SARS-CoV-2 (isolate 2019-nCoV/USA-WA1, NR52281) was obtained from BEI Resources. Virus was passaged twice on Vero E6 cells (ATCC CRL-1586) in 2% FBS-DMEM containing 10,000 IU/ml penicillin and streptomycin at 37°C under 5% CO₂ to generate stock virus used in these experiments. Virus was stored at −80°C. All work with infectious virus was performed at BSL-3 with approval from the Colorado State University Institutional Biosafety Committee.

All rodent work was approved by the CSU Institutional Animal Care and Use Committee (protocol 993). Deer mice were intranasally inoculated under inhalation of isoflurane anesthesia with 1x10⁴ (P. bairdii), 1.5x10⁴ (P. sonoriensis), or 2×10⁴ (P. polionotus and P. californicus) TCID₅₀ SARS-CoV-2 (dose based on appropriate inoculation volume for body size). On days 3, 6 and 15 post-challenge, all animals were orally swabbed and weighed, and three inoculated mice of each species were euthanized by inhalation anesthesia of 3% isoflurane to effect, and thoracotomy followed by cardiac exsanguination. Oral swabs and weights were taken for remaining mice on day 10 post-challenge. Control rodents were euthanized on day 0 using procedure described above. For all euthanized animals, necropsies were performed and samples (blood, lung) were collected for viral RNA detection (by RT-qPCR), virus isolation, and serology. Remaining whole carcasses were fixed in 10% neutral buffered formalin for histopathology and immunohistochemistry.

Eighteen California mice were intranasally inoculated under inhalation isoflurane anesthesia with 2×10⁴ TCID₅₀ SARS-CoV-2 and four were euthanized as controls on either day 3 or 10 post-challenge. All mice were orally swabbed, weighed, and observed for clinical signs daily. Necropsies were performed on 6 mice euthanized on days 3, 6, and 10 post-challenge, and tissues (blood, lung) were collected for viral RNA detection (by RT-qPCR), virus isolation, and serology. Remaining whole carcass was fixed in 10% neutral buffered formalin for histopathology and immunohistochemistry

Swabs were stored in 200 μL of viral transport media (VTM) at -80° C until processing and analysis. Swabs were vortexed for 10 seconds followed by centrifugation at 1,500 rcf for 10 minutes to pellet debris. Resulting supernatant was used in downstream assays. Tissues were stored in 500 μL of VTM and either flash frozen in LN₂ and stored at -80° C until processing and analysis (multi-species experiment) or immediately processed following collection and stored at -80° C until analysis (California mouse experiment). Processing of tissue samples included homogenization on a TissueLyser II (Qiagen) at 50 Hz for 5 minutes followed by centrifugation at 10,000 rcf to pellet debris. Resulting supernatants were used in downstream analysis including infectious virus isolation and RNA extraction for viral RNA quantification. Blood was collected in a serum separator tube, allowed to clot for 20 to 30 minutes, and then centrifuged at 20,000 rcf for 90 seconds. Sera were collected and stored at -80° C until analysis.

Supernatants from swabs or lung homogenates were plated in triplicate on 96-well plates seeded with Vero E6 cells in a 10-fold dilution series from 10⁰ to 10^-3. Samples that were above the limit of detection were re-plated in triplicate on 96-well plates seeded with Vero E6 cells in a 10-fold dilution series from 10^-2 to 10^-5. All plates were scored four days post-infection and titers were calculated using the Reed-Meunch method (18).

RNA was extracted from swab or tissue supernatant using the Mag-Bind Viral DNA/RNA 96 Kit (Omega Biotek) on the KingFisher FLEX System (ThermoFisher Scientific) according to manufacturer’s instructions. Reverse transcriptase quantitative PCR (RT-qPCR) was run using the GoTaq RT-qPCR System (Promega) with the IDT E Assay First Line Screening primers and probe (Cat #10006804) on a LightCycler 96 System (Roche Diagnostics, Cat# 05815916001).

Serum neutralization was performed starting at a 1:10 dilution with a 2-fold dilution series. An equal volume of SARS-CoV-2 containing 100 TCID₅₀s was added (final serum dilution of 1:20) and incubated for 1 hr at 37°C. The mixture was plated on Vero E6 cells and scored for cytopathic effect after four days. The titer was the reciprocal of the greatest dilution that conferred 100% protection.

Recombinant SARS-CoV-2 nucleocapsid antigen in PBS was coated onto 96-well plates overnight at 4°C. Plates were washed 3x with PBS-0.5% TWEEN-20 and 3x with PBS between each step, then blocked with 0.5% gelatin in PBS for 30 min. Serum samples were diluted 2-fold in 0.5% BSA-PBS starting 1:100 and incubated for 2 hours at room temperature. Detection antibody, anti-Peromyscus leucopus IgG H&L-HRP (SeraCare), was diluted 1:1000 and incubated for 1 hour, followed by ABTS substrate. Plates were read at 405 nm. Samples were considered positive if the OD was 0.200 above the mean of the negative control serum samples (uninfected deer mice).

After samples were collected for molecular analysis, the entire carcass was fixed in 10% neutral buffered formalin. Representative samples from the lungs, liver, heart, and the entire head (sagittal section) were collected and routinely processed for histopathology. Tissues were labeled with hematoxylin and eosin (H&E) for histologic analysis. Anti-SARS-CoV-2 immunohistochemistry was performed on embedded lung tissue from a representative subset of lung tissues at days 3 and 4 post-challenge, selected based on virus detection by RT PCR, using rabbit anit-SARS-CoV-2 nucleocapsid antibody (NP1189, ProSci) as a primary antibody with hematoxylin counterstain.

