Fig 2: Effects of SARS-CoV-2 on the olfactory receptor neuron lineage after short-term close contact. (A) Representative hematoxylin and eosin staining (HE) images of the olfactory epithelium in control (CT) hamsters and contact hamsters on days 3 and 7 (co-Day 3, co-Day7).(B–D): Representative images of immunohistological staining. The nasal septum (NS) area and lateral turbinate (LT) area are shown in magnified view. Sex-determining region Y-box 2 (SOX2)+ progenitor cells (B), growth-associated protein 4 (GAP43)+ immature olfactory receptor neurons (ORNs) (C), and olfactory marker protein (OMP)+ ORNs (D) are shown in brown. The basal layer is indicated by arrows. Tissue sections were counterstained with the nuclear dye hematoxylin (blue). Numbers of SOX2+ ORN progenitors per 300 µm of the basal layer and GAP43+ immature ORNs and OMP+ mature ORNs per 300 µm of olfactory epithelium in each area are counted in CT or contact hamsters. ****P < 0.0001. SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.

Fig 3: Hypothesis of temporal changes in the SARS-CoV-2 viral load in the lungs and the number of olfactory receptor neuron-related cells in different nasal areas. Changes over time in the ratio of the number of olfactory receptor neuron lineage cells divided by that of the control group at each time point are displayed in the graph. The dashed line shows the speculated future trend. In addition, the estimated viral load of SARS-CoV-2 in the lungs is overlaid on each graph. NS, nasal septum; LT, lateral turbinate; OMP, OMP+ mature olfactory receptor neurons; GAP43, GAP43+ immature olfactory receptor neurons; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; SOX2, SOX2+ olfactory progenitors.

Fig 4: (A,B) Representative images of hematoxylin and eosin (H&E)-stained sections of the olfactory epithelium from young adult mice (A, 40× magnification; B, 400× magnification). A black line in (A) indicates the range for counting the number of each cell type. The box in (A) indicates the region of the olfactory epithelium shown at a representative higher magnification in (B). Differences in the number of OMP+ mature olfactory receptor neurons (ORNs) (C), SOX2+ ORN progenitors (D), Ki-67+ proliferating cells (E), GAP43+ immature ORNs (F), and cleaved Cas3+ apoptotic cells (G) in the OE were evaluated by immunohistological staining (brown). Tissue sections were counterstained with the nuclear dye hematoxylin (blue). Representative images (400× magnification) of tissues stained with antibodies against olfactory marker protein (OMP), SRY (sex determining region Y)-box 2 (SOX2), Ki-67 (antigen identified by monoclonal antibody Ki-67), growth associated protein 43 (GAP43), and cleaved caspase 3 (CAS3) are shown. The number of cells per mm of basal layer length (C–G) was counted manually. Data represent the mean ± SD. **P < 0.01 (n = 6, Mann–Whitney U-test).

Fig 5: (A) Representative images of immunohistological staining (brown) of SOX2+ olfactory receptor neurons (ORN) progenitor cells, Gap43+ immature ORNs, Ki67+ proliferating cells and cleaved caspase-3+ (Cas3+) apoptotic cells. Tissue sections were counterstained with the nuclear dye hematoxylin (blue). Arrowheads indicate Cas3+ apoptotic cells in the OE (n = 6). (B) Numbers of SOX2+ ORN progenitors and Ki67+ actively proliferating cells per mm of the basal layer, and Gap43+ immature ORNs and Cas3+ apoptotic cells per mm of the OE in saline or CSS-treated mice. Data represent means ± SEM (n = 6). *P < 0.05; **P < 0.01; ***P < 0.001 compared with saline-treated mice (one-way ANOVA).