Persistence of viral RNA in North American elk experimentally infected with an ancestral strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)

Persistence of viral RNA in North American elk experimentally infected with an ancestral strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Persistence of viral RNA in North American elk experimentally infected with an ancestral strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Paola Boggiatto, Alexandra Buckley, Eric Cassmann, Hannah Seger, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3982475/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 14 May, 2024 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract White-tailed deer ( Odocoileus virginianus ) have emerged as a potential reservoir host for SARS-CoV-2 given their susceptibility to infection and demonstrated high rates of seroprevalence across the United States. As SARS-CoV-2 circulates within free-ranging white-tailed deer populations, there is the risk of transmission to other wildlife species and even back to the human population. The goal of this study was to determine the susceptibility, shedding, and immune response of North American elk ( Cervus elaphus canadensis ) to experimental infection with SARS-CoV-2, to determine if another wide-ranging cervid species could potentially serve as a reservoir host for the virus. Here we demonstrate that while North American elk do not develop clinical signs of disease, they do develop a neutralizing antibody response to infection, suggesting the virus is capable of replicating in this mammalian host. Additionally, we demonstrate SARS-CoV-2 RNA presence in the medial retropharyngeal lymph nodes of infected elk three weeks after experimental infection. Consistent with previous observations in humans, these data may highlight a mechanism of viral persistence for SARS-CoV-2 in elk. Biological sciences/Microbiology/Virology/Viral pathogenesis Biological sciences/Microbiology/Pathogens Biological sciences/Immunology/Adaptive immunity/Humoral immunity Biological sciences/Immunology Biological sciences/Immunology/Infection Figures Figure 1 Figure 2 Figure 3 Introduction Previous work from our laboratory identified white-tailed deer ( Odocoileus virginianus ) as a susceptible host for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), capable of transmission 1 , 2 . Subsequent field work demonstrated a high level of seroprevalence and infection in free-ranging white-tailed deer across the United States (US) 3 – 8 . Evidence of multiple 4 , 8 as well as new 9 variants within white-tailed deer populations has been presented. Additionally, intra- as well as inter-species transmission ( i.e. spillback into humans) 5 , 10 has shown that white-tailed deer could serve as an important reservoir host for SARS-CoV-2 evolution and transmission. Interest in determining whether white-tailed deer were susceptible to SARS-CoV-2 infection evolved from a comparative analysis of vertebrate angiotensin-converting enzyme 2 (ACE2), the main receptor for SARS-CoV-2. Based on these analyses, the white-tailed deer ACE2 receptor sequence showed a high degree of homology with the human ACE2 receptor and was classified as having a high propensity for binding the Spike protein of the virus 11 , 12 . Two additional cervid species, reindeer ( Rangifer tarandus ) and Pere David’s deer ( Elaphurus davidandus ) were also predicted to express ACE2 receptors with a high propensity for binding. North American elk ( Cervus elaphus canadensis ), another cervid species, was not reported as a possible susceptible host based on ACE2 receptor homology. Field surveillance of SARS-CoV-2 seroprevalance in red deer ( Cervus elaphus ), a closely related species to North American elk, found no evidence of exposure in Europe 13 – 15 . Data from these studies also suggested that other free-ranging deer species in Europe including Fallow ( Dama dama ), Muntjac ( Muntiacus reevesi ), Sika ( Cervus nippon ) and Roe deer ( Capreolus capreolus ) were also seronegative 13 . The high seroprevalence observed in white-tailed deer populations can be attributed to both susceptibility to infection but also to sources of infection from humans. As SARS-CoV-2 circulates in white-tailed deer, they may serve as a source of infection for other species. Therefore, we sought to determine the susceptibility of North American elk to SARS-CoV-2 infection. Recently, a study by Porter et al. 16 demonstrated that weanling elk are minimally susceptible to infection with the Delta variant of SARS-CoV-2. They did not develop clinical signs and were not capable of onward transmission. In the work presented here, we expand on this initial study in North American elk and assess the susceptibility of elk calves and adults to the ancestral Wuhan-like strain of SARS-CoV-2 (USA-WA1/2020). We provide further evidence that while North American elk do not develop clinical signs associated with infection, SARS-CoV-2 does elicit a neutralizing antibody response, suggesting that the virus is capable of establishing infection in this species. Additionally, we show that SARS-CoV-2 RNA persists in the lymph node of infected animals in the absence of viral protein. These findings provide additional information regarding host-pathogen interaction mechanisms for SARS-CoV-2 in one of its many hosts. Results Development of neutralizing antibody responses against SARS-CoV-2 Blood samples were collected on days 0, 7, 14 and 21 p.i. to assess neutralizing antibodies against SARS-CoV-2. In both calves (Fig. 1 A) and adult (Fig. 1 B) elk, antibody titers to SARS-CoV-2 measured by surrogate virus neutralization titers (sVNT) can be observed as early as 7 days p.i. and are sustained through day 21. Interestingly for elk calves, the mean sVNT inhibition value is around 70% at the peak of the response, while in the adult elk the percent inhibition is lower at 60%. Virus neutralization (VN) tests were also performed for both calves and adult elk. VN titers peaked for the elk calves at 14 days p.i. with the highest titer (1:256) observed in only one calf, while in the adult elk, VN titers peaked at day 7 p.i. and did not go above 1:16 (Table 1 ). Altogether, these data suggest that both calves and adult elk can mount neutralizing antibody responses to SARS-CoV-2 following challenge. However, this response appears to be more robust in the calves. Table 1 Virus neutralization test results for calves and adult elk Calves Adult Animal # D7 D14 D21 Animal # D7 D14 D21 4 < 8 32 32 2 8 8 < 8 5 16 256 8 3 16 < 8 < 8 6 8 64 16 7 8 < 8 < 8 Viral RNA detection in nasal, oral and rectal swabs Swabs (nasal, oral and rectal) were collected at various timepoints and analyzed via PCR for the presence of viral RNA. In elk calves, nasal swabs were only found positive in 2/7 animals at day 2 p.i., in 1/5 at day 3 p.i. and in 1/3 at days 7 and 10 p.i. (Table 2 ). We did not observe consistent positive PCR results in swabs collected from elk calves. In contrast, we observed PCR positive results in all (7/7) inoculated adult elk at day 2 p.i., and 2/5 at days 3 and 4 p.i. (Table 3 ). Oral and rectal swabs did not consistently show the presence of viral RNA. No oral or rectal swab samples collected from the elk calves were positive for SARS-CoV-2 RNA, and in samples collected from the adult elk, only one oral swab was found positive on day 4 p.i. and only 2 rectal swabs were found positive on day 2 p.i. (data not shown). Table 2 Elk calves nasal swab PCR results Animal # D0 D2 D3 D4 D5 D7 D10 D14 D21 1 -- -- 2 -- -- 3 -- 30.0/33.3 -- -- -- 4 -- -- -- -- -- 5 -- -- -- -- -- -- -- -- -- 6 -- 31.4/34.1 --/39.4 -- -- -- -- -- -- 7 -- -- -- -- -- 36.87/-- 37.9/-- -- -- Table 3 Adult elk nasal swab PCR Animal # D0 D2 D3 D4 D5 D7 D10 D14 D21 1 -- 36.3/-- 2 -- --/37.9 33.8/34.3 38.6/38.2 -- -- -- -- -- 3 -- 35.4/-- -- -- -- -- -- -- -- 4 -- 29.5/31.4 -- -- -- -- 5 -- 35.4/36.8 -- 36.0/-- -- -- 6 -- 34.4/36.2 7 -- 37.2/34.3 --/38.2 -- -- -- -- -- -- Viral RNA detection in lymphoid tissues At necropsy on days 2 and 21 p.i. mRPLN and palatine tonsil samples were collected and assessed for the presence of viral RNA via PCR, based on previous white-tailed deer studies (Table 4 ). At day 2 p.i., SARS-CoV-2 RNA was detected in the mRPLN of one elk calf and one adult elk. In contrast, no viral RNA was detected in tonsils collected from calves or adult elk. By day 21 p.i., viral RNA was detected in all three mRPLN from both calves and adult elk. SARS-CoV-2 was also detected in 2/3 tonsil samples collected from elk calves although Ct values were high (~ 38), but none was detected