’s multiple comparisons test. *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.
Fig 3.
Cytopathic effect (CPE) upon SARS-CoV-2 VOCs infection in Calu-3 cells.
The Calu-3 cells were infected with SARS-CoV-2 at MOI 0.1 for 48 hours. (a) Representative images of CPEs Calu-3 cells following infection with different SARS-CoV-2 variants; mock infection (a), wild-type strain (b), D614G/B.1.36.16 (c), Alpha/B.1.1.7 (d), Beta /B.1.351 (e), Delta/B.1.617.2 (f), and Omicron/ B.1.1.529.2.10 (BA.2.10) (g). All images were taken at 100× magnification using the Eclipse TS100 inverted microscope (Nikon, USA). All experiments were performed in triplicate.
Fig 4.
The measurement of pro-inflammatory cytokines/chemokines and interferons following infection Calu-3 cells with SARS-CoV-2 variants.
The Calu-3 cells were infected with different SARS-CoV-2 variants at MOI 0.1. At 48 hpi, supernatant of each well were collected and measured for pro-inflammatory cytokine/chemokine concentration. The values of IP-10 (a), TNF-α (b), IL-6 (c), IL-1β (d), IL-2 (e), IL-3 (f), IL-8 (g), IL-9 (h), IL-12p70 (i), IL-17A (j), IL-22 (k), MCP1 (l), MIP-α (m), MIP-β (n), IFN-α2 (o), and IFN-γ (p) were compared among variants. The geometric mean value of triplicate is shown with error bars representing the SD of the mean. Mock infection, wild-type, D614G, Alpha, Beta, Delta, and Omicron variants were presented in different bar motifs. Statistical significance was calculated using the one-way ANOVA with Dunnett’s multiple comparisons test. *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.
Fig 5.
The measurement of anti-inflammatory cytokines following infection Calu-3 cells with SARS-CoV-2 variants.
Different SARS-CoV-2 variants were used to infect the Calu-3 cells at a MOI of 0.1. Supernatants from each well were collected at 48 hpi, and their anti-inflammatory cytokine concentrations were analyzed. The values of IL-1RA (a) and IL-10 (b) were compared among VOCs. The geometric mean value of triplicates is shown with error bars representing the SD of the mean. Mock infection, wild-type, D614G, Alpha, Beta, Delta, and Omicron variants were presented in different bar motifs. Statistical significance was calculated using the one-way ANOVA with Dunnett’s multiple comparisons test. *p<0.05, ***p<0.001, and ****p<0.0001.
Fig 6.
The measurement of secreted growth factors following infection Calu-3 cells with SARS-CoV-2 variants.
The Calu-3 cells were infected with various SARS-CoV-2 variants at MOI 0.1. The supernatant of each well were taken at 48 hpi and measured for growth factors concentration. The values of FGF-2 (a), VEGF-A (b), G-CSF (c), PDGF-AA (d), and PDGF-BB (e) were compared among variants. The geometric mean value of triplicates is shown with error bars representing the SD of the mean. Mock infection, wild-type, D614G, Alpha, Beta, Delta, and Omicron variants were presented in different bar motifs. Statistical significance was calculated using the one-way ANOVA with Dunnett’s multiple comparisons test. *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.
Fig 7.
Heatmap of cytokine expression in Calu-3 cells infected with different SARS-CoV-2 VOCs at 48 hpi.
The cytokine concentrations were relatively transformed into log2 fold change value and scale in comparison to mock infection. Four main cytokine categories are presented, including pro-inflammatory cytokines/chemokines (a), interferons (b), anti-inflammatory cytokines (c), and growth factors (d).