Different smell loss and nose tissue changes in Syrian hamsters infected with SARS-CoV-2 variants

African green monkey kidney Vero E6 cells were grown in Dulbecco's modified Eagle's medium (DMEM; Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS; Gibco), 100 U/mL penicillin, and 0.1 mg/mL streptomycin (Gibco) at 37°C in a 5% CO₂ incubator. SARS-CoV-2 (USA/WA-1/2020) and Alpha (B.1.1.7; USA/CA-CDC5574/2020), Beta (B.1.351; MD-HP01542/2021), Gamma (P.1; MD-MDH-0841/2021), Delta (B.1.617.2; GNL-751), and Omicron (B.1.1.529; hCoV/EHC_C19_2811C) variants were received from the World Reference Center for Emerging Viruses and Arboviruses at the University of Texas Medical Branch (UTMB) and propagated in Vero E6 cells with DMEM supplemented with 2% FBS. Cell culture supernatants were stored at −80°C until use. All work with infectious SARS-CoV-2 was approved by the Institutional Biosafety Committee at UTMB and performed in BSL-3 facilities at UTMB in accordance with institutional guidelines.

Lung samples collected from SARS-CoV-2-infected hamsters were homogenized in DMEM supplemented with 2% FBS. Cell culture supernatants of SARS-CoV-2 variants or the lung homogenates were 10-fold serially diluted in DMEM with 2% FBS and inoculated into Vero E6 cells on 96-well plates. Four days after inoculation, the plates were fixed with 10% buffered formalin and stained with 0.25% crystal violet to visualize the cytopathic effect. The 50% tissue culture infectious dose (TCID₅₀) value was determined by the Reed and Muench method (18).

Five- to six-week-old female Syrian golden hamsters were purchased from Charles River Laboratories according to the previous studies (16, 17). We did not consider sex differences in this study. Seven groups of five hamsters were intranasally inoculated with either 10⁵ TCID₅₀ of SARS-CoV-2 WA-1, Alpha, Beta, Gamma, Delta, or Omicron variants diluted in 100 µL of sterile PBS or sterile PBS as mock infection. The body weight of animals was measured daily after the virus infection throughout the study for 5 days. All animals were subjected to a buried food detection test, as previously described (16), to investigate olfactory function at 5 days post-infection (dpi). After the behavioral test, the animals were euthanized to collect nasal turbinates and lungs for histopathological analysis or virus titration. All hamsters were housed in the animal biosafety level-2 (ABSL-2) and ABSL-3 facilities in the Galveston National Laboratory at UTMB. All animal studies are reviewed and approved by the Institutional Animal Care and Use Committee at UTMB and performed with the National Institutes of Health guidelines. This study is reported in accordance with ARRIVE guidelines (https://arriveguidelines.org/arrive-guidelines↗).

The buried food detection test was performed to examine olfactory function, as previously reported (16). A housing cage with at least 3 cm of bedding was prepared, and a honey-flavored teddy graham (Nabisco) was buried in one corner approximately 1 cm below the surface. A hamster was placed in the corner diagonal from the hidden cookie, the lid was closed, and the time until the hamster found and grasped the cookie was recorded. All hamsters were given a maximum of 300 s to find the buried cookie.

Collected nasal turbinates were fixed with 10% buffered formalin for 7 days with a formalin change after 24 h before removal from the BSL-3 facilities. The olfactory bulbs and nasal tissue were extracted from the skull and decalcified with 10% EDTA and embedded in paraffin. The paraffin-embedded tissues were then sectioned at 5 µm and stained with hematoxylin and eosin (HE). The scoring of olfactory epithelium damage was performed, as previously described (16). Briefly, each of the four turbinate zones including nasal septa (S), medial turbinate (MT), dorsal turbinate (DT), and lateral turbinate (LT) was assigned an epithelial damage score: 0—no damage (normal), 1—mild damage (damage reaches only through epithelial layer; basal layer remains untouched), 2—moderate damage (up to 75% of damage reaches basal cells), or 3—severe damage (over 75% of damage reaches basal cells). Scores were averaged between the four reviewers in a blinded manner. The HE-stained sections were imaged using an Olympus IX 71 microscope (Olympus) with a Nikon Digital Sight 1000 digital camera (Nikon) and Nikon NIS-Elements F software (Nikon).

