key: cord-0958832-3huh7iwn
authors: Martins, Mathias; Boggiatto, Paola M.; Buckley, Alexandra; Cassmann, Eric D.; Falkenberg, Shollie; Caserta, Leonardo C.; Fernandes, Maureen H.V.; Kanipe, Carly; Lager, Kelly; Palmer, Mitchell V.; Diel, Diego G.
title: From Deer-to-Deer: SARS-CoV-2 is efficiently transmitted and presents broad tissue tropism and replication sites in white-tailed deer
date: 2021-12-15
journal: bioRxiv
DOI: 10.1101/2021.12.14.472547
sha: 73ecb71f56e9eafd8f953aaed2ca00d15451cb5b
doc_id: 958832
cord_uid: 3huh7iwn

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19) in humans, has a broad host range, and is able to infect domestic and wild animal species. Notably, white-tailed deer (WTD, Odocoileus virginianus), the most widely distributed cervid species in the Americas, were shown to be highly susceptible to SARS-CoV-2 in challenge studies and reported natural infection rates approaching 40% in free-ranging WTD in the U.S. Thus, understanding the infection and transmission dynamics of SARS-CoV-2 in WTD is critical to prevent future zoonotic transmission to humans and for implementation of effective disease control measures. Here, we demonstrated that following intranasal inoculation with SARS-CoV-2, WTD fawns shed infectious virus up to day 5 post-inoculation (pi), with high viral loads shed in nasal and oral secretions. This resulted in efficient deer-to-deer transmission on day 3 pi. Consistent a with lack of infectious SARS-CoV-2 shedding after day 5 pi, no transmission was observed to contact animals added on days 6 and 9 pi. We have also investigated the tropism and sites of SARS-CoV-2 replication in adult WTD. Infectious virus was recovered from respiratory-, lymphoid-, and central nervous system tissues, indicating broad tissue tropism and multiple sites of virus replication. The study provides important insights on the infection and transmission dynamics of SARS-CoV-2 in WTD, a wild animal species that is highly susceptible to infection and with the potential to become a reservoir for the virus in the field. Author summary The high susceptibility of white-tailed deer (WTD) to SARS-CoV-2, their ability to transmit the virus to other deer, and the recent findings suggesting widespread SARS-CoV-2 infection in wild WTD populations in the U.S. underscore the need for a better understanding of the infection and transmission dynamics of SARS-CoV-2 in this potential reservoir species. Here we investigated the transmission dynamics of SARS-CoV-2 over time and defined the major sites of virus replication during the acute phase of infection. Additionally, we assessed the evolution of the virus as it replicated and transmitted between animals. The work provides important information on the infection dynamics of SARS-CoV-2 in WTD, an animal species that - if confirmed as a new reservoir of infection - may provide many opportunities for exposure and potential zoonotic transmission of the virus back to humans.

230 cap and mantle regions, virus positive cells were also noted in portions of the central germinal 231 centers (Fig 7 and 8) . Notably, high levels of expression of ACE2 and TMPRSS2 were detected 232 in crypt epithelial cells and in germinal centers in the tonsil (Fig 9C) , two areas in which active 233 virus replication was detected as demonstrated by antisense viral RNA staining (Fig 7 and 8) . By 234 day 20 pi, viral RNA staining was limited to germinal centers as was observed on days 2 and 5 pi 235 (data not shown).

Viral staining in the mandibular and/or retropharyngeal lymph nodes was observed on days 237 2 and 5 pi, with the interfollicular and paracortical regions of these lymph nodes containing large 238 foci of virus (Fig 8) . Additionally, a lower number of positive cells with less intense staining was 239 observed in the germinal center mantle and central regions (Fig 8) . On day 20 pi, viral RNA 240 staining within the medial retropharyngeal lymph node was characterized by low to moderate 241 numbers of single positive cells within germinal centers (data not shown). Importantly, no 242 evidence of viral replication (virus isolation) was observed in any of the tissues collected on day 243 20 pi ( Fig 6F) .

In the lung, moderate to abundant staining of bronchial epithelial cells was observed in 245 both animals on day 2 pi (Fig 7 and 8 ) and in only one animal euthanized on day 5 pi. Large foci 246 of virus positive cells (indicative of replication) were scattered throughout bronchial epithelium 247 with infrequent staining of individual cells within the submucosa. Staining was absent in 248 bronchioles, alveoli, or interstitial regions. Viral staining was also observed in tracheal epithelial 249 cells of one animal on day 2 pi, with foci of intense virus staining observed in the ciliated 250 epithelium (Fig 8) . In general, virus-specific staining was more broadly distributed and more 251 intense on day 2 pi with a noticeable decrease in the number of positive cells and staining intensity 252 being observed on day 5 pi. Together these results demonstrate active SARS-CoV-2 replication in 253 the upper and lower respiratory tract and lymphoid organs of WTD. Detection of higher viral loads 254 in URT are consistent with higher expression levels of ACE2 and TMPRSS2 in nasal turbinate, 255 when compared to tonsil and lung tissues (Fig 9A-C) .

256 Histological changes associated with SARS-CoV-2 replication. Tissue samples were processed 257 for standard histological examination. Rhinitis characterized by submucosal lymphoplasmocytic 258 infiltrates and frequent mucosal exudation of neutrophils (Fig 10) was observed in the nasal 259 turbinate. In lymphoid organs (tonsil and lymph nodes), follicles exhibited moderate lymphoid 260 depletion and lymphocytolysis (Fig 10) . Tonsil also contained multifocal hemorrhages in crypt 261 epithelium, crypts with variable numbers of neutrophils and cell debris, and congested vasculature 262 filled with neutrophils. In the lungs, diffuse congestion of alveolar capillaries and multifocal 263 hemorrhage in the submucosa of larger airways was observed (Fig 10) . Multifocally, there were 264 perivascular and interstitial lymphohistiocytic infiltrates. Occasionally, there were foci of mild 265 increased alveolar macrophages and type II pneumocyte hyperplasia. In general, these histological 266 changes were more pronounced in days 2 and 5 pi, with notable changes in histological features 

The transmissibility of SARS-CoV-2 is a major factor that contributed to the establishment 563 Statistical analysis was performed by 2way ANOVA followed by multiple comparisons and by 564 unpaired t-test. Statistical analysis and data plotting were performed using the GraphPad Prism 565 software (Version 9.0.1).

To assess ACE2 and TMPRSS2 expression levels, nasal turbinate and palatine tonsil from 567 controls (uninoculated) WTD were sectioned at 5 µm and subjected to IFA. Formalin-fixed 

Successful transmission and infection of SARS-CoV-2 to day 3 contact fawns was also 141 confirmed by rRT-PCR performed in tissues including nasal turbinate, palatine tonsil, and medial 142 retropharyngeal lymph nodes collected from these animals on day 21 pc (Fig 3C), which were 143 positive for SARS-CoV-2 RNA. Importantly, viral RNA loads in these tissues were comparable 144 to those observed in inoculated animals (Fig 3E)

Additionally, serological responses to SARS-CoV-2 (assessed by virus neutralization [VN] assay by day 21 pc (Fig 3D), presenting neutralizing titers comparable to those observed 149 in inoculated animals (Fig 3F)

Low genetic diversity observed following SARS-CoV-2 replication and transmission in

To investigate potential changes in the genome of SARS-CoV-2 following replication in

SARS-CoV-2 genome sequences were obtained directly from nasal secretions from all 156 the 6 inoculated fawns collected on days 3, 5, 7 and/or 9 pi and from oronasal secretions from the virus, a human SARS-CoV-2 isolate NYI67-20 of the B1 lineage (Fig 4A). Only minor 8 161 variant viral populations with frequencies below 40% were observed (Fig 4B). Only four of these 162 changes

Infection dynamics of SARS-CoV-2 in adult WTD is similar to that observed in WTD fawns

To gain insights into the sites of SARS-CoV-2 replication during acute infection, we conducted a 166 second study in adult WTD. For this, eight deer (3-4 years of age; n = 8) were used and kept in the 167 BSL-3Ag facility at the NADC

x 10 6.38 TCID 50 .ml -1 -same virus stock used in the transmission dynamics study) and housed in a 170 separate room (room B) (Fig 5A). Following inoculation, all animals were monitored daily for 171 clinical signs and body temperature was recorded daily until the day of euthanasia of each animal

Similar to results obtained in fawns inoculated, high viral loads were detected in nasal 179 secretions with titers ranging from 1.8 to 6.3 log10 TCID 50 .ml -1 in the early phase of infection (day 180 2 to 6 pi), while no infectious virus was detected in feces (Fig 5F-H). These results demonstrate 181 similar clinical outcome, infection dynamics and shedding patters of SARS-CoV-2 in fawns