in tonsils from adult elk. Table 4 SARS-CoV-2 RNA detection in tissues Calves Adult Necropsy mRPLN Tonsil Necropsy mRPLN Tonsil D2 25.5 -- -- -- D2 32.8 -- -- -- D21 32.2 27.9 23.6 38.7* -- 38.1* D21 32.2 22.7 28.0 -- -- -- *Denotes only of the two duplicate wells had a positive value. Detection of SARS-CoV-2 RNA via in situ hybridization (ISH) Microscopic analysis of tissues collected at necropsy revealed no lesions consistent with SARS-CoV-2 infection reported in other species. The presence of SARS-CoV-2 RNA was investigated through ISH on various tissues. Tissues were selected based on previous studies using white-tailed deer, these included palatine tonsil, mRPLN and lung. In elk calves, viral RNA was detected in the mRPLN of 2/2 calves examined at 2 days p.i., 2/2 calves examined at 5 days p.i., and 3/3 calves examined at 21 days p.i. In adult elk, viral RNA was detected in 2/2 cows examined 2 days p.i., 2/2 cows examined 5 days p.i. and 3/3 cows examined 21 days p.i. In all cases, labeling was limited to secondary lymphoid follicles, often within germinal centers (Fig. 2 ). Additionally, staining for viral RNA was primarily observed within the marginal zone of the follicle in both calves and adult elk on days 2 and 5 dpi (Fig. 2 a-b, d-e). However, on day 21 p.i., viral RNA staining was observed within the germinal center (Fig. 2 c, f). Viral RNA was not detected in palatine tonsils or lung from inoculated calves or cows. Additionally, viral RNA was not detected in tissues examined from non-inoculated control calves and cows. Detection of SARS-CoV-2 protein via immunohistochemistry (IHC) Given the detection of SARS-CoV-2 viral RNA via PCR and ISH, we performed IHC on mRPLN and tonsil samples in elk calves to determine if viral protein was also detectable within these tissues. In the mRPLN of elk calves, SARS-CoV-2 spike protein was detectable in 1/2 calves at 2 days p.i., 2/2 calves at 5 days p.i., and 0/3 calves at 21 days p.i (Fig. 3 ). No SARS-CoV-2 spike protein was detected in the tonsil of elk calves utilizing IHC. The mRPLN of adult elk cow was also analyzed by IHC. Immunolabeling for SARS-CoV-2 spike protein was observed in 0/2 cows at 2 days p.i., 2/2 cows at 5 days p.i., and 0/3 cows at 21 days p.i. Staining for viral Spike protein was primarily observed within the marginal zone of the follicle. Discussion Since the outbreak of the 2019 SARS-CoV-2 pandemic, concerns regarding the susceptibility of other vertebrate species, and their potential to act as reservoirs for the virus, have garnered attention. Field surveillance as well as various experimental infection studies have demonstrated a wide range of susceptibility across various species in the families Felidae, Canidae, Mustelidae, Cricetidae, and Cervidae 17 , 18 . Among these, white-tailed deer have emerged as a species of interest given their susceptibility to infection, the high seroprevalence in free-ranging populations, their ability to transmit the virus to other deer as well as back to humans, and the identification of novel variants within this species 1–3,7−10,13 . Characterization of susceptible wildlife species is critical for understanding not only the epidemiology of this virus, but also for our ability to implement intervention strategies to stop the spread of this disease. In this work, we sought to characterize the susceptibility of North American elk to SARS-CoV-2 infection, another cervid species with broad distribution in the US. Here, we demonstrate that both elk calves and adult elk are susceptible to infection with the ancestral Wuhan-like variant of SARS-CoV-2 (USA-WA1/2020), as characterized by the development of measurable neutralizing antibody responses and the detection of viral RNA and viral protein in the retropharyngeal lymph nodes of infected animals. However, this work demonstrates that there may be some differences in the quality of the responses between the two age groups. A previous study by Porter et al. demonstrated that weanling North American elk are minimally susceptible to infection with the Delta variant of SARS-CoV-2 19 . Post challenge, viral RNA could be detected from oral and nasal swabs between days 1 and 5 p.i. However, infected elk calves did not shed infectious virus, nor were they capable of transmission to an in-contact elk. Neutralizing antibody responses were observed in these animals; however, these responses were relatively low, with peak neutralizing titers at 1:20 at 21 days p.i. Additionally, no infectious virus was detected in tissues collected at necropsy. Similar to these findings, we observed the presence of viral RNA from nasal swabs and the development of neutralizing titers from infected animals. However, and in contrast to the results observed with Delta variant infection, we observed peak neutralizing titers at 14 days p.i., with values ranging from 1:32 up to 1:256 (Table 1 ). Interestingly, when we compared elk calves to adult virus neutralization titers, the elk calves appear to have a higher neutralization response; adult elk responses remained at 1:8 or 1:<8 at all timepoints except for one animal at day 7 p.i. with a 1:16 titer. Differences in viral neutralization responses may be related to the age of the animals and exposure to other viruses, including other coronaviruses. The high homology between SARS-CoV-2 and other coronaviruses, may result in cross-reactive immune responses. In humans, cross-reactive humoral and cellular immune responses developed prior to SARS-CoV-2 infection, have been characterized and demonstrated to play an important role in determining susceptibility to infection and disease progression (reviewed in 20 ). Pre-existing cross-reactive responses from previous viral infections may be beneficial or detrimental. For example, cross-reactive antibodies to one virus may bind a to another virus and provide neutralization and subsequent clearance. Alternatively, pre-existing cross-reactive antibodies to a virus may bind to another virus with low-avidity resulting in enhanced viral uptake via antibody-mediated internalization and/or inhibit the generation of de novo antibody responses to the latter, resulting in increased viral load 20 . Our data suggests that adult elk appear to have a lower magnitude of neutralizing antibody responses (both in sVNT and VN) as compared to the elk calves. These slight differences may be attributed to some level of pre-existing immunity in older animals, which allows them to control the infection more rapidly, thus decreasing viral load available for de novo antibody responses. Persistence of viral RNA, detectable by PCR and ISH, in tissues such as the mRPLNs is consistent with previous studies in white-tailed deer, including the location of labeling within secondary lymphoid follicles and germinal centers of lymphoid tissues 1 , 21 . Similarly, persistent SARS-CoV-2 RNA has been detected in human lymph nodes, tonsils and other tissues at autopsies conducted over 300 days post infection 22 . In such cases in deer species and humans it was not possible to isolate virus from these tissues, although viral RNA was detected using ISH or RT-PCR. A potential explanation for the detection of viral RNA in the absence of infectious virus is the integration of viral subgenomic RNA into the DNA of the host cell via reverse transcription. Ancestral evidence of non-retroviral RNA virus sequences in the genome of vertebrate species have been previously detected 23 , 24 . Additionally, DNA copies of nonretroviral RNA viruses including lymphocytic choriomeningitis virus (LCMV) and vesicular stomatitis virus have been shown to integrate into the DNA of their host cell 25 – 27 . Recently, using cultured human cells Zhang et al. demonstrated that SARS-CoV-2 can integrate into the genome of host cells 28 . Additionally, using published RNAseq data from cultured cells and organoid tissues, they demonstrate that SARS-CoV-2 sequences integrated into the host cell genome can be expressed as human-viral chimeric reads. RNA expression of viral subgenomic sequences could explain why some patients remain PCR positive for SARS-CoV-2 many weeks or months following recovery from infection. While infectious virus cannot be produced from this integrated material, it does raise the possibility that viral antigen could be expressed by host cells. In the work presented here we demonstrate the presence of viral RNA 21 days p.i. with peak viral Spike protein production at day 5 p.i. As detection of Spike protein wanes, viral RNA is consistently detected via ISH and PCR. The lack of correlation between viral RNA and protein production would suggest that active translation is not occurring. Additionally, based on nasal and oral swab data, we did not detect viral RNA in these secretions. These data would suggest that viral replication and shedding is not occurring. Consistent with continuous or recurrent SARS-CoV-2 PCR positive reports in recovered human patients 29 , 30 , the persistent viral RNA found in the lymph nodes of other susceptible species including white-tailed deer 1 , 2 and elk, as demonstrated here, may point to a common mechanism of viral persistence in susceptible hosts. However, the immunological implications of this phenomenon are not completely understood and warrant further study. The data presented here demonstrate that both calves and adult North American elk are susceptible to SARS-CoV-2 infection as they permit viral replication and develop virus neutralizing antibody responses following infection. However, no clinical signs of disease nor pathological changes in the lungs or lymph nodes of infected animals were observed, suggesting that they are not susceptible to disease. Interestingly, our findings suggest that there is persistence of SARS-CoV-2 viral RNA in the lymphoid tissues of infected animals in the presence of an immune response. The continued assessment of SARS-CoV-2 susceptibility in various species provides insights not only into potential reservoirs for the disease but also, as shown here, sheds light on the host-pathogen interactions that may be common across species and may drive immunity to infection and disease. Materials and Methods 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% CO 2 . 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 o C. Viral titer was determined by the Reed and Muench 31 . A viral suspension containing 10 5.5 tissue culture infectious dose 50 per ml (TCID 50 /ml) was used for elk calf inoculations and 10 6 TCID 50 /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 TCID 50 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. Declarations Acknowledgements The authors would like to thank the National Animal Disease Center (NADC) Animal Resources Unit (ARU) for the care of the animals used in this study. Specifically, the authors thank Dr. Rebecca Cox, Derek Vermeer, Jonathan Gardner, Tiffany Williams and Kolby Stallman for their animal husbandry, care, and assistance. Additionally, we would like to thank Sue Osorio and Sarah Anderson for their excellent technical assistance. We would also like to thank NVSL for their technical assistance with sample processing and testing. Author contribution P.B. performed the animal experiment, collected samples, prepared figure 1, and wrote the manuscript. A.B. prepared the virus for infections, assisted with animal work, and processed samples. E.C. and H.S. processed samples and prepared figure 3 for the manuscript. S.O. performed the animal experiment and collected samples. M.P. performed the animal experiment, collected samples, and prepared figure 2 for the manuscript. All authors designed the experiment and reviewed the manuscript. Data availability Data is provided within the manuscript. Funding This work was funded by intramural funding from the U.S. Department of Agriculture and by American Rescue Plan (ARP) funds through an interagency agreement between Agricultural Research Service (ARS) and the Animal and Plant Health Inspection Service (APHIS) Wildlife Service (WS). Additionally, this research was supported in part by an appointment to the ARS Research Participation Program administered by the Oak Ridge Institute for Science and Education (ORISE) through an interagency agreement between the U.S. Department of Energy (DOE) and the USDA. ORISE is managed by ORAU under DOE contract number DE-SC0014664. All opinions expressed in this paper are the authors’ and do not necessarily reflect the policies and views of USDA, DOE, or ORAU/ORISE. Ethics statement All animal work presented in this manuscript was performed under approval of the National Animal Disease Center (NADC) Institutional Animal Care and Use Committee (IACUC). The study is reported in accordance with ARRIVE guidelines. Conflicts of interest The authors report no conflicts of interest. References Martins, M. et al. From Deer-to-Deer: SARS-CoV-2 is efficiently transmitted and presents broad tissue tropism and replication sites in white-tailed deer. PLoS Pathog 18, e1010197 (2022). https://doi.org:10.1371/journal.ppat.1010197 Palmer, M. V. et al. Susceptibility of white-tailed deer (Odocoileus virginianus) to SARS-CoV-2. J Virol 95 (2021). https://doi.org:10.1128/JVI.00083-21 Chandler, J. C. et al. SARS-CoV-2 exposure in wild white-tailed deer (Odocoileus virginianus). Proc Natl Acad Sci U S A 118 (2021). https://doi.org:10.1073/pnas.2114828118 Hale, V. L. et al. SARS-CoV-2 infection in free-ranging white-tailed deer. Nature 602, 481–486 (2022). https://doi.org:10.1038/s41586-021-04353-x Kuchipudi, S. V. et al. Multiple spillovers from humans and onward transmission of SARS-CoV-2 in white-tailed deer. 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Microorganisms 10 (2022). https://doi.org:10.3390/microorganisms10040748 Porter, S. M. et al. Experimental SARS-CoV-2 Infection of Elk and Mule Deer. Emerg Infect Dis 30, 354–357 (2024). https://doi.org:10.3201/eid3002.231093 Frazzini, S., Amadori, M., Turin, L. & Riva, F. SARS CoV-2 infections in animals, two years into the pandemic. Arch Virol 167, 2503–2517 (2022). https://doi.org:10.1007/s00705-022-05609-1 Hobbs, E. C. & Reid, T. J. Animals and SARS-CoV-2: Species susceptibility and viral transmission in experimental and natural conditions, and the potential implications for community transmission. Transbound Emerg Dis 68, 1850–1867 (2021). https://doi.org:10.1111/tbed.13885 Porter, S. M., Hartwig, A. E., Bielefeldt-Ohmann, H., Root, J. & Bosco-Lauth, A. Experimental infection of elk (Cervus canadensis) and mule deer (Odocoileus hemionus) with SARS-CoV-2. bioRx (2023). https://doi.org:10.1101/2023.07.25.550568 Murray, S. M. et al. The impact of pre-existing cross-reactive immunity on SARS-CoV-2 infection and vaccine responses. Nat Rev Immunol 23, 304–316 (2023). https://doi.org:10.1038/s41577-022-00809-x Palmer, M. V. et al. Susceptibility of white-tailed deer ( Odocoileus virginianus ) to SARS-CoV-2. Journal of Virology 95, e00083-00021 (2021). https://doi.org:10.1101/2021.01.13.426628 Proal, A. D. et al. SARS-CoV-2 reservoir in post-acute sequelae of COVID-19 (PASC). Nat Immunol (2023). https://doi.org:10.1038/s41590-023-01601-2 Belyi, V. A., Levine, A. J. & Skalka, A. M. Unexpected inheritance: multiple integrations of ancient bornavirus and ebolavirus/marburgvirus sequences in vertebrate genomes. PLoS Pathog 6, e1001030 (2010). https://doi.org:10.1371/journal.ppat.1001030 Horie, M. et al. Endogenous non-retroviral RNA virus elements in mammalian genomes. Nature 463, 84–87 (2010). https://doi.org:10.1038/nature08695 Geuking, M. B. et al. Recombination of retrotransposon and exogenous RNA virus results in nonretroviral cDNA integration. Science 323, 393–396 (2009). https://doi.org:10.1126/science.1167375 Klenerman, P., Hengartner, H. & Zinkernagel, R. M. A non-retroviral RNA virus persists in DNA form. Nature 390, 298–301 (1997). https://doi.org:10.1038/36876 Shimizu, A. et al. Characterisation of cytoplasmic DNA complementary to non-retroviral RNA viruses in human cells. Sci Rep 4, 5074 (2014). https://doi.org:10.1038/srep05074 Zhang, L. et al. Reverse-transcribed SARS-CoV-2 RNA can integrate into the genome of cultured human cells and can be expressed in patient-derived tissues. Proc Natl Acad Sci U S A 118 (2021). https://doi.org:10.1073/pnas.2105968118 Li, N., Wang, X. & Lv, T. Prolonged SARS-CoV-2 RNA shedding: Not a rare phenomenon. J Med Virol 92, 2286–2287 (2020). https://doi.org:10.1002/jmv.25952 Yang, J. R. et al. Persistent viral RNA positivity during the recovery period of a patient with SARS-CoV-2 infection. J Med Virol 92, 1681–1683 (2020). https://doi.org:10.1002/jmv.25940 Reed, L. J. M., H.A. A simple method of estimating fifty percent endpoints. The American Journal of Hygiene, 493–497 (1938). Tan, C. W. et al. A SARS-CoV-2 surrogate virus neutralization test based on antibody-mediated blockage of ACE2-spike protein-protein interaction. Nat Biotechnol 38, 1073–1078 (2020). https://doi.org:10.1038/s41587-020-0631-z Jemersic, L. et al. Investigating the Presence of SARS CoV-2 in Free-Living and Captive Animals. Pathogens 10 (2021). https://doi.org:10.3390/pathogens10060635 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 14 May, 2024 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 10 Apr, 2024 Reviews received at journal 09 Apr, 2024 Reviews received at journal 03 Apr, 2024 Reviewers agreed at journal 25 Mar, 2024 Reviewers agreed at journal 24 Mar, 2024 Reviewers invited by journal 23 Mar, 2024 Editor assigned by journal 16 Mar, 2024 Editor invited by journal 13 Mar, 2024 Submission checks completed at journal 13 Mar, 2024 First submitted to journal 23 Feb, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3982475","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":279169878,"identity":"33d4b2be-4d58-40ef-8e3f-d05843d347dc","order_by":0,"name":"Paola Boggiatto","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABEElEQVRIiWNgGAWjYHACxgMMDAdAdAOIJwfEBgT1ILQcSGAwJkULiJXAkNhASIs5++EDB34w3JGT91/c+PnjD5v0DecPb/zA8MsmsQGHFsuetISDPQzPjA1vPGyWOJCQlrvhRlqxBGNfGk4tBgdyDA7wMBxO3DjjYANQy2GgFh4DCcaew8a4HGZw/o3BwT8QLc0/gFrSDc6fMf6BV8uNHIPDIFvm8ze2gWxJANprJsHw47AcLi2WM54lHJYxeGYMdEybxZm0NMOZN9LKLBIb0nBqMedPPvjwTQUwxPqPP75RYWMjz3f+8OYbH/7Y8OB0GIw0uJGAJJzYhksDUqzJ9x9AFv+DW8soGAWjYBSMOAAAs2ZqgPAG2SQAAAAASUVORK5CYII=","orcid":"","institution":"National Animal Disease Center, USDA, Agricultural Research Service","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Paola","middleName":"","lastName":"Boggiatto","suffix":""},{"id":279169879,"identity":"682bbd71-9a27-4748-bcb2-ca1c67071ee2","order_by":1,"name":"Alexandra Buckley","email":"","orcid":"","institution":"National Animal Disease Center, USDA, Agricultural Research Service","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Alexandra","middleName":"","lastName":"Buckley","suffix":""},{"id":279169880,"identity":"62849793-64b6-4cba-9c96-30e66ead0593","order_by":2,"name":"Eric Cassmann","email":"","orcid":"","institution":"National Animal Disease Center, USDA, Agricultural Research Service","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Eric","middleName":"","lastName":"Cassmann","suffix":""},{"id":279169882,"identity":"28e65e35-e531-4ff4-b4da-6281f81ca9f9","order_by":3,"name":"Hannah Seger","email":"","orcid":"","institution":"National Animal Disease Center, USDA, Agricultural Research Service","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hannah","middleName":"","lastName":"Seger","suffix":""},{"id":279169884,"identity":"7078b07e-103a-46eb-9976-42c3f9693a1f","order_by":4,"name":"Steven Olsen","email":"","orcid":"","institution":"National Animal Disease Center, USDA, Agricultural Research Service","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Steven","middleName":"","lastName":"Olsen","suffix":""},{"id":279169886,"identity":"c92e19a8-42d4-4c3e-8004-83ed2a81543e","order_by":5,"name":"Mitchell Palmer","email":"","orcid":"","institution":"National Animal Disease Center","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Mitchell","middleName":"","lastName":"Palmer","suffix":""}],"badges":[],"createdAt":"2024-02-23 16:15:28","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3982475/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3982475/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-024-61414-7","type":"published","date":"2024-05-15T00:38:26+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":52750884,"identity":"e358d23a-1e73-457d-a988-0aa3476dfe0b","added_by":"auto","created_at":"2024-03-15 10:20:19","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":13718,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePresence of SARS-CoV-2 neutralizing antibodies in serum from calves and adult elk.\u003c/strong\u003e Serum samples were collected at various timepoints following intranasal infection with SARS-CoV-2 and assessed via sVNT for antibodies against the virus. Shown are percent inhibition results for (A) elk calves and (B) adult elk. Bars indicate mean percent inhibition values, and error bars indicate ± SD. Dotted line indicates assay cut off for positive results.\u003c/p\u003e","description":"","filename":"Boggiattoetal.Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-3982475/v1/4e97b2b77a0180fd95f070dd.png"},{"id":52750887,"identity":"c482c4ba-ba85-4a98-93a6-e465b76319e0","added_by":"auto","created_at":"2024-03-15 10:20:19","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":14434410,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSARS-CoV-2 RNA detected in the germinal centers of medial retropharyngeal lymph nodes of infected elk calves and cows. \u003c/strong\u003eMedial\u003cstrong\u003e \u003c/strong\u003eretropharyngeal lymph nodes from elk calves (a-c) and elk cows (d-f) experimentally infected with SARS-CoV-2. In both elk calves and cows, labeling was observed on 2 (a,d), 5 (b,e), and 21 (c,f) days post inoculation (d.p.i). \u0026nbsp;Red labeling indicates presence of viral RNA within germinal centers (GC) or follicles (F). At days 2 and 5 p.i., this labeling is observed primarily within the marginal zone, while at day 21, labeling for SARS-CoV-2 RNA is seen within the GC.\u003c/p\u003e","description":"","filename":"Boggiattoetal.Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-3982475/v1/c21c5476b150ba00701c31bb.png"},{"id":52750888,"identity":"a7796bbb-0429-4f48-a3ed-468c93ae24ff","added_by":"auto","created_at":"2024-03-15 10:20:19","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":6907170,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSARS-CoV-2 Spike protein detected in the lymphoid follicles of the medial retropharyngeal lymph nodes of infected elk. \u003c/strong\u003eMedial retropharyngeal lymph nodes from elk calves (a-c) and elk cows (d-f) experimentally infected with SARS-CoV-2 at 2 (a,d), 5 (b, e), and 21 (c, f) days post inoculation (d.p.i). In calves, immunolabeling was present (dark brown color at the point of arrowheads) at day 2 (a) and 5 (b), but not at day 21 (c) d.p.i. In the elk cows, no immunolabeling for SARS-CoV-2 spike protein was present in the lymph nodes at 2 d.p.i. (d) or day 21 (f) d.p.i. but was present at 5 (e) d.p.i (arrowhead). In all cases, labeling was observed within lymphoid follicles, specifically, within the marginal zone (MZ) of follicles and not the germinal center (GC).\u003c/p\u003e","description":"","filename":"Boggiattoetal.Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-3982475/v1/b3f35bfa240e0bb9095f2835.png"},{"id":56566301,"identity":"ee5092bc-9e14-4864-b781-01a1586f0a4d","added_by":"auto","created_at":"2024-05-16 00:38:50","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":25504402,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3982475/v1/4c2ed40a-0842-46e6-aa49-5e772b44af4f.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Persistence of viral RNA in North American elk experimentally infected with an ancestral strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePrevious work from our laboratory identified white-tailed deer (\u003cem\u003eOdocoileus virginianus\u003c/em\u003e) as a susceptible host for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), capable of transmission \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Subsequent field work demonstrated a high level of seroprevalence and infection in free-ranging white-tailed deer across the United States (US) \u003csup\u003e\u003cspan additionalcitationids=\"CR4 CR5 CR6 CR7\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Evidence of multiple \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e as well as new \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e variants within white-tailed deer populations has been presented. Additionally, intra- as well as inter-species transmission (\u003cem\u003ei.e.\u003c/em\u003e spillback into humans) \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e has shown that white-tailed deer could serve as an important reservoir host for SARS-CoV-2 evolution and transmission.\u003c/p\u003e \u003cp\u003eInterest in determining whether white-tailed deer were susceptible to SARS-CoV-2 infection evolved from a comparative analysis of vertebrate angiotensin-converting enzyme 2 (ACE2), the main receptor for SARS-CoV-2. Based on these analyses, the white-tailed deer ACE2 receptor sequence showed a high degree of homology with the human ACE2 receptor and was classified as having a high propensity for binding the Spike protein of the virus \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Two additional cervid species, reindeer (\u003cem\u003eRangifer tarandus\u003c/em\u003e) and Pere David\u0026rsquo;s deer (\u003cem\u003eElaphurus davidandus\u003c/em\u003e) were also predicted to express ACE2 receptors with a high propensity for binding. North American elk (\u003cem\u003eCervus elaphus canadensis\u003c/em\u003e), another cervid species, was not reported as a possible susceptible host based on ACE2 receptor homology. Field surveillance of SARS-CoV-2 seroprevalance in red deer (\u003cem\u003eCervus elaphus\u003c/em\u003e), a closely related species to North American elk, found no evidence of exposure in Europe \u003csup\u003e\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Data from these studies also suggested that other free-ranging deer species in Europe including Fallow (\u003cem\u003eDama dama\u003c/em\u003e), Muntjac (\u003cem\u003eMuntiacus reevesi\u003c/em\u003e), Sika (\u003cem\u003eCervus nippon\u003c/em\u003e) and Roe deer (\u003cem\u003eCapreolus capreolus\u003c/em\u003e) were also seronegative \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe high seroprevalence observed in white-tailed deer populations can be attributed to both susceptibility to infection but also to sources of infection from humans. As SARS-CoV-2 circulates in white-tailed deer, they may serve as a source of infection for other species. Therefore, we sought to determine the susceptibility of North American elk to SARS-CoV-2 infection. Recently, a study by Porter \u003cem\u003eet al.\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e demonstrated that weanling elk are minimally susceptible to infection with the Delta variant of SARS-CoV-2. They did not develop clinical signs and were not capable of onward transmission. In the work presented here, we expand on this initial study in North American elk and assess the susceptibility of elk calves and adults to the ancestral Wuhan-like strain of SARS-CoV-2 (USA-WA1/2020). We provide further evidence that while North American elk do not develop clinical signs associated with infection, SARS-CoV-2 does elicit a neutralizing antibody response, suggesting that the virus is capable of establishing infection in this species. Additionally, we show that SARS-CoV-2 RNA persists in the lymph node of infected animals in the absence of viral protein. These findings provide additional information regarding host-pathogen interaction mechanisms for SARS-CoV-2 in one of its many hosts.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cem\u003eDevelopment of neutralizing antibody responses against SARS-CoV-2\u003c/em\u003e \u003c/p\u003e \u003cp\u003eBlood samples were collected on days 0, 7, 14 and 21 p.i. to assess neutralizing antibodies against SARS-CoV-2. In both calves (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) and adult (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) elk, antibody titers to SARS-CoV-2 measured by surrogate virus neutralization titers (sVNT) can be observed as early as 7 days p.i. and are sustained through day 21. Interestingly for elk calves, the mean sVNT inhibition value is around 70% at the peak of the response, while in the adult elk the percent inhibition is lower at 60%. Virus neutralization (VN) tests were also performed for both calves and adult elk. VN titers peaked for the elk calves at 14 days p.i. with the highest titer (1:256) observed in only one calf, while in the adult elk, VN titers peaked at day 7 p.i. and did not go above 1:16 (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Altogether, these data suggest that both calves and adult elk can mount neutralizing antibody responses to SARS-CoV-2 following challenge. However, this response appears to be more robust in the calves.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eVirus neutralization test results for calves and adult elk\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"11\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e \u003cp\u003eCalves\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"6\" nameend=\"c11\" namest=\"c6\"\u003e \u003cp\u003eAdult\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAnimal #\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eD7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eD14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eD21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003eAnimal #\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003eD7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e \u003cp\u003eD14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eD21\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c11\" namest=\"c10\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e256\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c11\" namest=\"c10\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e7\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c11\" namest=\"c10\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eViral RNA detection in nasal, oral and rectal swabs\u003c/em\u003e \u003c/p\u003e \u003cp\u003eSwabs (nasal, oral and rectal) were collected at various timepoints and analyzed via PCR for the presence of viral RNA. In elk calves, nasal swabs were only found positive in 2/7 animals at day 2 p.i., in 1/5 at day 3 p.i. and in 1/3 at days 7 and 10 p.i. (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). We did not observe consistent positive PCR results in swabs collected from elk calves. In contrast, we observed PCR positive results in all (7/7) inoculated adult elk at day 2 p.i., and 2/5 at days 3 and 4 p.i. (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Oral and rectal swabs did not consistently show the presence of viral RNA. No oral or rectal swab samples collected from the elk calves were positive for SARS-CoV-2 RNA, and in samples collected from the adult elk, only one oral swab was found positive on day 4 p.i. and only 2 rectal swabs were found positive on day 2 p.i. (data not shown).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eElk calves nasal swab PCR results\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"10\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAnimal #\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eD0\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eD2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eD3\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eD4\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eD5\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eD7\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eD10\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eD14\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003eD21\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e30.0/33.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e31.4/34.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e--/39.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e36.87/--\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e37.9/--\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAdult elk nasal swab PCR\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"10\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAnimal #\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eD0\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eD2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eD3\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eD4\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eD5\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eD7\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eD10\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eD14\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003eD21\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e36.3/--\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e--/37.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e33.8/34.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e38.6/38.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e35.4/--\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e29.5/31.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e35.4/36.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e36.0/--\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e34.4/36.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e37.2/34.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e--/38.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eViral RNA detection in lymphoid tissues\u003c/em\u003e \u003c/p\u003e \u003cp\u003eAt necropsy on days 2 and 21 p.i. mRPLN and palatine tonsil samples were collected and assessed for the presence of viral RNA via PCR, based on previous white-tailed deer studies (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). At day 2 p.i., SARS-CoV-2 RNA was detected in the mRPLN of one elk calf and one adult elk. In contrast, no viral RNA was detected in tonsils collected from calves or adult elk. By day 21 p.i., viral RNA was detected in all three mRPLN from both calves and adult elk. SARS-CoV-2 was also detected in 2/3 tonsil samples collected from elk calves although Ct values were high (~\u0026thinsp;38), but none was detected in tonsils from adult elk.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSARS-CoV-2 RNA detection in tissues\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"11\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e \u003cp\u003eCalves\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"5\" nameend=\"c11\" namest=\"c7\"\u003e \u003cp\u003eAdult\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNecropsy\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003emRPLN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTonsil\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e\u003cb\u003eNecropsy\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003emRPLN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e \u003cp\u003eTonsil\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c11\" namest=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eD2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e25.5\u003c/p\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e--\u003c/p\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e\u003cb\u003eD2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003e32.8\u003c/p\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c11\" namest=\"c10\"\u003e \u003cp\u003e--\u003c/p\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eD21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e32.2\u003c/p\u003e \u003cp\u003e27.9\u003c/p\u003e \u003cp\u003e23.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e38.7*\u003c/p\u003e \u003cp\u003e--\u003c/p\u003e \u003cp\u003e38.1*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e\u003cb\u003eD21\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003e32.2\u003c/p\u003e \u003cp\u003e22.7\u003c/p\u003e \u003cp\u003e28.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c11\" namest=\"c10\"\u003e \u003cp\u003e--\u003c/p\u003e \u003cp\u003e--\u003c/p\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"11\"\u003e*Denotes only of the two duplicate wells had a positive value.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eDetection of SARS-CoV-2 RNA via\u003c/em\u003e in situ \u003cem\u003ehybridization (ISH)\u003c/em\u003e\u003c/p\u003e \u003cp\u003eMicroscopic analysis of tissues collected at necropsy revealed no lesions consistent with SARS-CoV-2 infection reported in other species. The presence of SARS-CoV-2 RNA was investigated through ISH on various tissues. Tissues were selected based on previous studies using white-tailed deer, these included palatine tonsil, mRPLN and lung. In elk calves, viral RNA was detected in the mRPLN of 2/2 calves examined at 2 days p.i., 2/2 calves examined at 5 days p.i., and 3/3 calves examined at 21 days p.i. In adult elk, viral RNA was detected in 2/2 cows examined 2 days p.i., 2/2 cows examined 5 days p.i. and 3/3 cows examined 21 days p.i. In all cases, labeling was limited to secondary lymphoid follicles, often within germinal centers (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Additionally, staining for viral RNA was primarily observed within the marginal zone of the follicle in both calves and adult elk on days 2 and 5 dpi (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea-b, d-e). However, on day 21 p.i., viral RNA staining was observed within the germinal center (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec, f). Viral RNA was not detected in palatine tonsils or lung from inoculated calves or cows. Additionally, viral RNA was not detected in tissues examined from non-inoculated control calves and cows.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eDetection of SARS-CoV-2 protein via immunohistochemistry (IHC)\u003c/em\u003e \u003c/p\u003e \u003cp\u003eGiven the detection of SARS-CoV-2 viral RNA via PCR and ISH, we performed IHC on mRPLN and tonsil samples in elk calves to determine if viral protein was also detectable within these tissues. In the mRPLN of elk calves, SARS-CoV-2 spike protein was detectable in 1/2 calves at 2 days p.i., 2/2 calves at 5 days p.i., and 0/3 calves at 21 days p.i (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). No SARS-CoV-2 spike protein was detected in the tonsil of elk calves utilizing IHC. The mRPLN of adult elk cow was also analyzed by IHC. Immunolabeling for SARS-CoV-2 spike protein was observed in 0/2 cows at 2 days p.i., 2/2 cows at 5 days p.i., and 0/3 cows at 21 days p.i. Staining for viral Spike protein was primarily observed within the marginal zone of the follicle.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eSince the outbreak of the 2019 SARS-CoV-2 pandemic, concerns regarding the susceptibility of other vertebrate species, and their potential to act as reservoirs for the virus, have garnered attention. Field surveillance as well as various experimental infection studies have demonstrated a wide range of susceptibility across various species in the families Felidae, Canidae, Mustelidae, Cricetidae, and Cervidae \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Among these, white-tailed deer have emerged as a species of interest given their susceptibility to infection, the high seroprevalence in free-ranging populations, their ability to transmit the virus to other deer as well as back to humans, and the identification of novel variants within this species \u003csup\u003e1\u0026ndash;3,7\u0026minus;10,13\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eCharacterization of susceptible wildlife species is critical for understanding not only the epidemiology of this virus, but also for our ability to implement intervention strategies to stop the spread of this disease. In this work, we sought to characterize the susceptibility of North American elk to SARS-CoV-2 infection, another cervid species with broad distribution in the US. Here, we demonstrate that both elk calves and adult elk are susceptible to infection with the ancestral Wuhan-like variant of SARS-CoV-2 (USA-WA1/2020), as characterized by the development of measurable neutralizing antibody responses and the detection of viral RNA and viral protein in the retropharyngeal lymph nodes of infected animals. However, this work demonstrates that there may be some differences in the quality of the responses between the two age groups.\u003c/p\u003e \u003cp\u003eA previous study by Porter \u003cem\u003eet al.\u003c/em\u003e demonstrated that weanling North American elk are minimally susceptible to infection with the Delta variant of SARS-CoV-2 \u003csup\u003e19\u003c/sup\u003e. Post challenge, viral RNA could be detected from oral and nasal swabs between days 1 and 5 p.i. However, infected elk calves did not shed infectious virus, nor were they capable of transmission to an in-contact elk. Neutralizing antibody responses were observed in these animals; however, these responses were relatively low, with peak neutralizing titers at 1:20 at 21 days p.i. Additionally, no infectious virus was detected in tissues collected at necropsy. Similar to these findings, we observed the presence of viral RNA from nasal swabs and the development of neutralizing titers from infected animals. However, and in contrast to the results observed with Delta variant infection, we observed peak neutralizing titers at 14 days p.i., with values ranging from 1:32 up to 1:256 (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Interestingly, when we compared elk calves to adult virus neutralization titers, the elk calves appear to have a higher neutralization response; adult elk responses remained at 1:8 or 1:\u0026lt;8 at all timepoints except for one animal at day 7 p.i. with a 1:16 titer.\u003c/p\u003e \u003cp\u003eDifferences in viral neutralization responses may be related to the age of the animals and exposure to other viruses, including other coronaviruses. The high homology between SARS-CoV-2 and other coronaviruses, may result in cross-reactive immune responses. In humans, cross-reactive humoral and cellular immune responses developed prior to SARS-CoV-2 infection, have been characterized and demonstrated to play an important role in determining susceptibility to infection and disease progression (reviewed in \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e). Pre-existing cross-reactive responses from previous viral infections may be beneficial or detrimental. For example, cross-reactive antibodies to one virus may bind a to another virus and provide neutralization and subsequent clearance. Alternatively, pre-existing cross-reactive antibodies to a virus may bind to another virus with low-avidity resulting in enhanced viral uptake via antibody-mediated internalization and/or inhibit the generation of \u003cem\u003ede novo\u003c/em\u003e antibody responses to the latter, resulting in increased viral load \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Our data suggests that adult elk appear to have a lower magnitude of neutralizing antibody responses (both in sVNT and VN) as compared to the elk calves. These slight differences may be attributed to some level of pre-existing immunity in older animals, which allows them to control the infection more rapidly, thus decreasing viral load available for \u003cem\u003ede novo\u003c/em\u003e antibody responses.\u003c/p\u003e \u003cp\u003ePersistence of viral RNA, detectable by PCR and ISH, in tissues such as the mRPLNs is consistent with previous studies in white-tailed deer, including the location of labeling within secondary lymphoid follicles and germinal centers of lymphoid tissues \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Similarly, persistent SARS-CoV-2 RNA has been detected in human lymph nodes, tonsils and other tissues at autopsies conducted over 300 days post infection \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. In such cases in deer species and humans it was not possible to isolate virus from these tissues, although viral RNA was detected using ISH or RT-PCR. A potential explanation for the detection of viral RNA in the absence of infectious virus is the integration of viral subgenomic RNA into the DNA of the host cell via reverse transcription. Ancestral evidence of non-retroviral RNA virus sequences in the genome of vertebrate species have been previously detected \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Additionally, DNA copies of nonretroviral RNA viruses including lymphocytic choriomeningitis virus (LCMV) and vesicular stomatitis virus have been shown to integrate into the DNA of their host cell \u003csup\u003e\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Recently, using cultured human cells Zhang \u003cem\u003eet al.\u003c/em\u003e demonstrated that SARS-CoV-2 can integrate into the genome of host cells \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Additionally, using published RNAseq data from cultured cells and organoid tissues, they demonstrate that SARS-CoV-2 sequences integrated into the host cell genome can be expressed as human-viral chimeric reads. RNA expression of viral subgenomic sequences could explain why some patients remain PCR positive for SARS-CoV-2 many weeks or months following recovery from infection. While infectious virus cannot be produced from this integrated material, it does raise the possibility that viral antigen could be expressed by host cells. In the work presented here we demonstrate the presence of viral RNA 21 days p.i. with peak viral Spike protein production at day 5 p.i. As detection of Spike protein wanes, viral RNA is consistently detected via ISH and PCR. The lack of correlation between viral RNA and protein production would suggest that active translation is not occurring. Additionally, based on nasal and oral swab data, we did not detect viral RNA in these secretions. These data would suggest that viral replication and shedding is not occurring. Consistent with continuous or recurrent SARS-CoV-2 PCR positive reports in recovered human patients \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e, the persistent viral RNA found in the lymph nodes of other susceptible species including white-tailed deer \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e and elk, as demonstrated here, may point to a common mechanism of viral persistence in susceptible hosts. However, the immunological implications of this phenomenon are not completely understood and warrant further study.\u003c/p\u003e \u003cp\u003eThe data presented here demonstrate that both calves and adult North American elk are susceptible to SARS-CoV-2 infection as they permit viral replication and develop virus neutralizing antibody responses following infection. However, no clinical signs of disease nor pathological changes in the lungs or lymph nodes of infected animals were observed, suggesting that they are not susceptible to disease. Interestingly, our findings suggest that there is persistence of SARS-CoV-2 viral RNA in the lymphoid tissues of infected animals in the presence of an immune response. The continued assessment of SARS-CoV-2 susceptibility in various species provides insights not only into potential reservoirs for the disease but also, as shown here, sheds light on the host-pathogen interactions that may be common across species and may drive immunity to infection and disease.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e \u003cb\u003eCells and virus\u003c/b\u003e \u003c/p\u003e \u003cp\u003eVero E6 (ATCC\u0026reg; CRL-1586\u0026trade;) cells were cultured in Eagle\u0026rsquo;s Minimum Essential Medium (EMEM, ATCC) supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic-antimycotic 100X (Gibco\u0026trade;, Life Technologies, Carlsbad, CA, USA). The cell cultures were maintained at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e. 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 \u003csup\u003eo\u003c/sup\u003eC. Viral titer was determined by the Reed and Muench \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. A viral suspension containing 10\u003csup\u003e5.5\u003c/sup\u003e tissue culture infectious dose 50 per ml (TCID\u003csub\u003e50\u003c/sub\u003e/ml) was used for elk calf inoculations and 10\u003csup\u003e6\u003c/sup\u003e TCID\u003csub\u003e50\u003c/sub\u003e/ml for adult elk inoculations.\u003c/p\u003e\n\u003ch3\u003eAnimal Infection and Sampling\u003c/h3\u003e\n\u003cp\u003e 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 (~\u0026thinsp;5 months old; n\u0026thinsp;=\u0026thinsp;11) and adult elk cows (~\u0026thinsp;4 years old; n\u0026thinsp;=\u0026thinsp;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.\u003c/p\u003e \u003cp\u003eSeven elk calves and 7 cows were sedated with a combination of xylazine and ketamine and intranasally inoculated with an atomization device (LMA\u0026reg; MAD Nasal\u0026trade;, 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.\u003c/p\u003e\n\u003ch3\u003eSerology\u003c/h3\u003e\n\u003cp\u003eThe cPASS SARS-CoV-2 neutralization antibody detection kit (GenScript Biotech,\u003c/p\u003e \u003cp\u003eAmsterdam, Netherlands) was used as described \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e and according to the manufacturer\u0026rsquo;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\u0026deg;C. The virus had a TCID\u003csub\u003e50\u003c/sub\u003e of 100. Vero-76 cell culture was then added to the virus/serum mixture and incubated at 37\u0026deg;C for 3 days. Each well was observed for presence of absence of cytopathic effect.\u003c/p\u003e\n\u003ch3\u003eReal-time RT-PCR on swabs and tissues\u003c/h3\u003e\n\u003cp\u003eTo assess for viral shedding on nasal, oral and rectal samples, swabs were submitted to NVSL for processing and for rRT-PCR analysis.\u003c/p\u003e \u003cp\u003eTo determine the presence of viral RNA in tissue samples, tissues were thawed, cut into an approximately 50\u0026ndash;100 mg piece, and resuspended in 1\u0026ndash;2 mL of TRI-Reagent\u0026reg; (Life Technologies, Carlsbad, CA, USA) in individual gentle MACS\u0026trade; M tubes (Miltenyi Biotec, Bergisch Gladbach, Germany). Tissues were dissociated using a gentle MACS\u0026trade; Octo-Dissociator (Miltenyi Biotec) following the manufacturer\u0026rsquo;s recommendations. RNA was extracted from tissue homogenate samples using the MagMAX\u0026trade;-96 for Microarrays Total RNA Isolation Kit (Applied Biosystems, Waltham, MA, USA). Samples were run on a MagMAX\u0026trade; Express Magnetic Particle Processor (Applied Biosystems) following the manufacturer\u0026rsquo;s instructions. Next, 15 \u0026micro;L of extracted product was added to 5 \u0026micro;L of the AgPath-ID\u0026trade; 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\u0026deg;C for 10 min, followed by 1 cycle at 95\u0026deg;C for 10 min, 1 cycle at 95\u0026deg;C for 3 s, and 45 cycles at 55\u0026deg;C for 30 s. The forward primer sequence was 5\u0026rsquo;-GACCCCAAAATCAGCGAAAT-3\u0026prime;, the reverse primer sequence was 5\u0026rsquo;-TCTGGTTACTGCCAGTTGAATCTG-3\u0026rsquo;, and the probe sequence was 5\u0026rsquo;-FAM-ACCCCGCATTACGTTTGGTGGACC-BHQ1-3\u0026rsquo;. A positive control (2019-nCoV_N_Positive Control, Integrated DNA Technologies IDT, Coralville, IA, USA) and a negative control was run on every plate.