The deparaffinized sections were incubated for 20 min in a rice cooker in either 10 mM Tris-1 mM EDTA buffer (pH 9.0) for rabbit anti-Iba1 antibody (FUJIFILM Wako Chemicals, 013-27691) or in Target Retrieval Solution (Dako) for rabbit anti-SARS nucleocapsid protein (N) antibody (NOVUS, NB100-567576). The sections were soaked in 3% hydrogen peroxide solution and then incubated with protein blocking solution (Dako) for 15 min at room temperature. The sections were incubated overnight with anti-Iba1 antibody or anti-SARS N antibody at a dilution of 1:1,000 at 4°C. After washing with PBS, the sections were incubated with SignalStain Boost IHC detection reagent (HRP, Rabbit) (Cell Signaling Technology, 8114S) for 30 min. The immunohistochemical reactions were developed with 3,3′-diaminobenzidine tetrahydrochloride-hydrogen peroxide solution. The sections were lightly stained with hematoxylin and sealed with a coverslip in a mounting medium. The stained sections were examined and images were captured using an Olympus IX 71 microscope with a Nikon Digital Sight 1000 digital camera and Nikon NIS-Elements F software. The scoring of the distribution of viral antigen in each of four turbinate zones was assigned a score using IHC sections labeled with anti-SARS N antibody: 0—0% of immunoreactive positive cells, 1—<20% of immunoreactive positive cells, and 2—>20% immunoreactive positive cells in the olfactory epithelium composing cells. The scoring of the distribution of Iba1-positive macrophages in each of four turbinate zones was assigned a score using IHC sections labeled with anti-Iba1 antibody: 0—<10% of positive reaction, 1–10<30% of positive reaction, and 2—>30% positive reaction in the olfactory epithelium. Each score was averaged between the four reviewers in a blinded manner.

The alcian blue stained-sections were used for the detection and histoplanimetry of olfactory glands (Bowman's glands). The percentage of blue-stained area in the whole lamina propria in three randomly selected regions was calculated and averaged using Colour Deconvolution 1.7 plugin provided in Fiji (National Institutes of Health, Bethesda, MD, USA).

All data were analyzed using GraphPad Prism version 10.2.1 software. For comparison of weight change at each time point, we performed a two-way analysis of variance (ANOVA), followed by Dunnett's post hoc test. For comparison of virus titer in the lung, behavioral test, histopathological score, and histoplanimetry of olfactory gland, we performed one-way repeated-measures ANOVA, followed by Dunnett's test. Correlations were determined using a Pearson correlation test. P < 0.05 was deemed statistically significant for all analyses.

Syrian hamsters were inoculated intranasally with 10⁵ TCID₅₀ of SARS-CoV-2 (WA-1) or its variants; Alpha, Beta, Gamma, Delta, and Omicron variants, and monitored for signs of illness and body weight change (Fig. 1A). The WA-1, Beta, and Gamma variants-infected hamsters showed significant weight loss from 2 to 5 dpi compared to the negative control of infection (PBS). Animals infected with Alpha or Delta variants developed minor weight loss at 2 dpi and recovered by 3 dpi. The Omicron variant-infected hamsters did not show body weight loss. None of the SARS-CoV-2-infected animals or mock-infected animals showed any visible signs of illness. At 5 dpi, anosmia was investigated through a buried food detection test (Fig. 1B) (16). The PBS-inoculated animals found the hidden cookies in an average of 24.46 s. Cookie-finding time was significantly extended in WA-1, Alpha, Beta, or Gamma variant-infected hamsters, with average times of 118.55, 148.38, 133.41, and 122.86 s, respectively. One of the Alpha variant-infected animals was unable to find the cookie until the 300 s time limit. In the Omicron-infected animals, there was no significant increase in cookie-finding time compared to PBS inoculation, although one animal required longer than the others, at 85.26 s. After behavioral testing, lung and nasal samples were collected for virus titration and histological analysis. The lung samples of WA-1, Alpha, Beta, Gamma, and Delta variant-infected hamsters had detectable viral titers with mean viral loads measuring 3.57, 4.57, 4.90, 5.65, 3.47 log₁₀ TCID₅₀/g, respectively (Fig. 1C). Three of the five lung samples from Omicron variant-infected hamsters did not show viral titers above the detection limit (1.50 log₁₀ TCID₅₀/g), while the average of the two detectable samples was 3.30 log₁₀ TCID₅₀/g.