To investigate the susceptibility of different deer mouse species and subspecies, nine young, adult deer mice from each species or subspecies and of both sexes were intranasally challenged with SARS-CoV-2. Three unchallenged controls from each species group were euthanized and necropsied on day zero of the experiment. Three challenged mice from each species group were euthanized and necropsied on days 3, 6, and 15 post-challenge with one additional oral swab taken at day 10. Infectious virus was detected in 1/9 oral swabs from each species group on day 3 post-challenge, and viral RNA was detected in most lung samples from mice euthanized on days 3 and 6 post-challenge. Viral RNA was detected in oral swabs of all challenged mice on day 3, and infectious virus was detected in oral swabs from 3/4 species on day 3 post-challenge (prairie deer mouse, California mouse, and Oldfield mouse). Three of the four species (prairie deer mouse, Sonora white-footed mouse, and Oldfield mouse) had detectable neutralizing antibodies on day 15 post-challenge, with an average titer of 87 (SD ± 39). California mouse samples for this time point were compromised by mold contamination. A summary of virus detection and serology can be found in Table 1 , with exact values available in the Supplementary Materials .

No clinical signs, including weight loss (Figure 1), were observed in the prairie deer mouse, Sonora white-footed mouse, or Oldfield mouse at any timepoint in the study. On day 3 post challenge, two California mice (1 female, 1 male) were observed to have ruffled fur, a hunched posture, and lethargic behavior. When the mice were re-examined on day 4, the male appeared to have improved whereas the female was moribund and humanely euthanized for necropsy following the same methods for scheduled necropsies. This mouse had hepatomegaly and prominent hepatic reticular pattern. It was suspected that the mouse was suffering from hepatic lipidosis (or in humans, non-alcoholic fatty liver disease (NAFLD)). Three other California mice had a grossly prominent hepatic reticular pattern and hepatomegaly upon necropsy, including the male that displayed clinical signs on day 3. California mice lost significantly more weight 3 days post-challenge than any other group. To further investigate California mice as a model for SARS-CoV-2 infection, additional California mice were infected with SARS-CoV-2. Serial necropsies were performed on days 3, 6, and 10 post-challenge. No clinical signs were observed in these mice for the duration of the study.

We further developed California mice as an animal model of SARS-CoV-2 infection following our observation of clinical disease in two mice during our initial investigation. Our follow-up study indicated that the mice became infected, with infectious virus detectable in oral swabs on days 1, 2, and 3 post-challenge (average 3.16 x 10² ± 2.93 TCID₅₀/mL, Figures 2A, B ) and in all day 3 lungs (average 5.60 x 10⁴ ± 5.39 x 10⁴ TCID₅₀/mL, Figures 2C, D ). Viral RNA was detectable through day 10 post-challenge in both lungs and oral swabs. Serum IgG was detectable in all mice at 10 days post-challenge (geometric mean titer 400, 95%CI [180,887], Figure 2E ), and neutralizing antibody was detectable in 5 out of 6 mice (geometric mean titer 92, 95% CI [45,189], Figure 2F ). The SARS-CoV-2 challenged group showed significantly greater weight loss than the uninfected controls at days 1 and 3 post-challenge ( Figure 3 ). At day 10 the control mice had a significant decrease in weight compared to infected controls. The cause of the weight loss is unknown. As the infected mice had all cleared detectible infectious virus by day 10, the weight loss in the control group at this time point is not significant to the conclusions of this study.

Lung, liver, spleen, nasal skin and mucosa, and heart from all mice were evaluated for pathology. The most severe inflammation was observed in the lungs and nasal passages in all species. The nasal skin and mucosa, heart, and liver did not show histologic differences in any of the groups. Lung was the most affected tissue and infected mice showed variable severity of interstitial lymphohistiocytic and fibrinous pneumonia with edema, reactive vascular endothelium with neutrophilic margination and perivasculitis, and rarely minimal to mild bronchial epithelial hyperplasia ( Figures 4A, B ). Necrosuppurative rhinitis was the second most common finding in infected mice ( Figure 4C ). Other observed pathology included mild lymphohistiocytic myocarditis, microvascular hepatic lipidosis, necrotizing and lymphohistiocytic dermatitis of the muzzle and lymphoplasmacytic perineuritis. Lesions in all species, when present, were most severe at day 3 post-challenge versus days 6, 10, and 15. Pneumonia was present in all groups but was minimal to mild in the Sonora white-footed mouse. Immunohistochemistry for anti-SARS-CoV-2 nucleocapsid had minimal rare positive labeling of bronchiolar epithelium in mice with virus detectible by RT-PCR ( Figure 4D ).

In six of the twelve California mice, there was hepatic lipidosis that ranged from mild to severe. The mouse that became moribund during the study demonstrated the most severe hepatic lipidosis histologically ( Figure 5A ). Lymphohistiocytic pneumonia in this mouse was minimal to mild but there was increased bronchial epithelial hyperplasia compared to other animals and this animal had the strongest anti-SARS-CoV-2 IHC labeling of the hyperplastic epithelium and scattered histiocytes ( Figures 5B, C ).