Fig. 2

SARS-CoV-2 d16 mutant virus is attenuated in vivo. A Viral challenge scheme for the hamster model. Hamsters were intranasally inoculated with 10⁵ PFU (in 50 µl) of recombinant WT or d16 SARS-CoV-2. At 4 dpi, the lungs and nasal wash were harvested for viral yield detection. B Body weight changes of WT or d16 SARS-CoV-2-infected hamsters (n = 6). Viral loads determined assessment of the nasal wash and lung tissues of WT- or d16-challenged hamsters at 4 dpi by RT–qPCR (C) and plaque assay (D) (n = 4/group). The primers have been described elsewhere [67]. E Detection of the SARS-CoV-2 N protein in lung tissues infected with the indicated recombinant virus. The SARS-CoV-2 N protein (green) was labeled with rabbit anti-SARS-CoV-2 N antibodies followed by goat anti-rabbit antibodies conjugated to FITC. Nuclei were counterstained with DAPI (blue). Scale bars, 50 μm. F Effects of the WT virus and d16 on histopathological features of the lungs of hamsters. Representative hamster lung tissue sections harvested at 4 dpi were stained with H&E. Scale bars, 200 μm. G Proinflammatory cytokine and chemokine gene expression in WT virus- or d16-infected hamsters. The relative expression levels of representative chemokines and cytokines in lung tissue homogenates were quantified using RT–qPCR. The results are shown as the mean ± SEM. H Viral challenge scheme for the K18-hACE2 mouse model. Each mouse was intranasally inoculated with 10³ PFU (in 20 µl) of recombinant WT virus or d16. At 4 dpi, the viral loads in lung tissues were quantitated using RT–qPCR. Body weight changes (I) and survival rates (J) of WT virus- or d16-infected K18-hACE2 transgenic mice (n = 5/group). K Viral loads in lung tissues at 4 dpi (n = 4/group). L Detection of the SARS-CoV-2 N protein in the lung tissues of WT virus- or d16-infected mice. The SARS-CoV-2 N protein (green) was labeled with rabbit anti-SARS-CoV-2 N antibodies followed by goat anti-rabbit antibodies conjugated to FITC. Nuclei were counterstained with DAPI (blue). Scale bars, 50 μm. M Effects of WT and d16 SARS-CoV-2 on histopathological features of mouse lungs. Lung sections from uninfected mice (mock) were included as a negative control. Representative sections of lung tissues from K18-hACE2 mice harvested at 4 dpi were stained with H&E. Scale bars, 100 μm. N Pathological changes were scored as described in the Materials and Methods. Statistical analyses by Student’s t-test (*P < 0.05; **P < 0.01; ***P < 0.001)