Fig. 3: Mutations in S and nsp6 drive Omicron pathogenicity.

a–d, Replication kinetics of indicated mNeonGreen reporter viruses in ACE2/TMPRSS2/Caco-2 cells (MOI = 0.01) determined by flow cytometry (n = 3 replicates) (a,c) and plaque assay (n = 3 replicates) (b,d). Experiments were repeated twice. e, ACE2/TMPRSS2/Caco-2 cells were infected with virus mixtures at a 1:1 ratio to obtain the final MOI of 0.005 for each virus. The cells were fixed at the indicated times and analysed by flow cytometry. Percentage of uninfected, singly infected and doubly infected cells is shown. Singly infected cells were used for compensation. Individual data points are plotted along with the mean ± s.d. (n = 3 replicates). The experiment was repeated twice. f–h, K18-hACE2 mice were intranasally inoculated with 1 × 10⁴ PFU of viruses. Lung samples of infected mice were collected at 2 dpi for IHC detection of the N protein (n = 3 mice) (f) or for determination of viral titres (n = 4 mice) (g). In f, representative images of H&E staining of N-positive bronchioles are shown in insets. Bronchiolar epithelial necrosis is indicated with arrows. No evidence of necrosis was seen in bronchioles of mice infected with Omicron or Omi-S/nsp6. Scale bar, 100 µm. The right graph in f shows the percentage of N-positive bronchioles in the lungs of infected mice. Each dot represents an infected mouse. h, Survival of infected mice monitored daily for 14 days. Mice that lost 20% of their initial body weight were euthanized. Statistical significance was determined using a two-tailed, unpaired t-test with Welch’s correction (a–g) and log-rank (Mantel-Cox) test (h). *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; NS, not significant.

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