Cells and virus
Vero E6 (ATCC® CRL-1586™) cells were cultured in Eagle’s Minimum Essential Medium (EMEM, ATCC) supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic-antimycotic 100X (Gibco™, Life Technologies, Carlsbad, CA, USA). The cell cultures were maintained at 37°C with 5% CO2. The SARS-CoV-2 isolate (USA-WA1/2020) was obtained from BEI Resources (SARS-Related Coronavirus 2, Isolate hCoV-19/USA-WA1/2020, NR-52281, Lot#70036318). The stock virus was passaged 3 times in Vero E6 cells, clarified by centrifugation (1000 rpm for 5 min) and stored at -80 oC. Viral titer was determined by the Reed and Muench 31. A viral suspension containing 105.5 tissue culture infectious dose 50 per ml (TCID50/ml) was used for elk calf inoculations and 106 TCID50/ml for adult elk inoculations.
Animal Infection and Sampling
All animal work and procedures were approved prior to the experiment by the National Animal Disease Center (NADC) Institutional Animal and Care Use Committee (IACUC) (protocol #ARS-22-1047). Additionally, all methods were performed according to the IACUC and the Guide for Care and Use of Laboratory Animals guidelines and regulations. Elk calves (~ 5 months old; n = 11) and adult elk cows (~ 4 years old; n = 10) were obtained from a captive herd at the NADC in Ames, IA. Animals were housed in an agriculture biosafety level 3 (ABSL-3) facility at NADC and allowed to acclimate for a minimum of 2 weeks. All animals were sampled and screened for SARS-CoV-2 RNA by RT-PCR in oronasal secretions and by surrogate virus neutralization test (sVNT) and VN assays prior to virus inoculation.
Seven elk calves and 7 cows were sedated with a combination of xylazine and ketamine and intranasally inoculated with an atomization device (LMA® MAD Nasal™, Teleflex; Morrisville, NC, USA) for delivery of approximately 2.5 mL of inoculum into each nostril for a total of 5 mL. Following inoculation, the effects of xylazine were reversed using tolazoline. On days 0, 2, 3, 4, 5, 7, 10, 14 and 21 post-infection (p.i.) elk were sedated as described above and nasal, oral and rectal swabs collected for RT-PCR. Blood was collected on days 0, 7, 14 and 21 days p.i. for serologic assays. On days 2 and 5 p.i. 2 calves and 2 cows each were euthanized and examined. All other inoculated elk were euthanized and examined 21 days p.i. Four calves and 3 cows remained as non-inoculated controls and were euthanized and examined similar to inoculated elk.
Serology
The cPASS SARS-CoV-2 neutralization antibody detection kit (GenScript Biotech,
Amsterdam, Netherlands) was used as described 32,33 and according to the manufacturer’s recommendations. The assay detects the presence of specific anti-SARS-CoV-2 neutralizing antibodies against the S-protein in serum in a species and isotype-independent manner by blocking the interaction between the receptor-binding domains (RBD) of the viral spike glycoprotein with the ACE2 cell surface receptor. Spectrophotometry was conducted at 450 nm in a plate reader. The absorbance of the sample is inversely dependent on the titer of the anti-SARS-CoV-2 neutralizing antibodies in tested samples. To confirm the sVNT results, serum samples were submitted to the National Veterinary Services Laboratory (NVSL, Ames, Iowa) for testing via virus neutralization. Briefly, serum was serially diluted 2-fold with a starting dilution of 1:8. Each dilution was incubated with virus for one hour at 37°C. The virus had a TCID50 of 100. Vero-76 cell culture was then added to the virus/serum mixture and incubated at 37°C for 3 days. Each well was observed for presence of absence of cytopathic effect.
Real-time RT-PCR on swabs and tissues
To assess for viral shedding on nasal, oral and rectal samples, swabs were submitted to NVSL for processing and for rRT-PCR analysis.
