Fig 1.
SARS-CoV-2 was delivered via aerosol or IT/IN (multiroute) to RMs or AGMs at the noted doses. Animals were followed for 4 weeks, with biosampling performed as indicated above. Fig created in Biorender.com.
Fig 2.
Viral Loads Assessed via RT-qPCR post SARS-CoV-2 Challenge.
A)Viral loads in swabs and BAL supernatant were assessed via RT-qPCR post challenge for genomic (black) and subgenomic N (red) RNA. After necropsy, respiratory tissues were analyzed for the presence of genomic (B) and subgenomic (C) content.
Fig 3.
Antibody Responses to SARS-CoV-2 Challenge.
Antibody responses were followed post challenge by A)pseudovirus neutralization, B and C) ELISA for binding to RBD and NP, respectively, and D) surrogate virus neutralization test at necropsy. Route and species variability in antibody levels at time of necropsy were compared for each assay type (E-H). Comparisons were made using the Mann-Whitney or Welch’s t-test, depending on normality of data. Asterisks represent significant comparisons (*, p<0.05; ***, p<0.001).
Fig 4.
BAL Supernatant Cytokines post SARS-CoV-2 Challenge.
Cytokines in BAL supernatant were analyzed at indicated time points post challenge. Comparisons were made with two-way ANOVA using Tukey’s multiple comparisons test. Asterisks represent significant comparisons (*, p<0.05; **, p<0.01;***, p<0.001).
Fig 5.
Heat map of BAL cytokine expression dynamics.
Changes in the cytokine expression between time points demonstrates the delayed cytokine kinetics in the aerosol cohort (red). Similar dynamics are observed in both cohorts (arrows) occurring between 1 day post infection (DPI) and 1 week post infection (WPI) in the multiroute cohort compared to 1–2 WPI in the aerosol cohort.
Fig 6.
Clinical Disease Severity Scores.
Signs of disease present in each cohort over time. Number of animals showing signs of disease along with subgenomic viral loads in A) BAL supernatant and B) rectal swabs. Severity scores per group in C) both delivery cohorts and D) aerosol cohort split into species. E) Cumulative severity scores per delivery cohort. F) Simplified scoring system used for cohorts. Curves in figures were smoothed. Comparisons made using Welch’s t-test. Asterisks represent significant comparisons (*, p<0.05).
Fig 7.
Pleuritis in SARS-CoV-2 infected animals.
A) Normal pleura from a naïve animal. B-D). SARS-CoV-2 infected animals exhibited variable pleuritis (arrow) ranging from minimal (B) to mild (C) to moderate (D). Insets of the boxed regions show the magnitude of thickening and infiltration by inflammatory cells. H&E. Bar = 1 mm.
Fig 8.
A) Flow cytometry of BAL fluid showed a decrease in alveolar macrophages in SARS-CoV-2 infected animals over the course of the study. The decrease in alveolar macrophages was more pronounced in animals with pleuritic (red). B) Infiltrating interstitial macrophages (CD163+CD206-) were elevated compared to naïve animals both early (d1) and late (necropsy). C).
Fig 9.
Activated myofibroblasts are present within scarred lung tissue and persist long term.
A) KN90, lung alveoli. Myofibroblasts characterized by double-positive staining of αSMA (red) and cytokeratin 5 (green). Bar = 100μm. B) There was a >2 fold increase above baseline in TGF-B concentration in BAL in five of sixteen animals. TGF-B concentration was not siginifcantly different between animals that developed pleuritis.
Fig 10.
Absence of regenerative activity at 28 days post-exposure.
Immunofluorescence for the detection of bronchial epithelial progenitor cells. A) NJ48, lung. The epithelial lining of large airways (bronchi) has a basal layer containing progenitor cells characterized by cytoplasmic expression of cytokeratin 5 (green) and nuclear expression of p63 (red). B-D) KN90, lung alveoli. B) Progenitor cells are not observed in regions of pleuritis and pleural fibrosis with H&E (C) and trichrome (D). Bar = 100μm.