Fig 2: Detection of SARS-CoV-2 antigens in lung tissue of a fatal case with exudative fibrinous pneumonitis and diffuse pulmonary edema. A) Lung tissue in which the alveolus (*), artery (a), and pulmonary edema (£) are observed. The arrow indicates inflammatory cellular infiltrate in the alveolar space. The arrowhead indicates the interalveolar septum where gas exchange takes place, HE stain. B) Arrowheads indicate immunoreaction to SARS-CoV-2 antigens. Images at the right column show viral antigens mainly located in the cytoplasm of inflammatory infiltrate cells, likely macrophages and immune response cells. Immunohistochemistry was performed using the MACH4 Universal AP Polymer Kit™ and visualized with Warp Red. For the detection, a preparation of rabbit anti-SARS nucleocapsid protein antibody (Novus Biologicals-NB100-56576, 1:100 dilution in DaVinci green diluent - Biocare Medical) was used. Scales in A and B = 50 µm; scales in the enlarged right column images = 10 µm

Fig 3: SARS-CoV-2 detection in the brain.Representative single-label IHC shows infrequent SARS-CoV-2 nucleocapsid (SARS-N) positivity in a cerebellar blood vessel of RM1 (a). Positivity was not detected in non-infected controls (AGM6 cerebellum b). SARS-CoV-2 spike (SARS-S) mRNA expression was assessed with in situ hybridization (RNAscope) but not seen in control animal tissue (RM6 cerebellum f-h). Rare positivity was seen in infection (AGM1 cerebellum c–e). RNAscope was performed 7 times on the brain regions investigated. A majority of brain tissue does not show any virus in infected animals which can be seen in the neighboring vessel (RM3 basal ganglia l–n) to a vessel with suggestive virus positivity (i–k). Endothelial cell infection is suggested by double fluorescent labeling of SARS-N with von Willebrand factor (vWF) (RM3 basal ganglia i–k). Merged images show colocalization of SARS-N (red; j) with vWF (green; i), indicated by white arrows. Blue color represents DAPI labeled cell nuclei. Immunohistochemical staining for SARS-N was performed 12 times on the brain regions investigated. SARS-CoV-2 RNA was detected in the different brain regions of infected animals via a CRISPR-based fluorescent method (o) where n = 3 repeats of independent samples. Dotted line indicates the cut-off value of positivity equal to 3.6 × 106 photoluminescence (PL) intensity. Data are expressed as mean ± SEM. Source data are provided as a Source Data file. Abbreviations: BG basal ganglia, CER cerebellum, BS brainstem, FL frontal lobe, CSF cerebrospinal fluid, BC blank control, arb. units arbitrary units, AGM African green monkey, RM Rhesus macaque. Scale bars = 50 µm (a and b), 20 µm (c–h), and 10 µm (i–n). Fluorescence images are at 100×.

Fig 4: SARS-Cov-2 WA1 selectively targets human olfactory neuroepitheliuma,b, Confocal images of SARS-CoV-2 viral antigen NP (red, Novus, NB100–56576) and olfactory neuronal marker ß-III Tubulin (Tuj1, green) in superior turbinate biopsies from 2 separate patients. Images were obtained under tile scan mode, which covered olfactory and adjacent respiratory epithelium in the same piece of tissue. Boxed area in (b) was highlighted in Extended Data Fig. 1c, d.c, Co-staining of NP and Tuj1 in human biopsy collected from the olfactory cleft.d, Representative image of NP overlapped with Tuj1-negative ciliated cell (brightfield). Confocal image was obtained from a biopsy which contains only respiratory epithelium.e, Quantification of NP+ cells per mm tissue. 24 independent specimens have exclusively respiratory epithelium (RE), while 7 specimens contained both respiratory and olfactory epithelium (OE).Arrowheads (a-c) indicate the detachment of infected cells. Data in (e) are represented as mean ± S.D. p value was calculated by one-way ANOVA. Scale bars, 50 µm (a and b); 20 µm (c,d).