Fig 1.
Growth curve analysis of B6, B.1.617.1, and B.1.617.2 variants in Calu-3 cells.
Calu-3 cells were infected with 0.01 MOI of SARS-CoV-2 virus B.6, B.1.617.1 (Kappa), and B.1.617.2 (Delta) variants. (A) Virus titer in the culture supernatant collected at indicated time-points were estimated by plaque assay. Error bars represent geometric mean with 95% CI. (B) Sub-genomic RNA of N gene was estimated by qRT-PCR. RNAse P was used as internal control for normalization. Error bars represent geometric mean with 95% CI. (C) Calu-3 cells grown on transwell inserts were infected with above variants at 0.3 MOI and TEER was monitored at indicated time points. Values (Mean ± SD) are represented as relative to time zero before infection and statistical significance was determined by comparing the zero hour and 48 h readings by two-way ANOVA with Dunnett’s multiple comparisons test. (D) Viral titers were measured in supernatants at 48 h pi by focus-forming units. Data are from two or three independent experiments (Mean ± SD). ns: non-significant; ** P<0.01; **** P<0.0001.
Fig 2.
Kappa and Delta variants of SARS-CoV-2 disrupt epithelial junctions.
Calu-3 cells were grown on transwell inserts and infected with indicated variants of SARS-CoV-2 at 0.3 MOI. At 48 h pi, cells were fixed and stained with occludin and β-catenin antibodies. Nucleocapsid antibody was used to visualize the SARS-CoV-2 infection. Appropriate Alexa Fluor dye-conjugated secondary antibodies were used for visualization. Nuclei were stained with DAPI. Images were captured at 100X magnification. Images were analyzed using cellSens software and Z-projection at maximum intensity images are shown in the figure. Scale bar is 10 μM.
Fig 3.
Growth kinetics of B6, B.1.617.2, and B.1.1.529 variants in Calu-3 cells.
(A) Calu-3 cells were infected with above variants of SARS-CoV-2 at 0.3 MOI. Cells were collected at indicated time points and total RNA was isolated to estimate N gene copy numbers by RT-qPCR. RNase P was used as a housekeeping control for normalization. Error bars represent geometric mean with 95% CI. (B) Viral titers were measured in supernatants by plaque assay. Error bars represent geometric mean with 95% CI. Statistical significance was estimated by two-way ANOVA with Dunnett’s multiple comparisons test. (C) Western blot analysis of cell lysates prepared from infected cells at indicated time points to detect the expression of SARS-CoV-2 nucleocapsid. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. Numbers on the left indicate the size of the bands in molecular weight marker. (D) Calu-3 cells were infected at 5 MOI and the amount of internalized RNA was estimated by RT-qPCR as described above. Data are from two independent experiments. Error bars represent geometric mean with 95% CI. Statistical significance was estimated by Kruskal-Wallis test with Dunn’s multiple comparison test. ns: non-significant, * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001.
Fig 4.
Antiviral response with B.6, B.1.617.2, and B.1.1.529 variants.
Calu-3 cells were infected with indicated variants of SARS-CoV-2 at 0.3 MOI. Cells were collected at indicated time-points and total RNA was isolated. RT-qPCR was set up to determine the expression of (A) IFN-β. (b) IFN-λ1, (C) ISG-15, and (D) OAS1. GAPDH was used as house-keeping control for normalization. Data are from two independent experiments (Mean ± SD). Statistical significance was estimated by two-way ANOVA with Tukey’s multiple comparisons test. ns: non-significant, * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001.
Fig 5.
Sub-lineages of Omicron variant have milder effects on epithelial junctions.
Calu-3 cells were grown on transwell inserts under air-liquid interface (ALI) conditions. Cells were infected with the indicated SARS-CoV-2 variants at 0.3 MOI. (A) Graph indicates TEER values relative to mock infection at indicated time points from two independent experiments (Mean and error with range). Statistical significance was estimated by two-way ANOVA with Tukey’s multiple comparisons test. Transwells with delta variant were discontinued at 36 h pi and therefore are indicated as NA at later points. (B) Viral titers in apical and (C) basolateral supernatants collected at indicated time points was estimated by focus-forming unit assay. Error bars represent geometric mean with 95% confidence intervals. ND: Not detected. * indicates P <0.05 relative to BA.1 sub-lineage obtained by two-way ANOVA with Dunnett’s multiple comparison test. (D) Viral RNA levels in 1000 PFU of stock viruses of indicated SARS-CoV-2 variants was quantitated by qRT-PCR. (E) Calu-3 cells were infected with 105 genome equivalents of indicated SARS-CoV-2 variants and the amount of internalized RNA was estimated by RT-qPCR 1 h pi. Data are from two independent experiments. Error bars represent geometric mean with 95% CI. Statistical significance was estimated by Kruskal-Wallis test with Dunn’s multiple comparison test. ns: non-significant, * P<0.05, ** P<0.01.
Fig 6.
Omicron infection induces cross-protective antibodies.
(A) Binding antibodies to nucleocapsid (N) and (B) RBD were estimated by quantitative ELISA for the three time-points post-vaccination as indicated (n = 15). (C) Neutralizing antibody titers to Delta variant and (D) Omicron variant was determined in the same samples by FRNT assay. Statistical significance was estimated by Kruskal-Wallis test with Dunn’s multiple comparison test. ns: not significant. * P<0.05, **** P<0.0001. (E) Neutralizing antibody titers to the BA.1, BA.2 and BA.2.75 sub-lineages were determined in samples from participants that were infected with Omicron variant in early 2022 (n = 20) and (F) indicates the same in individuals unexposed to the Omicron variants (n = 4). Numbers indicate the geometric mean NT50 values. Statistical significance was estimated by paired analysis using Friedman test with Dunn’s multiple comparison test. ns: not significant. * P<0.05, **** P<0.0001. Paired analysis for neutralizing antibodies in six month follow-up convalescent samples which were tested for neutralization of (G) Delta variant, (H) BA.1 (I) BA.2 and (J) BA.2.75. P values were obtained by Wilcoxon matched-pairs signed rank test. * P<0.05. ns: Not significant.
Fig 7.
Iron and copper salts significantly inhibit SARS-CoV-2 infection.
(A) SARS-CoV-2 RdRp activity assay was performed in the presence of indicated concentrations of CaCl2/CuCl2/FeSO4/ZnSO4 in the standard reaction mix containing MgCl2 and MnCl2 and the RdRp complex (nsp7-nsp8-nsp12) of SARS-CoV-2. (B) RdRp assays were performed in the presence of varying of concentrations of ZnSO4 or (C) FeSO4 to determine the IC50 values. To determine the effect of metal ions in infection, Calu-3 cells were infected with indicated variants at 0.3 MOI and media containing 50 μM each of the indicated salts were added after virus adsorption. At 24 h pi, supernatants were collected and viral titers were measured by plaque assay. The graph indicates the virus titers of (D) Delta (E) BA.1 and (F) BA.2.75 variants. Data are from two or three independent experiments. Error bars represent geometric mean with 95% CI. Statistical significance was estimated by Kruskal-Wallis test with Dunn’s multiple comparison test. ns: non-significant, * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001.