key: cord-0922461-7e6xy81o
authors: Palmer, Mitchell V.; Martins, Mathias; Falkenberg, Shollie; Buckley, Alexandra; Caserta, Leonardo C.; Mitchell, Patrick K.; Cassmann, Eric D.; Rollins, Alicia; Zylich, Nancy C.; Renshaw, Rendall W.; Guarino, Cassandra; Wagner, Bettina; Lager, Kelly; Diel, Diego G.
title: Susceptibility of white-tailed deer (Odocoileus virginianus) to SARS-CoV-2
date: 2021-01-14
journal: bioRxiv
DOI: 10.1101/2021.01.13.426628
sha: 08a176c8fef22b4d6718a8669ebc0fe60608d5c9
doc_id: 922461
cord_uid: 7e6xy81o

The origin of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus causing the global coronavirus disease 19 (COVID-19) pandemic, remains a mystery. Current evidence suggests a likely spillover into humans from an animal reservoir. Understanding the host range and identifying animal species that are susceptible to SARS-CoV-2 infection may help to elucidate the origin of the virus and the mechanisms underlying cross-species transmission to humans. Here we demonstrated that white-tailed deer (Odocoileus virginianus), an animal species in which the angiotensin converting enzyme 2 (ACE2) – the SARS-CoV-2 receptor – shares a high degree of similarity to humans, are highly susceptible to infection. Intranasal inoculation of deer fawns with SARS-CoV-2 resulted in established subclinical viral infection and shedding of infectious virus in nasal secretions. Notably, infected animals transmitted the virus to non-inoculated contact deer. Viral RNA was detected in multiple tissues 21 days post-inoculation (pi). All inoculated and indirect contact animals seroconverted and developed neutralizing antibodies as early as day 7 pi. The work provides important insights into the animal host range of SARS-CoV-2 and identifies white-tailed deer as a susceptible wild animal species to the virus. IMPORTANCE Given the presumed zoonotic origin of SARS-CoV-2, the human-animal-environment interface of COVID-19 pandemic is an area of great scientific and public- and animal-health interest. Identification of animal species that are susceptible to infection by SARS-CoV-2 may help to elucidate the potential origin of the virus, identify potential reservoirs or intermediate hosts, and define the mechanisms underlying cross-species transmission to humans. Additionally, it may also provide information and help to prevent potential reverse zoonosis that could lead to the establishment of a new wildlife hosts. Our data show that upon intranasal inoculation, white-tailed deer became subclinically infected and shed infectious SARS-CoV-2 in nasal secretions and feces. Importantly, indirect contact animals were infected and shed infectious virus, indicating efficient SARS-CoV-2 transmission from inoculated animals. These findings support the inclusion of wild cervid species in investigations conducted to assess potential reservoirs or sources of SARS-CoV-2 of infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel coronavirus, within the genus Betacoronavirus (subgenus Sarbecovirus) of the family Coronaviridae, that causes coronavirus disease 2019 in humans (1) . COVID-19 was first reported in Wuhan, Hubei province, China in December 2019 (2) . The early clusters of the disease in humans had an epidemiological link to the Huanan Seafood Wholesale market in Wuhan, where several live wild animal species were sold (2) (3) (4) . Genome sequence analysis of SARS-CoV-2 revealed a high degree of similarity to coronaviruses circulating in bats (3, 5, 6) , with current evidence pointing to horseshoe bats as the most likely source of the ancestral virus that crossed the species barrier to cause the global COVID-19 pandemic in humans (7, 8) .