To determine the presence of viral RNA in tissue samples, tissues were thawed, cut into an approximately 50–100 mg piece, and resuspended in 1–2 mL of TRI-Reagent® (Life Technologies, Carlsbad, CA, USA) in individual gentle MACS™ M tubes (Miltenyi Biotec, Bergisch Gladbach, Germany). Tissues were dissociated using a gentle MACS™ Octo-Dissociator (Miltenyi Biotec) following the manufacturer’s recommendations. RNA was extracted from tissue homogenate samples using the MagMAX™-96 for Microarrays Total RNA Isolation Kit (Applied Biosystems, Waltham, MA, USA). Samples were run on a MagMAX™ Express Magnetic Particle Processor (Applied Biosystems) following the manufacturer’s instructions. Next, 15 µL of extracted product was added to 5 µL of the AgPath-ID™ One step RT-PCR master mix (Applied Biosystems). Samples were run in duplicate, The RT-qPCR reactions were performed on an ABI 7500 Fast instrument (Applied Biosystems) run in standard mode with the following conditions: 1 cycle at 45°C for 10 min, followed by 1 cycle at 95°C for 10 min, 1 cycle at 95°C for 3 s, and 45 cycles at 55°C for 30 s. The forward primer sequence was 5’-GACCCCAAAATCAGCGAAAT-3′, the reverse primer sequence was 5’-TCTGGTTACTGCCAGTTGAATCTG-3’, and the probe sequence was 5’-FAM-ACCCCGCATTACGTTTGGTGGACC-BHQ1-3’. A positive control (2019-nCoV_N_Positive Control, Integrated DNA Technologies IDT, Coralville, IA, USA) and a negative control was run on every plate.
Necropsy and Sample Collection
Two inoculated calves and 2 inoculated adult cows were euthanized on days 2 and 5 p.i. and the remaining animals were euthanized on day 21 p.i. Following necropsy, multiple tissues (palatine tonsil, nasal turbinate, medial retropharyngeal lymph node [mRPLN], cerebellum, cerebrum, olfactory lobes, caudate nucleus, trachea, lung [ right and left cranial and caudal lobes], heart, tracheobronchial lymph node, mediastinal lymph node, liver, spleen, kidney) were collected. Samples were individually bagged, placed on dry ice, and transferred to a -80°C freezer until testing. Additionally, tissue samples were collected and processed for standard microscopic examination, a subset were also processed by in situ hybridization (ISH) and immunohistochemistry (IHC). For this, tissue sections of approximately ≤ 0.5 cm in width were fixed by immersion in 10% neutral buffered formalin (≥ 20 volumes fixative to 1 volume tissue) for approximately 24 h, and then transferred to 70% ethanol, followed by standard paraffin embedding techniques. Slides for standard microscopic examination were stained with hematoxylin and eosin (HE).
In situ hybridization (ISH)
Paraffin-embedded tissues were sectioned at 5 µm and subjected to ISH using the RNAscope ZZ probe technology (Advanced Cell Diagnostics, Newark, CA). In situ hybridization was performed to detect tissue distribution of SARS-CoV-2 RNA in tissues. Nasal turbinate, palatine tonsil, mRPLN, and lung were tested by RNAscope 2.5 HD Reagents–RED kit (Advanced Cell Diagnostics) as previously described 21. Proprietary ZZ probes targeting SARS-CoV-2 RNA (V-nCoV2019-S probe) designed and manufactured by Advance Cell Diagnostics were used for detection of viral RNA. A positive control probe targeted the Bos taurus –specific cyclophilin B (PPIB) or ubiquitin (UBC) housekeeping genes, while a probe targeting dapB of Bacillus subtilis was used as a negative control.
Immunohistochemistry
Immunohistochemical staining for SARS-CoV-2 was performed on palatine tonsils from calves and mRPLN from adult and calf elk. To prepare the formalin-fixed paraffin-embedded tissues for staining they were heated for 45 minutes at 57°C. Tissues were then deparaffinized with xylene and rehydrated through a series of graded alcohol solutions. Slides were submerged in a 1X citrate unmasking solution (Abcam) until boiling was initiated, then maintained in the unmasking solution at a sub-boiling temperature (95°C-98°C) for ten minutes to perform epitope retrieval. A 3% hydrogen peroxide solution (Fischer Bioreagents, catalog no. BP2633500) was used to quench endogenous peroxidases. Slides were then immersed in a blocking solution of Tris Buffered Saline (Thermo Scientific) and Tween20® (Sigma-Aldrich) with 5% normalized goat serum. A rabbit monoclonal antibody targeting the spike protein of SARS-CoV-2 (S1) at a concentration of 1:800 was used as the primary antibody (Cell Signaling Technologies, Boston, MA). Tissues were then incubated in a SignalStain® Boost IHC Detection Reagent (HRP, Mouse, Cell Signaling, catalog no. 8125S) followed by SignalStain® DAB substrate to produce a brown reaction product (Cell Signaling Technologies). Finally, counterstaining was performed using hematoxylin stain solution and Bluing Agent (Ventana). Nasal turbinate tissue from a single mink inoculated with SARS-CoV-2 served as a positive control.