SARS-CoV-2 RNA was 187 consistently detected in 17 out of 24 tissues sampled from both deer euthanized on day 2 pi, with 188 the highest viral RNA loads detected in the nasal turbinate and palatine tonsil (Fig 6A). Similarly, 189 from the tissues collected on day 5 pi, several tested positive by rRT-PCR, confirming a broad 190 tissue distribution of SARS-CoV-2 RNA. As observed on day 2 pi

By day 20 pi, viral RNA loads decreased when compared to early times pi, and viral RNA was 193 more consistently detected in lymphoid tissues such as palatine and pharyngeal tonsil, as well as 194 medial retropharyngeal-and mandibular lymph nodes (Fig 6C)

Replication of SARS-CoV-2 was investigated in rRT-PCR positive tissues by viral

Notably, infectious virus was consistently detected in a broad range of tissues on days 2 198 and 5 pi including nasal turbinate, tonsil (palatine and pharyngeal), lymph nodes (medial 199 retropharyngeal, mandibular and tracheobronchial) and/or in the low respiratory tract tissues 200 (trachea, bronchus and lung). Interestingly, infectious virus was also detected in olfactory lobe

with the highest viral 203 loads detected in the nasal turbinate of the two animals euthanized on day 2 pi (6.3 and 6.5 log10 204 TCID 50 .g -1 ) (Fig 6D)

Infectious virus was still detected in nasal turbinate (titers of 3.3 and 4.0 log10 TCID 50 .g -1 ) and in 207 the olfactory lobes (titers 1.0 log10 TCID 50 .g -1 ) in the two deer euthanized on day 5 pi

No infectious virus was detected in tissues collected from inoculated animals euthanized on day 209 20 pi (Fig 6F)

The replication of SARS-CoV-2 in tissues was confirmed by in situ hybridization (ISH)

and immunohistochemistry (IHC) staining. Nasal turbinate, palatine tonsil, medial retropharyngeal 212 lymph nodes, and lung from inoculated deer euthanized on days 2 and 5 pi were examined by ISH 213 using the RNAscope ZZ technology and positive and negative sense RNA probes to detect SARS

Epithelial cells 218 with contact to turbinate lumens were most often virus positive. Some cells in the middle and 219 basilar portions of the epithelium were also sporadically stained. Additionally, moderate punctate in nasal turbinate epithelial cells was consistent with detection of high levels of 223 expression of the virus receptor ACE2

Numerous tonsillar follicles and overlying epithelium presented virus specific RNA and 225 NP staining on day 2 pi (Fig 7 and 8)

CoV-2 RNA-and NP staining in crypt epithelial cells, tonsillar reticular epithelium and lymphoid 229 follicles was similar to that observed on day 2 pi. However, in addition to staining of the follicular 389 screened for SARS-CoV-2 RNA by real-time reverse transcriptase PCR (rRT-PCR) in oronasal separate room (room A). Body temperatures were recorded daily. Nasal swab (NS) and rectal 396 swabs (RS) were

After 48 hours, contact pair 1 fawns were sampled again (ONS) 406 and placed into a separate room until necropsy on 21 days post-contact (d pc). This contact process 407 was repeated using cleaned room B or C with two fawns (contact pair 2), which were added on 408 day 6 pi and two additional fawns (contact pair 3) added on day 9 pi (Fig 1A). For contact animals, 409 swab samples were collected in viral transport media on 0, 2, 3, and 21 d pc. Blood was collected 21 pc (inoculated and contact animals, respectively) with an 413 intravenous dose of barbiturate (Fatal-Plus ® Solution

Samples were processed for real-time reverse transcriptase PCR (rRT-PCR) and virus isolation 417 (VI) were individually bagged, placed on dry ice, and transferred to a -80 °C freezer until testing

Tissue tropism of SARS-CoV-2 in white-tailed deer

After acclimation, six adult deer were inoculated intranasally with 5 ml (2.5 ml per nostril) of 10 6

Two animals were maintained as non-inoculated 421 controls. Body temperatures were recorded daily. NS and RS were collected on 0

