Evolution of nasal and olfactory infection characteristics of SARS-CoV-2 variants

Abstract

SARS-CoV-2 infection of the upper airway and the subsequent immune response are early, critical factors in COVID-19 pathogenesis. By studying infection of human biopsies in vitro and in a hamster model in vivo, we demonstrated a transition in nasal tropism from olfactory to respiratory epithelium as the virus evolved. Analyzing each variant revealed that SARS-CoV-2 WA1 or Delta infect a proportion of olfactory neurons in addition to the primary target sustentacular cells. The Delta variant possessed broader cellular invasion capacity into the submucosa, while Omicron displayed enhanced nasal respiratory infection and longer retention in the sinonasal epithelium. The olfactory neuronal infection by WA1 and the subsequent olfactory bulb transport via axon were more pronounced in younger hosts. In addition, the observed viral clearance delay and phagocytic dysfunction in aged olfactory mucosa were accompanied by a decline of phagocytosis-related genes. Further, robust basal stem cell activation contributed to neuroepithelial regeneration and restored ACE2 expression postinfection. Together, our study characterized the nasal tropism of SARS-CoV-2 strains, immune clearance, and regeneration after infection. The shifting characteristics of viral infection at the airway portal provide insight into the variability of COVID-19 clinical features, particularly long COVID, and may suggest differing strategies for early local intervention.

Keywords: COVID-19; Neurological disorders; Neuroscience.

Figure 1. SARS-Cov-2 WA1 selectively targets human olfactory neuroepithelium.

(A and B) Confocal images of SARS-CoV-2 viral antigen NP (red, Novus, NB100-56576) and olfactory neuronal marker β-III Tubulin (TUBB3, green) in superior turbinate biopsies from 2 separate patients. Images were obtained under z stack (4 μm) and tile scan mode, which covered olfactory and adjacent respiratory epithelium in the same piece of tissue. Boxed area in (B) was highlighted in Supplemental Figure 1, C and D. (C and D) Costaining of NP and TUBB3 in human biopsy collected from the olfactory cleft (C) or in a biopsy contains only respiratory epithelium (D). In panel D, NP overlapped with TUBB3^– ciliated cell (brightfield). Images were obtained under z stack (4 μm) and tile scan mode. (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 ± SD. P value was calculated by 1-way ANOVA. Scale bars: 50 μm (A and B); 20 μm (C and D).

Figure 2. Omicron variant shows tropism transition from olfactory to respiratory epithelium.

(A) Scheme of the tissue section. To avoid variability across different animals, frozen sections were collected and examined at 3 consistent levels (L1–3) representing the anterior (mainly respiratory epithelium), middle (respiratory + olfactory epithelium), and posterior (mainly olfactory epithelium). (B) Confocal images of NP and TUBB3-labeled hamster nasal sections at L2. WA1, Delta, and Omicron-infected hamsters were examined at 4 dpi. Images were obtained under z stack (12 μm) and tile scan mode. Boxed areas are highlighted at bottom. Scale bars: 500 μm. (C) Percentage of the infected olfactory epithelium. The total length of TUBB3⁺ or NP⁺/TUBB3⁺ epithelium in each section at L1–3 were quantified using Image J. (D) Quantification of NP⁺ cells in nasal respiratory epithelium. The total NP⁺ cells in TUBB3^– respiratory epithelium including paranasal sinuses of each section were counted. The total length of TUBB3^– respiratory epithelium in each section was measured using Image J. (E) qPCR analysis of Ace2 expression in mouse nasal respiratory or olfactory epithelium at age of 2 weeks, 2 months, and 19 months. The entire nasal respiratory or olfactory epithelium from the same animal were isolated separately. Data are represented as mean ± S.D. Statistical significance was determined by unpaired 2-tailed t test. Each data point represents an individual animal (n = 3).

Figure 3. Delta variant infects cells in submucosa of the nose.

(A) Representative image shows NP⁺/Pan-Keratin⁺ Bowman’s glands in Delta-treated hamsters. (B) Quantification of infected Bowman’s glands. The average number of NP⁺ Bowman’s glands in each section was calculated. 3 sections per animal were counted. (C) Confocal image shows NP⁺/α-SMA⁺ myofibroblasts. Hamsters infected with Delta variant at 4 dpi were examined. (D) Costaining of TUBB3 and NP in nasal sections at 7 dpi. Whole nasal cavity images were captured using a tile scan and z stack mode on a 14 μm section. Boxed area in Omicron-infected hamster is highlighted on the right. Scale bars: 500 μm. (E) Quantification of NP⁺ respiratory epithelial cells in paranasal sinuses. 3 sections per animal were counted. The total length of TUBB3^– respiratory epithelium in each section was measured using Image J. SRE, sinus respiratory epithelium. Images were obtained under 3 μm z stack (A and C) mode or (12 μm) z stack plus tile scan mode (D). Data are represented as mean ± S.D. Statistical significance was determined by unpaired 2-tailed t test. Each data point represents an individual animal (n = 3).

Figure 4. Age-associated SARS-Cov-2 infection in olfactory sensory neurons.

(A–C) Confocal images showing WA1-infected hamster olfactory epithelium at 4 dpi. Insert in A highlighting a NP-stained axon bundle (horizontal section). Arrowheads indicate virus-infected TUBB3⁺ immature (B) or OMP⁺ mature (C) sensory neurons (coronal sections). White line indicated the basal layer of epithelium. (D and E) NP⁺ axon travel from neuroepithelium to laminar propria and merge into TUBB3⁺ axon bundle. (F–H), Quantification of NP⁺ axons in young and old hamsters at 6 dpi. Representative images show horizontal (F) or coronal sections (G). NP⁺ axons were quantified per μm of the diameter of axon bundle. (I and J) Representative images showing NP located in TUBB3⁺ human olfactory neurons (J) and the percentage of NP⁺ cells in TUBB3⁺ population (I). Dotted line in J indicates virus infected NP⁺ axon. Arrowheads denote NP⁺/TUBB3⁺ neurons compared with uninfected cells (empty arrowhead). Infected biopsies from 3 young donors (age 25–33 years) and 5 biopsies from older donors (age 54–72 years) were quantified for TUBB3⁺ neuronal infection. (K and L) Representative images of NRP1 expression in human olfactory epithelium (K) and quantification of NRP1⁺ cells in TUBB3⁺ population (L). 3 biopsies from young (age 20–30 years) and 4 biopsies from older donors (age 68–79 years) were examined for NRP1 expression. Images in A–G were captured with 2 μm Z-stack and exported by maximum intensity projections. Each data point represents an individual sample from hamster (H, n = 3), or human (I and L, n = 3-5). Details of human biopsies can be found in Supplemental Table 1. Data are represented as mean ± SD. Statistical significance was determined by unpaired 2-tailed t test. Scale bars: 20 μm.

Figure 5. Increased olfactory bulb transport of SARS-CoV-2 in young hamsters.

(A–C) Confocal images of IBA1 and NP costaining in hamster olfactory bulbs. Arrowheads indicate infected axon. (D) Costaining of NP and TUBB3 in a serial section next to panel B. (E) Quantification of IBA1⁺microglials in hamster olfactory bulb. Each data point in E (n = 3) represents an individual hamster sample. Data are represented as mean ± SD. Statistical significance was determined by unpaired 2-tailed t test. (F) Confocal image of cleaved caspase-3⁺/NEUN^– apoptotic cells (arrowheads) in the glomerular layer at 4 dpi. Images were captured with 3 μm (A–D) or 4 μm (F) Z-stack and exported by maximum intensity projections. Olfactory bulb tissues were collected from young and old hamsters at 6 dpi (A–D) or from mock control. Scale bars: 50 μm (A–D); 20 μm (F). ONL, olfactory nerve layer; GL, glomerular layer; EPL, external plexiform layer; MCL, mitral cell layer. Boxed areas are highlighted at bottom. Dotted circles indicate glomeruli.

Figure 6. Nasal inflammatory severity is correlated with the tropism of variants.

(A) Representative images showing CD45 and IBA1 costaining in olfactory mucosa. Mock or maSARS-Cov2 infected WT mice were examined at 6 dpi. Scale bars: 20 μm. (B and C) Confocal images of NP and IBA1-labeled hamster nasal sections. WA1, Delta, and Omicron-infected hamsters were examined at 4 dpi. Images were obtained under z stack (12 μm) and tile scan mode (B). White boxed areas are highlighted at bottom. Red boxed area is highlighted in C. Scale bars: 500 μm. (D) Quantification of IBA1⁺ cells in the olfactory epithelium and lamina propria. IBA1⁺ cells inside of sloughed-off debris in the nasal lumen were not included. 2 mm tissue of each section were counted. Data are represented as mean ± S.D. Statistical significance was determined by unpaired 2-tailed t test. Each data point represents an individual animal (n = 3).

Figure 7. Age-associated delay in viral clearance in olfactory mucosa.

(A) Coimmunostaining shows IBA1⁺ macrophages engulfing NP⁺ debris in hamster olfactory mucosa at 4dpi. (B) Representative image of IBA1 and cleaved caspase-3 costaining in hamster at 4 dpi. Arrowheads highlight the IBA1⁺ macrophages undergoing apoptosis. (C) Representative images showing IBA1 or NP staining in serial sections. Each panel combines 6 40× images acquired under tile scan mode. Young or old hamsters’ olfactory tissues were examined at 6dpi. (D and E) Quantification of IBA1⁺ (D) or NP⁺ (E) cells in hamster nasal olfactory lumen at 6dpi. Serial sections (C) from 4 different levels were quantified. (F) Violin plots showing the differentially expressed Clec4n (DECTIN-2) or Fpr2 in young and old macrophage/dendritic lineage. (G) Confocal images of IBA1 and DECTIN-2 costaining in mouse olfactory mucosa. Each data point represents an individual hamster sample (D and E, n = 3) or an individual cell (F). Statistical significance was determined by an unpaired 2-tailed t test. The white dotted line indicates the basement membrane. Scale bars: 20 μm (A, B, and G); 50 μm (C).

Figure 8. Regeneration of olfactory epithelium and reexpression of ACE2.

(A) Confocal images showing ACE2 (red) and KRT5⁺ horizontal basal cells (green) in olfactory epithelium of mock or SARS-CoV-2 infected hamster at 4 dpi. (B and C) qPCR analysis of Ace2 (B) or Omp (C) mRNA expression in SARS-CoV-2 infected hamster turbinate lysate at indicated time points. (D and E) Representative images of KRT5⁺ cells in newly regenerated olfactory epithelium (D) at 6 dpi, and quantification of epithelium thickness (E). The thickness of septal olfactory epithelium was measured using Zen lite “line” function. For each section, 8 spots were measured randomly. (F) Confocal image showing regenerated hamster olfactory epithelium expression of ACE2 at 28 dpi. (G) Representative image shows ACE2 and TUBB3⁺olfactory neurons in an olfactory biopsy from a patient with COVID-19 on day 12 after diagnosis. Dots in graph represent independent animal (B and C, n = 3–5; E, n = 3). Data are represented as mean ± SD. P value was calculated by unpaired 2-tailed Student’s t test. Scale bars: 20 μm.