Other pathogenic zoonotic coronaviruses, including severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) are also believed to have originated in bat reservoirs. However, there is no evidence of direct bat-tohuman transmission, and current data indicate that human infections with SARS-CoV and MERS-CoV resulted from interactions with intermediate animal hosts, such as palm civet cats (Paguma larvata) and dromedary camels (Camelus dromedaries), respectively (9) (10) (11) . The epidemiological link of the first reported human cases of COVID-19 with the Huanan animal market in Wuhan, suggest that SARS-CoV-2 may have spilled over into humans from an animal host (3) (4) (5) (11) (12) (13) . Early studies proposed that pangolins (Manis sp.) may have served as an intermediate host for SARS-CoV-2, as they are a natural reservoir for SARS-CoV-2-like coronaviruses (14) . However, phylogenetic analyses and amino acid sequence analysis of the S gene of SARS-CoV-2 did not support the hypothesis of the virus arising directly from the closely related pangolin betacoronaviruses (15) . A better understanding of the host range and species susceptibility of SARS-CoV-2 is critical to elucidate the origin of the virus and to identify potential animal reservoirs and routes of transmission to humans.

The tropism and host range of coronaviruses is largely determined by the Spike (S) glycoprotein, which binds to host cell receptors triggering fusion and virus entry into susceptible cells (16) .

The SARS-CoV-2 S protein binds host cells through the angiotensin-converting enzyme 2 (ACE2) protein receptor (17) . Comparison of the human ACE2 protein to that of over 400 vertebrate species demonstrated that the ACE2 protein of several animal species presents a high degree of amino acid conservation to the human protein (18) . Further analysis of the ACE2/S binding motif and predictions of the SARS-CoV-2 S-binding propensity led to the identification of several animal species with an ACE2 protein with a high binding probability to the SARS-CoV-2 S protein (18) . Not surprisingly, the majority of species with the highest S/ACE2 binding propensity are non-human primates (18) . Notably, the ACE2/S protein binding motif of three species of deer, including Père David's deer (Elaphurus davidianus), reindeer (Rangifer tarandus), and white-tailed deer (Odocoileus virginianus) share a high homology to the human ACE2 (18) . These observations suggest a putative broad host range for SARS-CoV-2, however, the susceptibility of most of these animal species to SARS-CoV-2 infection remains unknown.

Natural SARS-CoV-2 infections have been reported in dogs, cats, mink, tigers and lions in Hong Kong, Netherlands, China and the United States (19) (20) (21) (22) . The increased interest in the virus host range, in understanding the array of susceptible animal species, and the need to develop reliable animal models for SARS-CoV-2 infection, led to several experimental inoculations in domestic and wild animal species. Experimentally infected non-human primates, ferrets, minks, cats, dogs, raccoon dogs, golden Syrian hamsters, and deer mice have displayed mild to moderate clinical disease upon SARS-CoV-2 infection (23) (24) (25) (26) (27) (28) . Whereas experimental inoculation of swine, cattle, poultry, and fruit bats have shown that these species are either not susceptible to SARS-CoV-2 or that inoculation on these studies did not result in productive infection and sustained viral replication (23, (29) (30) (31) . Here we assessed the susceptibility of white-tailed deer to SARS-CoV-2.

Viral infection, clinical outcomes, shedding patterns and tissue distribution were evaluated.

Additionally, transmission of SARS-CoV-2 to indirect contact animals was also investigated.

The susceptibility of deer cells to SARS-CoV-2 infection and replication were assessed in vitro. Deer lung (DL) cells were inoculated with SARS-CoV-2 isolate TGR/NY/20 (32) and the ability of SARS-CoV-2 to infect these cells was compared to Vero-E6 and Vero-E6/TMPRRS2 cells. Virus infection and replication were assessed by immunofluorescence (IFA) staining using a SARS-CoV-2 Nspecific monoclonal antibody. As shown in Fig. 1A , SARS-CoV-2 N expression was detected in DL cells at 24 h post-inoculation (pi), indicating that these cells are susceptible to SARS-CoV-2 infection. Importantly, staining for ACE2 confirmed expression of the SARS-CoV-2 receptor in DL cells (Fig. 1A) .

The replication kinetics of SARS-CoV-2 was investigated in DL cells. For comparison we included Vero-E6 and Vero-E6/TMPRSS2 cells, which are known to support efficient SARS-CoV-2 replication (33, 34) . All cells were inoculated with SARS-CoV-2 at a multiplicity of infection (MOI) of 0.1 and 1, and harvested at 0, 12, 24, 36, 48 and 72 h pi. Consistent with previous studies showing efficient replication of SARS-CoV-2 in Vero-E6 and Vero-E6/TMPRSS2 cells (33, 34) , SARS-CoV-2 replicated well in these cells, reaching peak titers (~10 6 to 10 7 TCID 50 .ml -1 ) within 12-24 h pi (Fig. 1B) . Interestingly, while SARS-CoV-2 also replicated to high titers in DL cells (~10 6 to 10 7 TCID 50 .ml -1 ), replication kinetics was delayed with peak viral titers being reached by 72 h pi (Fig. 1B) . These results show that DL cells are susceptible to SARS-CoV-2 infection and support productive virus replication in vitro.

Given that white-tailed-deer have been recently identified as one of the species with an ACE2 protein with high binding probability to the SARS-CoV-2 S protein (18), we investigated the susceptibility of white-taileddeer fawns to SARS-CoV-2 infection. Six-week-old fawns (n = 4) were inoculated intranasally with 5 ml (2.5 ml per nostril) of a virus suspension containing 10 6.3 TCID 50 .ml -1 of SARS-CoV-2 TGR/NY/20, an animal SARS-CoV-2 strain that is identical to human viral strains (22) . To assess the potential transmission of SARS-CoV-2 between white-tailed-deer, two fawns (n = 2) were maintained as non-inoculated contacts in the same biosafety level 3 (BSL-3Ag) room. The inoculated and indirect contact animals were kept in separate pens divided by a plexiglass barrier, to prevent direct nose-to-nose contact between inoculated and contact animals ( Fig. 2A) . pi and decreasing thereafter through day 21 pi (Fig. 3A) . Notably, high levels of viral RNA were also detected in nasal secretions from indirect contact animals throughout the experimental period (Fig. 3B ). Viral RNA from the feces was detected in all inoculated and indirect contact animals ( Fig. 3A and B) , however, intermittent and short duration fecal shedding was observed, with most animals (4/5) only transiently shedding detectable SARS-CoV-2 in feces through days 6-7 pi ( Fig. 3A and B) . Tissues were also analyzed using the anti-genomic negative sense RNA probe (V-nCoV2019-Ssense probe) to determine virus replication. While intense labeling was noted with the genomic probe ( Fig 6E) , no labeling was observed with the anti-sense probe in any of the tissues (Fig.   6F ), suggesting lack of virus replication. These findings are consistent with lack of virus isolation from RT-rPCR positive tissues. The ability of the anti-sense probe to bind actively replicating virus, was confirmed in Vero cells infected with SARS-CoV-2 isolate TGR/NY/20 (Suppl Fig. 1 ). One of the most remarkable characteristics of SARS-CoV-2 is its highly efficient transmissibility (35, 36) . Consistent with this, rapid SARS-CoV-2 transmission was observed in several experimentally infected animal species (27, 31, (37) (38) (39) (40) (41) (42) (43) . Similarly, results here show efficient SARS-CoV-2 transmission between white-tailed-deer. Indirect contact fawns became infected and supported productive SARS-CoV-2 replication as evidenced by virus shedding in nasal secretions and seroconversion. Evidence to date indicates that transmission of SARS-CoV-2 occurs mainly through direct, indirect, or close contact with infected individuals through infected secretions such as respiratory-, salivary-, and/or fecal droplets (44) . Under experimental conditions, transmission via direct contact has been demonstrated in ferrets, minks, raccoon dogs, hamsters, cats, and deer mice (28, 31, 37, 39, 40, 43, 45) , while indirect/aerosol transmission was observed in ferrets and hamsters (37, 39) . The experimental setup in the present study prevented direct contact between animals suggesting that transmission of SARS-CoV-2 from inoculated to room indirect contact animals most likely occurred via aerosols or droplets generated by inoculated animals.

Despite the potential for severe illness and mortality due to SARS-CoV-2 infection in humans (46, 47) , asymptomatic and mild cases represent approximately 80% of human COVID-19 cases, while severe and critical disease outcomes account for approximately 15 and 5% of the cases, respectively (48) . Notably, experimental infections in several animal species, including nonhuman primates (26) , cats (19, 23, 49) , ferrets (23, 31, 50, 51) , minks (27) , deer mice (40) (41), rhesus macaques (25, 56, 57) , hACE2 mice (58, 59), ferrets (37) , and minks (27) . It is also notable that no SARS-CoV-2 viral RNA was associated with lesions in these white-tailed deer, which could be a result of earlier viral clearance from these sites, or potent immunological responses following SARS-CoV-2 infection.

In summary, our study shows that white-tailed-deer are susceptible to SARS-CoV-2 infection and can transmit the virus to indirect contact animals. These results confirmed in silico predictions describing a high propensity of interaction between SARS-CoV-2 S protein and the cervid ACE2 receptor. Our findings indicate that deer and other cervids should be considered in investigations conducted to identify the origin and potential intermediate host species that may have served as the link host reservoir to humans. 

All animals were handled in accordance with the Animal Welfare Act Amendments (7 U.S. Code §2131 to §2156) and all study procedures were reviewed and approved by the Institutional Animal Care and Use Committee at the National Animal Disease Center (IACUC approval number ARS-2020-861). White-tailed deer fawns (n = 6) were obtained from a breeding herd maintained at the National Animal Disease Center in Ames, IA. Within 24 hrs of birth, fawns were removed from the pasture, moved to indoor housing, and bottle-fed. Hand-raising fawns has been found to greatly decrease animal stress when moved to containment housing (62) . At approximately 4 weeks of age, fawns were moved to the biosafety level 3 (Agriculture) (BSL-3Ag) facility at NADC, and were microchipped (subcutaneously; SC) for identification and body temperature monitoring. Fawns were fed white-tailed deer milk replacer and hay was also available.

After a 2-week acclimation period, four fawns were inoculated intranasally with 5 ml (2.5 ml per nostril) of a virus suspension containing 10 6.3 TCID 50 .ml -1 of SARS-CoV-2, isolated from respiratory secretions from a tiger at the Bronx Zoo (TGR/NY/20) (32) . Two fawns were maintained as non-inoculated indirect contact animals to evaluate potential transmission of SARS-CoV-2 from inoculated to indirect contact animals. All fawns were maintained in a 3.7m

x 3.7m room, and inoculated and room indirect contact animals were kept in two pens separated by a plexiglass barrier approximately 0.9 m (~3-feet) in height, to prevent direct nose-to-nose contact ( Fig. 2A) . Airflow in the room was maintained at 10-11 air exchanges per hour, at a standard exchange rate for BSL-3Ag housing of large animals. Body temperatures were recorded daily, nasal swabs (NS) and rectal swabs (RS) were collected on days 0, 1, 2, 3, 4, 5, 6, 7, 10, 12, 14 and 21 post-inoculation (pi). Upon collection, swabs were placed individually in sterile tubes containing 2 ml of viral transport media (MEM with 1,000 U.ml -1 of penicillin, 1,000 µg.ml -1 of streptomycin, and 2.5 µg.ml -1 of amphotericin B) and stored at -80 °C until analysis. Blood was collected through jugular venipuncture in EDTA and serum separator tubes on days 0, 7, 14, and 21 pi. The tubes were centrifuged for 25 min at 1200 x g and buffy coat (BC) was collected from EDTA tubes and stored at -80 °C. Serum from the serum separator tubes was aliquoted and stored at -80 °C until analysis.

Fawns were humanely euthanized on day 21 pi. Following necropsy, multiple specimens including tracheal wash (TW), lung lavage (LL) and several tissues (nasal turbinates, palatine tonsil, thymus, trachea, lung, bronchi, kidney, liver, spleen, ileum, ileocecal junction, spiral colon, cerebellum, cerebrum, olfactory bulbs and medial retropharyngeal, mandibular, tracheobronchial, mediastinal and mesenteric lymph nodes) were collected. Samples were processed for RT-rPCR and virus isolation (VI) and 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 and in situ hybridization (ISH). For this, tissue fragments 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).

Nucleic acid was extracted from nasal secretions, feces, BC, serum tracheal wash, lung lavage and all the tissue samples collected at necropsy. Before extraction, 0.5 g of tissues were minced with a sterile scalpel and resuspended in 5 ml DMEM (10% w/v) and homogenized using a 

Nasal and rectal swabs collected on days 0, 1, 2, 3, 4, 5, 6, 7, 10, 12, 14 and 21, and tissues collected during the necropsy that tested positive for SARS-CoV-2 by RT-rPCR were subjected to virus isolation under biosafety level 3 conditions at Cornell University. Twenty-four well plates were seeded with ~75,000 Vero E6/TMPRSS2 cells per well 24 h prior to sample inoculation. Cells were rinsed with phosphate buffered saline (PBS) (Corning ® ) and inoculated with 150 µl of each sample and inoculum adsorbed for 1 h at 37 °C with 5% CO 2 . Mockinoculated cells were used as negative controls. After adsorption, replacement cell culture media supplemented as described above was added, and cells were incubated at 37 °C with 5% CO 2 and monitored daily for cytopathic effect (CPE) for 3 days. SARS-CoV-2 infection in CPE-positive cultures was confirmed with an immunofluorescence assay (IFA) as described above. Cell cultures with no CPE were frozen, thawed, and subjected to two additional blind passages/inoculations in Vero E6/TMPRSS2 cell cultures. At the end of the third passage, the cells cultures were subjected to IFA as above. Positive samples were subjected to end point titrations by limiting dilution using the Vero E6/TMPRSS2 cells and virus titers were determined using the Spearman and Karber's method and expressed as TCID 50 .ml -1 .

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 nucleic acid in palatine tonsil, medial retropharyngeal-tracheobronchial-mediastinal and mesenteric lymph nodes, nasal turbinate, brain, lung, and kidney, using the RNAscope 2.5 HD Reagents-RED kit (Advanced Cell Diagnostics) as previously described (63) . Proprietary ZZ probes targeting SARS-CoV-2 RNA (V-nCoV2019-S probe ref# 8485561) or anti-genomic RNA (V-nCoV2019-S-sense ref#845701) 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 Cat# 3194510) or ubiquitin (UBC Cat # 464851) housekeeping genes, while a probe targeting dapB of Bacillus subtilis (Cat # 312038) was used as a negative control. The distribution of mRNA of ACE2 was assessed using the BaseScope 2.5 HD Reagents-RED kit (Advanced Cell Diagnostics) as previously described (63) . Proprietary ZZ probes targeting the region spanning AA 31-82 for the ACE2 receptor specific to Odocoileus virginianus (BA-Ov-ACE2-1zz-st probe; ref# 900101) designed and manufactured by Advance Cell Diagnostics. Slides were counterstained with hematoxylin, and examined by light microscopy using a Nikon Eclipse Ci microscope. Digital images were captured using a Nikon DE-Ri2 camera.