Following necropsy, multiple specimens including a tracheal swab, bronchial swabs, and several 425 tissues (palatine tonsil, pharyngeal tonsil, nasal turbinate, medial retropharyngeal lymph node, 426 mandibular lymph node, cerebellum, cerebrum, olfactory lobes, caudate nucleus, parotid salivary 427 gland, thymus, trachea, bronchus, lung [4 sites], heart, tracheobronchial lymph node, mediastinal 428 lymph node, liver, spleen, kidney, ileum, ileocecal valve, spiral colon, and mesenteric lymph node) 429 were collected. Samples were individually bagged, placed on dry ice, and transferred to a -80 °C 430 freezer until testing. Additionally, tissue samples were collected and processed for standard 431 microscopic examination, a subset were also processed by in situ hybridization (ISH)

Nucleic acid extraction and real-time RT-PCR (rRT-PCR)

Nucleic acid was extracted from nasal and oral secretions, feces, and all the tissue samples 439 collected at necropsy. Before extraction, 0.5 g of tissues were minced with a sterile scalpel and 440 resuspended in 5 ml DMEM (10% w/v) and homogenized using a stomacher (Stomacher ® 80 MagMax Core extraction kit

which detects 447 both genomic and subgenomic viral RNA targeting the virus nucleocapsid protein (N) gene. The 448 limit of detection (LOD) for this assay was previously establish as 250 viral genome copies per 449 mL, and 1.75 viral genome copies per reaction [39]. An internal inhibition control was included in 450 all reactions

Virus Isolation and titrations

Nasal and oral secretions, feces, and tissues collected during the necropsy that tested positive for 453 SARS-CoV-2 by rRT-PCR were subjected to virus isolation under biosafety level

Twenty-four well plates were seeded with ~75,000 Vero E6/TMPRSS2 cells 455 per well 24 h prior to sample inoculation. Cells were rinsed with phosphate buffered saline

Corning ® ) and inoculated with 150 µl of each sample and inoculum adsorbed for 1 h at 37 °C with

After adsorption, replacement cell 458 culture media supplemented as described above was added, and cells were incubated at 37 °C with 459 5% CO 2 and monitored daily for cytopathic effect (CPE) for 3 days. SARS-CoV-2 infection in 460 CPE-positive cultures was confirmed with an immunofluorescence assay (IFA) as described 461 previously

In situ hybridization was performed 470 to detect tissue distribution of SARS-CoV-2 RNA in tissues from tissue tropism study (tissue 471 distribution and tropism of SARS-CoV-2 in WTD). Nasal turbinate, palatine tonsil, medial 472 retropharyngeal

RNAscope 2.5 HD Reagents-RED kit (Advanced Cell Diagnostics) as previously described

Proprietary ZZ probes targeting SARS-CoV-2 RNA (V-nCoV2019-S probe ref # 8485561) or anti-475 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 477 -specific cyclophilin B (PPIB Cat# 3194510) or ubiquitin (UBC Cat # 464851) housekeeping

Paraffin-embedded tissues were sectioned at 5 µm and subjected to IHC using Vectastain Elite 482 ABC Peroxidase (HRP) Kit (Vector Laboratories Cat # PK-6102) as previously described

483 Tissues from study 2 (distribution and tropism of SARS-CoV-2 in WTD) including nasal turbinate, 484 palatine tonsil, mandibular lymph nodes, medial retropharyngeal lymph nodes, trachea, lung, 485 olfactory lobe, caudate nucleus of brain, and cerebellum, were subjected to IHC. Formalin-fixed 486 paraffin-embedded (FFPE) tissues were deparaffinized with xylene and rehydrated through a series solution (Abcam Cat # ab64218). A mouse monoclonal antibody targeting nucleoprotein 491 (N) of SARS-CoV-2

For SARS-CoV-2 detection, tissue sections were incubated with 493 anti-mouse biotinylated secondary antibody followed by incubation with the Vectastain Elite ABC 494 HRP reagent. Finally, tissues sections were incubated with Vector DAB peroxidase substrate 495 (Vector Laboratories Cat # SK-4100)

VNA) performed under BSL-3 conditions at Cornell. Twofold serial dilutions (1:8 to 1:1,024) of 37 °C. Following incubation of serum and virus, 50 µl of a cell suspension of Vero cells was 502 added to each well of a 96-well plate and incubated for 48 h at 37 °C with 5% CO 2 . The cells were 503 fixed and permeabilized as described above and subjected to IFA