\u003c/p\u003e\n\u003ch3\u003eNecropsy and Sample Collection\u003c/h3\u003e\n\u003cp\u003eTwo 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\u0026deg;C freezer until testing. Additionally, tissue samples were collected and processed for standard microscopic examination, a subset were also processed by \u003cem\u003ein situ\u003c/em\u003e hybridization (ISH) and immunohistochemistry (IHC). For this, tissue sections of approximately\u0026thinsp;\u0026le;\u0026thinsp;0.5 cm in width were fixed by immersion in 10% neutral buffered formalin (\u0026ge;\u0026thinsp;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).\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn situ\u003c/b\u003e \u003cb\u003ehybridization (ISH)\u003c/b\u003e\u003c/p\u003e \u003cp\u003eParaffin-embedded tissues were sectioned at 5 \u0026micro;m and subjected to ISH using the RNAscope ZZ probe technology (Advanced Cell Diagnostics, Newark, CA). \u003cem\u003eIn situ\u003c/em\u003e 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\u0026ndash;RED kit (Advanced Cell Diagnostics) as previously described \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. 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 \u003cem\u003eBos taurus\u003c/em\u003e \u0026ndash;specific cyclophilin B (PPIB) or ubiquitin (UBC) housekeeping genes, while a probe targeting dapB of \u003cem\u003eBacillus subtilis\u003c/em\u003e was used as a negative control.\u003c/p\u003e\n\u003ch3\u003eImmunohistochemistry\u003c/h3\u003e\n\u003cp\u003eImmunohistochemical 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\u0026deg;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\u0026deg;C-98\u0026deg;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\u0026reg; (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\u0026reg; Boost IHC Detection Reagent (HRP, Mouse, Cell Signaling, catalog no. 8125S) followed by SignalStain\u0026reg; 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.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank the National Animal Disease Center (NADC) Animal Resources Unit (ARU) for the care of the animals used in this study. Specifically, the authors thank Dr. Rebecca Cox, Derek Vermeer, Jonathan Gardner, Tiffany Williams and Kolby Stallman for their animal husbandry, care, and assistance. Additionally, we would like to thank Sue Osorio and Sarah Anderson for their excellent technical assistance. We would also like to thank NVSL for their technical assistance with sample processing and testing.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eP.B. performed the animal experiment, collected samples, prepared figure 1, and wrote the manuscript. A.B. prepared the virus for infections, assisted with animal work, and processed samples. E.C. and H.S. processed samples and prepared figure 3 for the manuscript. S.O. performed the animal experiment and collected samples. M.P. performed the animal experiment, collected samples, and prepared figure 2 for the manuscript. All authors designed the experiment and reviewed the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData is provided within the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was funded by intramural funding from the U.S. Department of Agriculture and by American Rescue Plan (ARP) funds through an interagency agreement between Agricultural Research Service (ARS) and the Animal and Plant Health Inspection Service (APHIS) Wildlife Service (WS). Additionally, this research was supported in part by an appointment to the ARS Research Participation Program administered by the Oak Ridge Institute for Science and Education (ORISE) through an interagency agreement between the U.S. Department of Energy (DOE) and the USDA. ORISE is managed by ORAU under DOE contract number DE-SC0014664. All opinions expressed in this paper are the authors\u0026rsquo; and do not necessarily reflect the policies and views of USDA, DOE, or ORAU/ORISE.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal work presented in this manuscript was performed under approval of the National Animal Disease Center (NADC) Institutional Animal Care and Use Committee (IACUC). The study is reported in accordance with ARRIVE guidelines.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors report no conflicts of interest.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMartins, M. \u003cem\u003eet al.\u003c/em\u003e From Deer-to-Deer: SARS-CoV-2 is efficiently transmitted and presents broad tissue tropism and replication sites in white-tailed deer. PLoS Pathog 18, e1010197 (2022). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1371/journal.ppat.1010197\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1371/journal.ppat.1010197\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePalmer, M. 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Pathogens 10 (2021). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.3390/pathogens10060635\u003c/span\u003e\u003cspan address=\"https://doi.org:10.3390/pathogens10060635\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-3982475/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3982475/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eWhite-tailed deer (\u003cem\u003eOdocoileus virginianus\u003c/em\u003e) have emerged as a potential reservoir host for SARS-CoV-2 given their susceptibility to infection and demonstrated high rates of seroprevalence across the United States. As SARS-CoV-2 circulates within free-ranging white-tailed deer populations, there is the risk of transmission to other wildlife species and even back to the human population. The goal of this study was to determine the susceptibility, shedding, and immune response of North American elk (\u003cem\u003eCervus elaphus canadensis\u003c/em\u003e) to experimental infection with SARS-CoV-2, to determine if another wide-ranging cervid species could potentially serve as a reservoir host for the virus. Here we demonstrate that while North American elk do not develop clinical signs of disease, they do develop a neutralizing antibody response to infection, suggesting the virus is capable of replicating in this mammalian host. Additionally, we demonstrate SARS-CoV-2 RNA presence in the medial retropharyngeal lymph nodes of infected elk three weeks after experimental infection. Consistent with previous observations in humans, these data may highlight a mechanism of viral persistence for SARS-CoV-2 in elk.\u003c/p\u003e","manuscriptTitle":"Persistence of viral RNA in North American elk experimentally infected with an ancestral strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-15 10:20:14","doi":"10.21203/rs.3.rs-3982475/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-04-10T06:54:40+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-04-09T16:00:27+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-04-03T21:36:54+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"8cedc40c-d615-48e6-9a8d-e6e092af7aef","date":"2024-03-25T19:18:50+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"0cc60494-4a4f-429b-907d-94d80232616c","date":"2024-03-24T12:05:48+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-03-23T18:39:10+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-03-16T13:32:02+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-03-13T06:14:52+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-03-13T06:01:23+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-02-23T15:55:19+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"cdb61b03-6538-44e5-a1d4-4d5995fc5fff","owner":[],"postedDate":"March 15th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":29400971,"name":"Biological sciences/Microbiology/Virology/Viral pathogenesis"},{"id":29400973,"name":"Biological sciences/Microbiology/Pathogens"},{"id":29400974,"name":"Biological sciences/Immunology/Adaptive immunity/Humoral immunity"},{"id":29400975,"name":"Biological sciences/Immunology"},{"id":29400976,"name":"Biological sciences/Immunology/Infection"}],"tags":[],"updatedAt":"2024-05-16T00:38:26+00:00","versionOfRecord":{"articleIdentity":"rs-3982475","link":"https://doi.org/10.1038/s41598-024-61414-7","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2024-05-15 00:38:26","publishedOnDateReadable":"May 15th, 2024"},"versionCreatedAt":"2024-03-15 10:20:14","video":"","vorDoi":"10.1038/s41598-024-61414-7","vorDoiUrl":"https://doi.org/10.1038/s41598-024-61414-7","workflowStages":[]},"version":"v1","identity":"rs-3982475","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3982475","identity":"rs-3982475","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}