Fig. 1: OTS constructs exhibit similar replication kinetics to WT in vitro but are more sensitive to mutagenic drugs.
a, Overview of mutations introduced into the SARS-CoV-2 genome to generate LAVs. Fragments 4, 5, 7 and 8 were modified to generate one-to-stop codons. Specific changes are indicated for each fragment. OTS-206 also has additional Nsp1 mutations (K164A/H165A) and deletions of ORF6 to ORF8. OTS-228 is additionally missing the PCS. b, Violin plot of individual plaque sizes in Vero E6/TMPRSS2 cells. Mean plaque sizes (indicated by a line) were comparable between OTS and WT viruses (n = 10 plaques measured per group). No significant difference was found using ordinary one-way ANOVA and P values were adjusted using Tukey’s multiple-comparisons test. c–e, Growth kinetics of WT and OTS viruses in Vero E6/TMPRSS2 cells (c) (n = 3 independent biological replicates per group), hNECs (d) (n = 3 independent biological replicates per group) and hBECs (e) (n = 6; 3 independent biological replicates from 2 donors per group). Samples were collected at designated timepoints and assessed for infectious particle titres using TCID50. Graphs show each biological replicate. Statistical analysis was performed using two-way ANOVA. f,g, Treatment of Vero E6/TMPRSS2 cells with 5-fluorouracil (5-FU) and molnupiravir (n = 6 independent biological replicates per group), followed by infection with WT or OTS4-5-7-8, indicating a higher sensitivity of OTS-4-5-7-8 to 5-FU and molnupiravir. Statistical significance was assessed using two-sided, unpaired, non-parametric multiple t-test with Mann–Whitney test (compared ranks). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Additional data in Extended Data Fig. 1.
