CELLS AND VIRUS
Vero E6 cells were maintained using Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin, and L-glutamine. SARS-CoV-2 (USA/WA-1/2020) was propagated in Vero E6 cells with DMEM supplemented with 2% FBS. Cell culture supernatant was stored in the − 80°C freezer until use.
ANIMAL EXPERIMENTS
Five- to six-week-old female Syrian golden hamsters were purchased from Charles River and inoculated intranasally with 100 µL of SARS-CoV-2 diluted in phosphate-buffered saline (PBS) or PBS as a mock control. Body temperatures were measured using subcutaneously implanted BMDS IPTT-300 transponders and the DAS-8007 transponder reader from Bio Medic Data Systems. Body weights were measured with an Ohaus CX1201 Portable Scale. All hamsters were housed in the animal biosafety level-2 (ABSL-2) and ABSL-3 facilities within the Galveston National Laboratory at the University of Texas Medical Branch (UTMB). A high-flow rate of CO2 followed by thoracotomy were used at the time of euthanasia. All animal studies are reviewed and approved by the Institutional Animal Care and Use Committee at UTMB and are conducted according to the National Institutes of Health guidelines. This study is reported in accordance to ARRIVE guidelines (https://arriveguidelines.org/arrive-guidelines).
BEHAVIORAL TESTING
Our behavioral test was developed using the buried food test based on Yang et al in 2009 [15]. On each sampling day (2, 3, 5, 8, 17, 21, 35, and 42 dpi), four hamsters were tested for any signs of anosmia. An empty housing cage was prepared with at least 3cm of bedding. In one corner, a honey-flavored Teddy Graham (Nabisco) was buried about 1cm below the surface of the bedding. In the corner diagonal to the buried cookie, a hamster was placed, the lid closed, and a timer started. The timer was stopped once the hamster had revealed and grasped the cookie, and the time recorded. All hamsters were given a maximum of 5 min (300 s) to find the cookie.
VIRAL TITRATION
Collected lung samples were homogenized with DMEM supplemented with 2% FBS to make a 10% homogenate. These samples were then diluted 10-fold serially and inoculated into Vero E6 cells on 96-well plates. Infected cells were allowed to incubate for 72 hours at 37°C with 5% CO2, then fixed with 10% formalin and stained with 0.25% crystal violet to visualize the presence of cytopathic effect (CPE). TCID50 values were then calculated and recorded using the Reed and Muench method [16].
HISTOLOGICAL ANALYSIS
At the time of sampling, collected muzzles were fixed in 10% buffered formalin for 7 days before removal from the BSL-3 facilities. Samples were prepared for histological analysis as previously described [14]. Briefly, olfactory bulbs and nasal tissue including the olfactory epithelium (OE) were extracted from the skull and decalcified with EDTA (10% w/v) and embedded in paraffin. Thin sections with 5µm thickness were mounted on glass slides and stained with Hematoxylin and Eosin. To analyze the OE, coronal sections of the OE were divided into four areas along zonal organization as previously described [14] and two independent analyses were performed examining the histopathology of the OE for each group. Each of the four turbinate zones were examined and assigned a pathology 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 two reviewers. To investigate the changes in OE thickness, two independent measurements were performed for the four different nasal turbinate regions as previously described [14].
STATISTICAL ANALYSIS
Statistical analyses were performed using the GraphPad Prism software (ver 9.1.2). Statistical significance for weight changes between SARS-CoV-2-infected and uninfected hamsters were determined using a two-way ANOVA followed by Fisher’s LSD test. Statistical significance for the behavioral test and olfactory epithelium thickness was determined using a one-way ANOVA followed by Dunnett’s post hoc test. Correlations were determined using a Pearson correlation test. p < 0.05 was deemed statistically significant.