Fig 1.
Inoculation of K18-hACE2 mice results in lethal infection and virus shedding.
. a. Relative weight loss in mice after SARS-CoV-2 inoculation. The lines represent mean ± SEM. Wilcoxon matched-pairs rank test was conducted to compare weight differences between groups. b. Survival curves of mice inoculated with 104 or 105 TCID50 SARS-CoV-2, or 105 γ-irradiated SARS-CoV-2. c. Violin plot of viral load in nasal, oropharyngeal and rectal swabs with median as centre. Blue: 104 TCID50 (low dose animals, n = 6); red: 105 TCID50 (high dose animals, n = 6); green: 105 TCID50 γ-irradiated (control animals, n = 2); dotted line = limit of detection.
Fig 2.
SARS-CoV-2 tissue tropism in K18-hACE mice.
a. Violin plot of viral load in tissues quantified by RT-qPCR with median as centre. b. Violin plot of infectious SARS-CoV-2 titers in tissues, with median as centre. Blue: 104 TCID50 (low dose animals, n = 6); red: 105 TCID50 (high dose animals, n = 6); green: 105 TCID50 γ-irradiated (control animals, n = 2); dotted line = limit of detection.
Fig 3.
Pathological changes in lungs of K18-hACE mice inoculated with SARS-CoV-2 at 3 and 7 DPI. a, b, c, d.
γ-irradiated SARS-CoV-2 inoculated control lungs appear normal and lack SARS-CoV-2 antigen immunoreactivity. e, f, g. Inflammation at 3 DPI, characterized by perivascular and septal infiltration by neutrophils, macrophages, lymphocytes, and edema. i, j, k. Multifocal interstitial pneumonia at 7 DPI, characterized by type II pneumocyte hyperplasia (arrowheads), alveolar and perivascular inflammation, fibrin, edema, syncytial cells (inset arrowheads) and single cell necrosis. h, l. SARS-CoV-2 antigen immunoreactivity in type I (arrowheads) and type II pneumocytes (arrows) at 3 and 7 DPI. HD: high dose (105 TCID50 SARS-CoV-2). Magnification: a, e = 40 x; b, f = 100 x; c, g, h = 400 x, inset 1000 x.
Fig 4.
Infiltration of innate and adaptive immune-cell populations in the lungs of SARS-CoV-2 infected mice.
a, e, i, non-infected control. b, f, j,105 TCID50 3 DPI. c, g, k, 105 TCID50 7 PDI d, h, l, survivor animal 21 DPI. a. γ-irradiated SARS-CoV-2 inoculated controls with few macrophages (brown). b, c. Increased macrophages at 3 and 7 DPI. d. Macrophages present at end of study. e. Scattered T cells (brown) in the γ-irradiated SARS-CoV-2 inoculated controls. f, g. T cells are increased in perivascular tissue and alveolar septa at 3 and 7 DPI. h. T cells forming lymphoid aggregates with B cells in perivascular tissues. i. B-cells (brown) are few in the γ-irradiated SARS-CoV-2 inoculated controls. j,k. B cells are increased in alveolar septa at 3 and 7 DPI. l. B cells forming lymphoid aggregates with T cells in perivascular tissues. Magnification: a-l = 400 x.
Fig 5.
Histologic features of nasal turbinates of SARS-CoV-2 infected mice.
a. Normal nasal turbinates from γ-irradiated SARS-CoV-2 inoculated control mouse lined by olfactory and respiratory epithelium. b. Absence of SARS-CoV-2 antigen immunoreactivity in respiratory and olfactory epithelial cells of low dose inoculated animal at 3 DPI. c. 3 DPI nasal turbinates lack inflammation but d. exhibit cytoplasmic immunoreactivity (brown) of respiratory epithelial cells for SARS-CoV-2 antigen. e. Higher magnification of respiratory epithelium. f. Higher magnification showing SARS-CoV-2 antigen immunoreactivity in ciliated respiratory epithelial cells. Magnification: a, b, c, d = 100 x; e, f = 400 x.
Fig 6.
Neurotropism of SARS-CoV-2 in infected mice at 7 DPI.
a-b Normal hippocampus in a γ-irradiated SARS-CoV-2 inoculated control mouse. c. No inflammation is noted and d. there is no detection of SARS-CoV-2 antigen at 3 DPI. e. Generalized increase in cellularity of the cerebral cortex and hippocampus and the meninges are mildly expanded by edema and inflammatory cells at 7 DPI. f. SARS-CoV-2 antigen immunoreactivity (brown) throughout the cerebral cortex and hippocampus. g and h. SARS-CoV-2 antigen immunoreactivity in neurons of the hippocampus and cerebral cortex. i. A small caliber vessel in the cerebral cortex contains a microthrombus (arrowhead) surrounded by hemorrhage and inflammatory cells which infiltrate the adjacent neuropil; there are increased glial cells throughout the image. j. Another microthrombus (arrowhead) in a small caliber vessel. Magnification: a, b, c, d, e, f = 100 x; g, h, i = 400 x; and j = 1000 x.
Fig 7.
Humoral and cytokine/chemokine responses to SARS-CoV-2 infection in K18-hACE mice.
a. IgM and IgG antibody titres against SARS-CoV-2 spike ectodomain by ELISA in serum. White line represents geometric mean of end point dilutions per study group. Dotted line represents limit of detection. b, c, d. Four-fold serial-diluted brain homogenate, lung homogenate and serum of selected cytokines/chemokines in K18-hACE mice challenged with SARS-CoV-2 measured on Bio-Plex 200 instrument (Bio-Rad) using Milliplex Mouse Cytokine/Chemokine MAGNETIC BEAD Premixed 25 Plex Kit (Millipore). Violin plots are depicted with individual values and median of all mice. Two-tailed Mann-Whitney’s rank test was used to compare differences between groups. e. Heatmap showing cytokine titers clusters based on DPI and dose of inoculation.
Fig 8.
Effect of NK-cell and Gr-1 depletion on SARS-CoV-2 infection in K18-hACE mice.
a. Relative weight loss in mice inoculated with 102 TCID50 SARS-CoV-2 after depletion of NK cells or neutrophils (Gr-1+ cells). The lines represent mean ± SEM. Area under the curve (AUC) was determined for each weight curve. b. Violin plot of IgG antibody titres against SARS-CoV-2 spike ectodomain by ELISA in serum. White line represents geometric mean of end point dilutions per study group. Dotted line represents limit of detection. c. Violin plot of viral load in tissues quantified by RT-qPCR with median as centre. d. Violin plot of infectious SARS-CoV-2 titers in brain at 7DPI, with median as centre. Dotted line represents limit of detection. e. Violin plot of viral load in oropharyngeal swabs with median as centre. Viral RNA was quantified using RT-qPCR; Dotted line represents limit of detection. Area under the curve (AUC) was determined for each group. Differences were determined by ordinary one-way ANOVA. Blue: Gr-1 depletion, n = 4); yellow: NK1.1. depletion, n = 4/5); black: control, n = 6).