Antibody responses to SARS-CoV-2 was assessed by a Luminex and virus neutralization assays facility. Four fawns were inoculated intranasally with a virus suspension containing 5x10 6.3 TCID 50 of SARS-CoV-2 isolate TGR/NY/20, and two fawns were maintained as non-inoculated room contact animals. All fawns were maintained in a 3.7m x 3.7m room, and inoculated and room contact animals were kept in two pens separated by a plexiglass barrier approximately 0.9 m (~3-feet) in height, to prevent direct nose-to-nose contact. Airflow in the room was maintained at 10-11 air exchanges per hour and was directional from the contact pen towards the inoculated pen. (B) Fawns were microchipped subcutaneously for identification and monitored daily for clinical signs and body temperature starting on day 1 before inoculation or contact day (day -1). Body temperatures are expressed in degrees Celsius (°C). In situ hybridization using BaseScope technology was used to detect mRNA for the O. virginianus ACE2 receptor. (A and B) In nasal turbinates, positive labeling for the ACE2 receptor was detected at low levels within nasal epithelial cells and cells associated with submucosal glands. (C) Within the palatine tonsil, labeling for the receptor was seen in keratinized and non-keratinized tonsillar epithelium, including regions of lymphoepithelium overlying follicles, where the epithelial layer was heavily infiltrated by lymphoid cells. In addition, labeling was also observed in interstitial and mucous cells associated with submucosal glands (D). (E) Within the kidney, labeling was observed in proximal tubular epithelial cells, but not glomeruli, distal tubules, or collecting ducts. No ACE2 labeling was detected in lung, medial retropharyngeal lymph nodes or tracheobronchial lymph nodes. Sections of palatine tonsil (A & C), nasal turbinate (B & D) and kidney (E) from white-tailed deer fawns inoculated intranasally with SARS-CoV-2 and examined 21 days (tonsil, kidney) or 8 days (turbinate) later. Note distinct punctate magenta labeling of ACE2 receptor mRNA in tonsillar and turbinate epithelium and submucosal glands and renal tubular epithelial cells. ISH-Basescope.

Sections of lung from white-tailed deer fawns intranasally inoculated with SARS-CoV-2 and examined 21 days later. A) Note the well demarcated focus of congestion. Alveolar septal capillaries are engorged with blood surrounded by normal appearing alveolar septa. HE. B) Multiple alveolar septa are lined by bands of eosinophilic hyalinized proteinaceous material (arrows) consistent with hyaline membranes. Multiple alveoli contain flocculent to fibrillar eosinophilic material (arrow head) consistent with fibrin. HE. C) Expanded alveolus contains a large collection of fibrin, inflammatory cells, and cell debris (arrow). HE. D) Alveolar septa are expanded by an inflammatory infiltrate (interstitial pneumonia) composed primarily of lymphocytes (arrows) and macrophages (E). HE. F) Within a field of congested alveolar septa are irregular regions characterized by hypocellular septa containing few erythrocytes. Septal stroma is fibrillar and lightly eosinophilic. Multiple alveoli within these regions contain flocculent strands of fibrin. HE. G) There is type II pneumocyte hyperplasia and an increase in alveolar macrophages (arrow). HE. H) Lumens of cortical tubules in the kidney are filled with necrotic cellular debris. Renal tubules are variably lined by attenuated epithelium, occasionally have hypereosinophilic cytoplasm and pyknotic nuclei (degeneration and necrosis), and overall exhibit increased cytoplasmic basophilia (regeneration). Tubules are divided by interstitial edema and a cellular infiltrate composed of lymphocytes, plasma cells, and fewer macrophages. 

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At 24 h post-inoculation (h pi), cells were fixed and subjected to an immunofluorescence assay using a monoclonal antibody (MAb) anti-ACE2 (green), and with a MAb anti-SARS-CoV-2-nucleoprotein (N) (red). Nuclear counterstain was performed with DAPI (blue)

The authors thank clinical veterinarian, Dr. Rebecca Cox and animal caretakers, Tiffany Williams, Kolby Stallman, Derek Vermeer and Robin Zeisneiss for excellent animal care and Patricia Federico for excellent technical assistance. Antibody reagents to SARS-CoV-2 and the virus neutralization assay used to assess serological responses were developed with support from the Cornell Feline Health Center. Mention of tradenames or commercial products is solely for the purpose of providing specific information and does not imply recommendation of endorsement by the US Department of Agriculture.

 Supplementary Fig. 2 Supplementary Fig. 3