Histopathological analysis was performed on nasal samples collected from SARS-CoV-2 variant-infected hamsters to quantify tissue damage (Fig. 2 and 3; Fig. S1). In WA-1-infected hamsters, nasal tissue damage including degeneration of olfactory epithelial cell alignment, olfactory epithelium cell disruption, and shortening of cilia was severe in S, MT, DT, and LT regions. In particular, the tissue damage in the LT region was the most severe with an average histopathology score of 2.56. In Alpha-infected animals, the tissue damage in S, MT, and DT was less severe, but the damage in LT was also significant with an average histopathology score of 2.16. Tissue damage in the LT region tended to be most severe of the four regions, even in the Beta-, Gamma-, and Delta-infected hamsters with average histopathology scores of 1.84, 2.24, and 2.12, respectively. In Omicron-infected hamsters, there was no or very little tissue damage in the S, MT, and LT regions, though minor tissue damage was observed in the DT region with disruption of olfactory epithelium cell alignment.

The distribution of viral antigen in the olfactory epithelium of SARS-CoV-2 variant-infected hamsters was investigated using immunohistochemistry (Fig. 2; Fig. S2; Table S1). Viral antigen-positive olfactory epithelial cells were found in both retained and detached olfactory epithelium. In WA-1-infected animals, more virus-infected olfactory epithelial cells were observed in the DT and LT regions compared to the S and MT regions. The Alpha-, Beta-, Gamma-, Delta-, and Omicron variant-infected animals also showed viral antigen-positive cells in the DT and LT regions. In all groups, the number of viral antigen-positive cells in the LT region was similar to or higher than in the DT region. Viral infection of the S region was most commonly observed in the Beta variant-infected group, followed by the Gamma variant-infected group. In the other variant-infected groups, the distribution of the viral antigen-positive cells in this region was absent or minor. In the MT region, more viral antigens were detected in the Beta and Gamma variant-infected groups. In contrast, viral antigens were not detectable in the MT region in Delta and Omicron-infected animals. Interestingly, in the Omicron-infected group, there were areas of viral antigen-positive cells distributed from cilia to epithelial cells in the DT and LT regions. Macrophage infiltration into the lamina propria and the olfactory epithelium was observed in the S, MT, DT, and LT regions to a similar degree in WA-1-, Alpha-, Beta-, and Gamma-infected animals (Fig. 2; Fig. S3; Table S2). On the other hand, macrophage infiltration in the DT region was notable compared to the S, MT, and DT regions in Delta and Omicron-infected animals.

Through secreting mucus, the olfactory glands in the olfactory tissue play a crucial role in maintaining the health of the olfactory epithelium, ensuring proper olfactory function, and protecting against pathogens (19–21). We compared the extent of damage to these glands in hamsters infected with different SARS-CoV-2 variants by measuring the percentage of olfactory gland area stained by alcian blue (Fig. 2 and 4; Fig. S4). In the mock-infected group, the mean blue area percentage was 3.7%, 4.5%, 4.9%, and 3.9% in the S, MT, DT, and LT regions, respectively. In all SARS-CoV-2-infected animals, significant olfactory gland shrinkage ranging from 27% to 80% was observed. In WA-1, Alpha, Beta, and Gamma-infected hamsters, the area of olfactory glands was reduced to less than half, especially in the DT and LT regions of the Gamma-infected group, which lost 80% and 77% of their olfactory glands, respectively.