Unbound antibodies were washed 506 from cell cultures by rinsing the cells PBS, and virus infectivity was assessed under a fluorescence 507 microscope. Neutralizing antibody titers were expressed as the reciprocal of the highest dilution 508 of serum that completely inhibited SARS-CoV-2 infection/replication

The genetic make-up of SARS-CoV-2 following replication in WTD over viral transmission was 513 investigated by whole genome sequencing. For this, nasal secretions collected on days 2 to 9 pi 514 from inoculated and oronasal secretions on days 2 and 3 pc in contact animals were subjected to 515 MinION-based targeted SARS-CoV-2 WGS. A multiplex RT-PCR was developed as previously 516 described [41] following the amplicon-based approach used by the ARTIC Network for 517 sequencing SARS-CoV-2

Oxford Nanopore demultiplexed with the MinIT device (ONT) and then processed through the artic-ncov2019-525 medaka conda environment

LoFreq [42] and subsequently filtered using Variabel

528 ACE2 and TMPRSS2 transcription and expression

RNA samples extracted from nasal turbinate, 530 palatine tonsil, and lungs from all 15 fawns from the transmission dynamics study (euthanized on 531 days 21 or 22) and from 4 deer from the pathogenesis study (euthanized on day 20) were included. 532 were treated with DNA-free TM Kit DNase Treatment & Removal (Invitrogen) according the /ul and 10 ng/ul, 536 respectively

Custom primers and probe were designed to angiotensin-converting enzyme

transmembrane serine protease 2 (TMPRSS2) and glyceraldehyde 3-phosphate dehydrogenase 539 (GAPDH) of WTD using PrimerQuest Tool from Integrated DNA Technologies website. The 540 primers and probe sequence for WTD ACE2 were 5'-GGATCTTGGCGTACAGAGAAAG-3

/3IABkFQ/ based on Odocoileus virginianus 543 texanus ACE2 accession number XM_020913306.1; for WTD TMPRSS2 were 5'-544 CCTGTATGTCTTGGCCCTTT-3

/3IABkFQ/ based on Odocoileus virginianus 546 texanus TMPRSS2 accession number XM_020907939.1; and for WTD GAPDH were 5'-547 TGAGATCAAGAAGGTGGTGAAG-3

PBS]) at room temperature (rt), and 30 min blocking using a goat normal serum (0.2% in PBS) at

rt, tissues subjected to IFA. Then, were incubated for 45 min at rt using a rabbit polyclonal antibody 574 (pAb) anti-ACE2 (Abcan ref # ab15348) and a mouse monoclonal antibody (mAb

Inc. ref # sc-515727). Followed by 30 min incubation at rt with a goat 576 anti-rabbit IgG (goat anti-rabbit IgG, Alexa Fluor® 594) and a goat anti-mouse IgG antibody (goat 577 anti-mouse IgG

Dihydrochloride (DAPI) (10 min at rt) and visualized under a confocal microscopy 579 (LSM710 Confocal -Zeiss)

The authors thank the animal caretakers and veterinarians at NADC for their help and

The 589 species Severe acute respiratory syndrome-related coronavirus: classifying 2019-nCoV 590 and naming it SARS-CoV-2. Nature Microbiology

A pneumonia outbreak 593 associated with a new coronavirus of probable bat origin

Detection and 596 Characterization of Bat Sarbecovirus Phylogenetically Related to SARS-CoV-2

Emerg Infect Dis

Possible Bat Origin of 599 Severe Acute Respiratory Syndrome Coronavirus 2

Coronaviruses with a SARS-CoV-2-like receptor-binding domain allowing ACE2-603 mediated entry into human cells isolated from bats of Indochinese peninsula. Res Sq

A new coronavirus associated 606 with human respiratory disease in China

Functional assessment of cell entry and receptor usage for 609 SARS-CoV-2 and other lineage B betacoronaviruses

SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a 613 Clinically Proven Protease Inhibitor

Broad host 615 range of SARS-CoV-2 predicted by comparative and structural analysis of ACE2 in 616 vertebrates

619 Susceptibility of white-tailed deer ( Odocoileus virginianus ) to SARS-CoV-2

CoV-2 exposure in wild white-tailed deer (Odocoileus virginianus)

SARS-CoV-2 Neutralizing Antibodies 625 in White-Tailed Deer from Texas

Multiple spillovers and onward 627 transmission of SARS-CoV-2 in free-living and captive white-tailed deer

CoV-2 infection in free-ranging white-tailed deer ( Odocoileus virginianus )

