Histopathological, immunohistochemical, immunofluorescence, and TUNEL staining findings of respiratory tract tissues at days 2 and 4 after SARS-CoV-2 infection. A, Images of H&E- and immunofluorescence-stained nasal turbinate sections at 2 dpi and 4 dpi. (a) At 2 dpi, the epithelial layer was relatively intact with submucosal infiltration. (b) SARS-CoV-2 N protein was present almost throughout the entire epithelial layer and in some submucosal glands (insert). Cell nuclei were stained by DAPI. (c) At 4 dpi, epithelial cell death and desquamation were mild, but the intraepithelial and submucosal infiltration increased. (d) Viral N protein was expressed in the epithelial cells and cell debris. B, Trachea. (a) At 2 dpi, epithelial desquamation and loss of mucosal integrity with plugs of cell debris in the tracheal lumen were observed (black arrows) along with lamina propria connective tissue infiltration. The black square area was magnified showing detached epithelial cells mixed with mononuclear cells (arrowheads). (b) Viral N protein (white arrows and magnified images) was mostly found in intraluminal cell debris. (c) The same section of trachea was positive by TUNEL staining indicating apoptotic cell death. At 4 dpi, (d) tracheal epithelial desquamation and submucosal infiltration are mild and (e) viral N protein was only found in intraluminal cell debris. C, Histopathological changes in the lungs at 2 dpi. Images (a) and (b) represent lung tissues of two virus-challenged hamsters at low magnification (4x), showing patches of focal inflammation with intense color and pleural invaginations (arrows) were seen. (c and d) Varying severities of diffuse lung damage, including (e) protein-rich exudates and hyaline membrane filled alveolar space; (f) large amount of mononuclear cells infiltrating alveolar sac; (g) cell debris in bronchiolar lumen (arrows); and (h) alveolar collapse with cell death and acute hemorrhage (arrows). D, Viral N protein expression and TUNEL staining in the lungs. (a) SARS-CoV-2 N protein distribution in the lung (“Br” = bronchiole; “Al” = alveoli; “V” = blood vessel); (b) and (c) are magnified images illustrating viral N protein–positive staining in alveolar macrophages (thin arrows in b and c) and type I (arrowheads in c) and type II pneumocytes (thick arrows in b); (d) to (f) show TUNEL labeling in representative fields of the lung tissues. E, Histopathological changes in the lungs at 4 dpi. (a and b) Low-magnification images (4×) showing diffuse inflammation, large areas of consolidation, pleural invaginations (thin arrows), and severe hemorrhage (thick arrows). (c) Representative images of patchy consolidation and (d) pulmonary hemorrhage. High magnification showing (e) intense peribronchiolar infiltration, epithelial cell swelling, intraepithelial layer infiltration, mixture of cell debris and exudates filling in the bronchiolar lumen, and a gigantic multinucleated syncytial body in the magnified image (arrows); (f) and (g) show various sizes and locations of syncytial bodies (arrows); (h) alveolar collapse with mononuclear cell infiltrate and dead epithelial cells in alveolar space (arrows); and (i) marked hemorrhagic exudates with tissue destruction. F, (a) Extensive pulmonary cellular proliferation forming multiple layers of thickened alveolar wall. (b) to (d) Immunohistochemical staining showed extensive expression of proliferation marker Ki67 in bronchiolar epithelium and alveoli. G, Diffuse distribution of (a) viral N protein and (b) TUNEL staining showed diffuse apoptotic cell death in the lungs. Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; dpi, days postinoculation; H&E, hematoxylin and eosin; NP, nucleocapsid protein; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; TUNEL, terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling.