Evaluation of body weight, lung weight, lung histopathology and nucleocapsid immunohistochemistry

Mouse survival was monitored and body weights recorded at the indicated timepoints. Specifically, 20-week-old mice (n = 5) infected with the SARS-CoV-2 gamma strain and 40-week-old mice (n = 4–8) infected with MA-P10 were examined for survival and weighed daily from Day 0 through to Day 14. Whole-lung weight of 40-week-old mice infected with MA-P10 (n = 5–10) was measured 4 days post-infection. For histopathological analysis, left lungs of 40-week-old mice infected with MA-P10 (n = 3–5) were fixed in 10% neutral buffered formalin 4 days post-infection, paraffin-embedded, sectioned at 3 μm thickness, and stained with haematoxylin and eosin (H&E). Pathological evaluation was performed by modifying the Owen’s report.⁷ Blinded histopathological evaluations for alveolar epithelial degeneration or necrosis, bronchial or bronchiolar epithelial degeneration or necrosis, vascular endothelial degeneration or necrosis, alveolar/interstitial inflammation, bronchial or bronchiolar inflammation, perivascular inflammation and thrombosis were independently performed by two pathologists. The lung pathology parameters were scored 0 (normal), 1 (minimal), 2 (mild), 3 (moderate) and 4 (marked). Total histopathological scores for individual mice were calculated by adding the individual histopathological scores, and mean total histopathological scores were calculated for each group. Disparate scores between the pathologists were resolved by the first pathologist rescoring the specimen.

Immunohistochemical (IHC) staining of the left lungs of 40-week-old mice infected with MA-P10 was also performed 4 days post-infection. Briefly, deparaffinized sections were treated with citric acid buffer (pH 6.0) for 10 min at 121°C using an autoclave for antigen retrieval. The sections were incubated with 3% hydrogen peroxide followed by blocking with normal goat serum. The sections were incubated overnight at 4°C with a primary rabbit polyclonal antibody against the SARS-CoV-2 nucleocapsid protein (1:1000; Cat. No. ab281297; Abcam, Cambridge, UK). The sections were then incubated for 30 min at room temperature with a horseradish peroxidase-labelled secondary polyclonal antibody (Cat. No. 414341; Nichirei Biosciences Inc., Tokyo, Japan). The target antigen was detected using 3,3′-diaminobenzidine as a chromogen. The sections were counterstained with haematoxylin and evaluated under a microscope. Semi-quantitative grading of the IHC staining was performed using the following outcomes: −, no staining detected; ±, minimal staining; +, mild staining; ++, moderate staining; +++, marked staining.