Severe SARS-CoV-2 Infection in a Cat with 631

Tracking 633 Changes in SARS-CoV-2 Spike: Evidence that D614G Increases Infectivity of the 634 COVID-19 Virus

SARS-CoV-2 636 spike D614G change enhances replication and transmission

639 Infection and transmission of SARS-CoV-2 and its alpha variant in pregnant white-tailed 640 deer

642 Susceptibility of Raccoon Dogs for Experimental SARS-CoV-2 Infection

SARS-CoV-2 645 infection and transmission in the North American deer mouse

SARS-CoV-2 648 infection, neuropathogenesis and transmission among deer mice: Implications for 649 spillback to New World rodents

From people 652 to panthera: Natural sars-cov-2 infection in tigers and lions at the bronx zoo

Transmission of SARS-CoV-2 on mink farms between humans and 656 mink and back to humans. Science (80-)

CoV-2 evolution in animals suggests mechanisms for rapid variant 5 selection

Coronavirus biology and replication: 665 implications for SARS-CoV-2

Virology, transmission, and pathogenesis 668 of SARS-CoV-2

SARS-CoV-2 670 infection of the oral cavity and saliva

Age-related susceptibility of ferrets to 673 SARS-CoV-2 infection

Neuroinvasion and 676 encephalitis following intranasal inoculation of sars-cov-2 in k18-hace2 mice

CoV-2 in Transgenic Mice Expressing Human Angiotensin-Converting Enzyme 2

A Mouse Model of SARS-682

CoV-2 Infection and Pathogenesis

Neurologic Manifestations of 685 Hospitalized Patients with Coronavirus Disease

Olfactory 688 transmucosal SARS-CoV-2 invasion as a port of central nervous system entry in 689 individuals with COVID-19

Evidence of central nervous system 692 infection and neuroinvasive routes, as well as neurological involvement, in the lethality of 693 SARS-CoV-2 infection

695 Estimating infectiousness throughout SARS-CoV-2 infection course. Science (80-)

Elizabeth Plocharczyk DGD. Viral load and 699 infectivity of SARS-CoV-2 in paired respiratory and oral specimens from symptomatic, 700 asymptomatic or post-symptomatic individuals

Infection and Rapid Transmission of SARS-CoV-2 in Ferrets

Clinical 705 evaluation of a multiplex real-time RT-PCR assay for detection of SARS-CoV-2 in 706 individual and pooled upper respiratory tract samples

Severe 709 SARS-CoV-2 Infection in a Cat with Hypertrophic Cardiomyopathy

Primer3 on the WWW for general users and for biologist 712 programmers

LoFreq: a sequence-quality 714 aware, ultra-sensitive variant caller for uncovering cell-population heterogeneity from 715 high-throughput sequencing datasets

Rescuing Low 717 Frequency Variants within Intra-Host Viral Populations directly from Oxford Nanopore 718 sequencing data

), which were housed in the separate rooms E and F, 750 respectively. Respiratory nasal secretion, feces and serum were collected on the days indicated in 751 the figure. B) Inoculated and control fawns were microchipped subcutaneously with temperature 752 chips prior to the experiment, and monitored daily for body temperature starting on day 1 before 753 inoculation until day 11 pi. Additionally, daily clinical observations were performed

SARS-CoV-2 RNA load assessed in nasal and oral 759 secretions and feces of controls (animals no. 34, 67, 84, no shown) and six inoculated fawns 760 (animals no. 33, 65, 79, 80, 88, and 116). Nasal, oral and rectal swabs collected on days 0, 2-7, 9, 761 11, 14, and 22 post-inoculation (pi) were tested for the presence of SARS-CoV-2 RNA by real-762 time reverse transcriptase PCR (rRT-PCR). B) Infectious SARS-CoV-2 load in nasal and oral 763 secretions and feces was assessed by virus titration in RT-rPCR-positive samples. Virus titers were 764 determined using end point dilutions and expressed as TCID 50

