Extended Data Fig. 2: Attenuation of OTS2, OTS7, OTS8, OTS4-5 and OTS7-8 in K18-hACE2 mice.
a, K18-hACE2 mice (7–16 weeks old, n=4 mice/group, n=2 mice/mock group) were infected with 5,000 PFU of either OTS2, OTS7, OTS8, or SARS-CoV-2 WT virus, or only with medium for 5 days. Overview created with BioRender.com. b, c, Mice were monitored for body weight change (mean ± SEM) (Mock vs WT, p=0.0442) and clinical symptoms over the 5-day course of infection. On day 5 post-infection (dpi), mice were euthanized, and samples from the nose, lungs, brain, and olfactory bulbs were collected for evaluation of infectious virus titers, viral genome copy numbers, and pathology. d, Infectious virus titers from the nose, lung, and brain samples were determined using plaque assays in VeroE6 cells. e, Genome copy numbers in the nose, lung, brain, and olfactory bulb samples of infected mice were quantified using probe-specific RT–qPCR. f, Histopathological lung score was given for characterization and comparison of the severity of lung lesions. g, Hematoxylin and eosin stain (left panel) and immunohistochemical analysis (right panel) specific for SARS-CoV-2 nucleocapsid protein of lung and brain sections. Scale bar, 500 μm. h, Experimental setup of comparison of OTS4-5, OTS7-8 to WT infection in short-term (7–16 weeks-old, n=4 mice/group). Overview created with BioRender.com. i, j, Mice were monitored for body weight (mean ± SEM) (Mock vs WT, p<0.0001) change and clinical symptoms over the 5-day course of infection. k, Infectious virus titers from the nose, lung, and brain samples were determined using plaque assays. l, m, Genome copy numbers in the nose, lung, brain, and olfactory bulb samples of infected mice were quantified using RT–qPCR. n, Histopathological lung score was given for characterization and comparison of the severity of lung lesions. o, Hematoxylin and eosin stain (left panel) and immunohistochemical analysis (right panel) specific for SARS-CoV-2 nucleocapsid protein of lung and brain sections (n=4 per group) (magnification 50x). Scale bar, 500 μm. Statistical significance was determined using two-sided, two-way ANOVA (b-f, i-m), and P values were adjusted using Tukey’s multiple-comparison test or was assessed by two-sided, unpaired, nonparametric multiple t-test with Mann-Whitney test (compared ranks, CI: 95%); *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Data were obtained from one experiment, with each data point representing one biological replicate. Body weight changes, clinical scores, and histopathological scores of all K18-hACE2 mice experiments are shown in Supplementary Table 6.
