Fig 2: Infection of the olfactory mucosa by SARS-CoV-2, related to Figure 5Confocal images of sections through the olfactory mucosa of COVID #8 (A and B), COVID #7 (C-H), COVID #57 (I), and COVID #25 (J). (A and B) SARS-CoV-2-N puncta occur throughout the apical-basal width of the olfactory epithelium. KRT8-IR signal labels a patch of uninfected sustentacular cells (stippled line) and cells lining gland ducts in the lamina propria (A). Infected sustentacular cells are low on or negative for UGT2A1 puncta and ERMN-IR signal, in contrast to uninfected sustentacular cells (B). (C-E) SARS-CoV-2-N puncta occur throughout the apical layer of sustentacular cells, in a mutually exclusive manner with CNGA2 puncta and TUBB3-IR signal (C and D) or GNAL puncta (E) in the middle layer of OSNs. (F) Sustentacular cells harbor SOX2 puncta across their apical-basal width. The stippled lines outline two sustentacular cells harboring perinuclear SARS-CoV-2-orf1ab-sense puncta reflecting ongoing viral replication. (G and H) Three infected sustentacular cells harbor densely packed SARS-CoV-2-N puncta, reflecting a high viral load. The stippled lines in H outline two sustentacular cells that are in the plane of focus of this confocal image: their KRT18-IR signal is depleted, in contrast to the strong KRT18-IR signal in nearby uninfected sustentacular cells. (I) Remnants of TUBB3-IR OSNs do not contain nucleocapsid-IR signal. (J) A patch of disintegrating olfactory epithelium containing nucleocapsid-IR signal is flanked by two areas of olfactory epithelium that do not contain nucleocapsid-IR signal but contain numerous TUBB3-IR OSNs.

Fig 3: Spatial whole-transcriptome profiling of the olfactory epithelium in COVID-19(A) Scanned image of a section through the OM of COVID #8. RNAscope for S was combined with IHC for KRT8/18 and pan-KRT. Four AOIs in the OE are indicated, with a low viral load (13 and 14) or a high viral load (20 and 7). SYTO 83 served as nuclear stain.(B) Magnification of AOI 13 and AOI 7.(C) Boxplots of nucleus counts of AOIs with low versus high viral load, with the numbers of the four AOIs shown in (A). NS, not significant.(D) Boxplots of log2 normalized expression counts for orf1ab (p = 7.96e-07).(E) Regression curve between normalized expression counts for S (x axis) and orf1ab (y axis).(F) Volcano plot with the magnitude expressed as log2 FC (x axis) and significance expressed as –log10 of the unadjusted p value (y axis) of differential expression of 9,262 genes (dots) in WTA data between the ten ORF1ab High AOIs and the seven ORF1ab Low AOIs. Dashed vertical lines represent a |log2 FC| of one. Genes of interest are labeled. Dots with gray outlines are the 26 OR genes. Dots with p values with a false discovery rate (FDR) <5% are shown in red.(G) Log2 FC values of markers for sustentacular cells (bottom) and OSNs (middle) and of OR genes (top). The asterisk indicates the average of the 26 differentially expressed OR genes.(H) Boxplots of log2 normalized expression counts for the 26 differentially expressed OR genes (26 OR) and the 8 OSN markers (OSN).

Fig 4: Infection of the respiratory mucosa by SARS-CoV-2(A–C) Confocal images of sections through the RM of control #12. FOXJ1 and KRT7 are markers for ciliated cells, and KRT5/6 for basal cells (A). EPCAM is a marker for epithelial cells in the RE and the LP, MUC5AC for goblet cells in the RE, and cells lining a transversely cut gland duct in the LP (B). The ACE2-IR signal caps the surface of the RE, and TMPRSS2 puncta cluster with FOXJ1 puncta in ciliated cells (C).(D–L) Confocal images of sections through the RM of COVID #7, #27, #51, #39, #29, and #63. Widespread nucleocapsid-IR signal occurs apically within the RE, and orf1ab-sense puncta reflect ongoing viral replication (D). The dashed square in (D) is the area magnified in (E). N-sense puncta cluster with orf1ab-sense puncta in nucleocapsid-IR cells (F) and with orf1ab puncta (G). Ciliated cells harbor N-sense, M, and FOXJ1 puncta (H). An individual ciliated cell harbors orf1ab-sense and FOXJ1 puncta (I). Ciliated cells harbor S-sense and FOXJ1 puncta and contain KRT7-IR signal (J). KRT8-IR cells lining gland ducts in the LP harbor N puncta (K) or orf1ab-sense puncta (L). DAPI served as nuclear stain.See also Figure S2.

Fig 5: Negative control stainings, related to Figures 3, 5, and 7(A) Confocal image of a section of the respiratory mucosa of COVID #63. A negative control for the RNAscope protocol was performed on a section adjacent to the section of which a confocal image is shown in Figure 3L. The specificity of the densely packed SARS-CoV-2-orf1ab-sense puncta in cells lining gland ducts in the lamina propria in Figure 3L is demonstrated by the absence of puncta for the dapB gene of Bacillus subtilis in any of the three Opal channels (3-plex). (B) Confocal image of a section of the respiratory mucosa of COVID #51. The SARS-CoV-2-S probe gives densely packed red puncta. The SARS-CoV-2003-S probe, specific for S of SARS-CoV (now known as SARS-CoV-1) causing an outbreak in 2002–2004, gives no green puncta. (C) Confocal image of a section of the respiratory mucosa of control #12. This negative control for the SARS-CoV-2-M probe and the nucleocapsid antibody reveals no red puncta or blue IR signal. KRT8 marks epithelial cells in the respiratory epithelium and the lamina propria. (D) Confocal image of a section of the respiratory mucosa of control #12. This negative control for the SARS-CoV-2-N-sense and SARS-CoV-2-orf1ab probes reveals no red or blue puncta. Ciliated cells harbor FOXJ1 puncta. (E) Confocal image of a section of the olfactory mucosa of control #15. This negative control for the SARS-CoV-2-S and SARS-CoV-2-orf1ab-sense probes reveals no red or blue puncta. TUBB3 marks OSNs in the olfactory epithelium and labels OSN axon bundles in the lamina propria. (F) Confocal image of a section of the olfactory bulb of control #15. This negative control for the SARS-CoV-2-N and SARS-CoV-2-S-sense probes reveals no red or blue puncta. TUBB3 marks axons and olfactory bulb neurons. DAPI served as nuclear stain.