64 and 103), and day 9 contact (animals no. 86 and 770 107) fawns. Oronasal and rectal swabs collected on days 0 (before contact), days 2, 3 and 21 post-771 contact (pc) were tested for the presence of SARS-CoV-2 RNA by real-time reverse transcriptase 772 PCR (rRT-PCR). B) Shedding of infectious SARS-CoV-2 in oronasal secretions and feces was 773 assessed by virus titrations in rRT-PCR-positive samples. Virus titers were determined using end 774 point dilutions and expressed as TCID 50 .ml -1 . C) Tissue distribution of SARS-CoV-2 RNA 775 assessed in nasal turbinate, palatine tonsil, retropharyngeal lymph node, and lung collected and 776 processed for rRT-PCR 21 d pc of contact fawns. D) Antibody responses following contact of 777 fawns with inoculated animals was assessed by virus neutralization (VN) assay. Neutralizing 778 antibody titers were expressed as the reciprocal of the highest dilution of serum that completely 779 inhibited SARS-CoV-2 infection/replication. E) Tissue distribution of SARS-CoV-2 RNA 780 assessed in nasal turbinate, palatine tonsil

Neutralizing antibody titers were expressed as the 783 reciprocal of the highest dilution of serum that completely inhibited SARS-CoV-2 784 infection/replication

Genome sequences were obtained directly from nasal secretions from all the 788 6 inoculated fawns collected on days 3, 5, 7 and/or 9 post-inoculation (pi) and from oronasal 789 secretions from the day 3 contact animals (n = 2) collected on days 2 and 3 post-contact (pc). A) 790 Whole genome sequence analyses of SARS-CoV-2 sequences recovered from all inoculated and 791 contact fawns revealed no amino acid differences in the consensus SARS-CoV-2 genome in 792 comparison to the genome sequence of the inoculum virus isolate NYI67-20. B) Minor variant 793 viral populations distributed throughout the virus genome from inoculated fawns on days

Adult deer were kept in two rooms of a biosafety 799 level 3 (agriculture) (BSL-3Ag) facility. Two deer were maintained as control (uninoculated) in a 800 separate rom (room A), and six deer were

1705 and 1810) and two control 804 (animals no. 1754 and 1815) deer were euthanized on day 20 pi. B) Inoculated and control deer 805 were microchipped subcutaneously for temperature monitoring. Temperature and clinical signs 806 were monitored daily starting on day 1 before inoculation until day 20 pi or until euthanasia. Body 807 temperatures are expressed in degrees Celsius. C-E) SARS-CoV-2 RNA load assessed in 808 respiratory secretions and feces by real-time reverse transcriptase PCR (rRT-PCR) in deer until 809 days 2 (C), 5 (D), or 20 pi (E). F-H) Infectious SARS-CoV-2 assessed in respiratory secretions 810 and feces assessed by virus titration in rRT-PCR-positive samples until days 2 (F), 5 (G), or 20 pi 811 (H)

Tissue distribution of SARS-CoV-2 RNA and infectious virus in white-tailed deer

Tissues were collected and processed for real-time reverse transcriptase PCR (rRT-PCR) and virus 816 titrations. A-C) Tissue distribution of SARS-CoV-2 RNA assessed in twenty-four tissues collected 817 and processed for rRT-PCR in deer on days 2 (A), 5 (B), or 20 post-inoculation (pi

Virus titers were determined using end point dilutions 820 and expressed as TCID 50 .ml -1 . All the controls fawns

situ hybridization (ISH) in tissues from white-tailed deer inoculated with SARS

Intense labeling of viral RNA (all viral RNA) highlighted on the three tissues on days 2 and 827 5 pi. Labeling using the antisense genome probe demonstrate genome RNA replication in the nasal 828 turbinate

Immunohistochemistry (IHC) in tissues from white-tailed deer inoculated with SARS

SARS-CoV-2 labeling in the tissues of the inoculated WTD by IHC showing staining for 832 the SARS-CoV-2 N protein (brown stain) in several tissues on days 2 and 5 post-inoculation

Tissues from all 15 839 fawns from the transmission study (euthanized on days 21 or 22) and 4 deer from the pathogenesis 840 study (euthanized on day 20) were included. Expression levels of ACE2 (A) and TMPRSS2 (B) 841 in nasal turbinate, palatine tonsil and lung. C) Expression of ACE2 and TMPRSS2 proteins in the 842 nasal turbinate, palatine tonsil, and lung are presented. Paraffin-embedded tissues from a control 843 deer (uninoculated) were subjected to an immunofluorescence assay using a polyclonal antibody 844 anti-ACE2 (red) and a

Histological examination of nasal turbinate from white-tailed deer inoculated with 848 SARS-CoV-2. Upper respiratory tract (URT) after inoculation, there was submucosal