Susceptibility of White-Tailed Deer (Odocoileus virginianus) to SARS-CoV-2
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Abstract
Given the presumed zoonotic origin of SARS-CoV-2, the human-animal-environment interface of the COVID-19 pandemic is an area of great scientific and public and animal health interest. Identification of animal species that are susceptible to infection by SARS-CoV-2 may help to elucidate the potential origin of the virus, identify potential reservoirs or intermediate hosts, and define the mechanisms underlying cross-species transmission to humans.
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SciScore for 10.1101/2021.01.13.426628: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IACUC: Animals, inoculation and sampling: All animals were handled in accordance with the Animal Welfare Act Amendments (7 U.S. Code §2131 to §2156) and all study procedures were reviewed and approved by the Institutional Animal Care and Use Committee at the National Animal Disease Center (IACUC approval number ARS-2020-861).
IRB: Fetal bovine serum (FBS) and convalescent human serum (kindly provided by Dr. Elizabeth Plocharczyk, Cayuga Medical Center [CMC]; under CMC’s Institutional Review Board protocol number 0420EP) were used as a negative and positive controls, respectively.Randomization not detected. Blinding Cell cultures with no CPE were frozen, thawed, and … SciScore for 10.1101/2021.01.13.426628: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IACUC: Animals, inoculation and sampling: All animals were handled in accordance with the Animal Welfare Act Amendments (7 U.S. Code §2131 to §2156) and all study procedures were reviewed and approved by the Institutional Animal Care and Use Committee at the National Animal Disease Center (IACUC approval number ARS-2020-861).
IRB: Fetal bovine serum (FBS) and convalescent human serum (kindly provided by Dr. Elizabeth Plocharczyk, Cayuga Medical Center [CMC]; under CMC’s Institutional Review Board protocol number 0420EP) were used as a negative and positive controls, respectively.Randomization not detected. Blinding Cell cultures with no CPE were frozen, thawed, and subjected to two additional blind passages/inoculations in Vero E6/TMPRSS2 cell cultures. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources At 24 h post-inoculation, cells were fixed with 3.7% formaldehyde for 30 min at room temperature, permeabilized with 0.2% Triton X-100 (in Phosphate Buffered Saline [PBS]) and subjected to an immunofluorescence assay (IFA) using a monoclonal antibody (MAb) anti-ACE2 (Sigma-Aldrich), and then incubated with a goat anti-rabbit IgG (goat anti-rabbit IgG, Alexa Fluor 488®), and using a monoclonal antibody (MAb) anti-SARS-CoV-2 nucleoprotein (N) (clone B6G11) produced and characterized in Dr. anti-ACE2suggested: Noneanti-rabbit IgGsuggested: Noneanti-SARS-CoV-2 nucleoprotein (Nsuggested: NoneDiel’s laboratory, and then incubated with a goat anti-mouse IgG secondary antibody (goat anti-mouse IgG, Alexa Fluor® 594), and Nuclear counterstain was performed with DAPI, and visualized under a fluorescence microscope. anti-mouse IgGsuggested: NoneFluorescent beads were coupled with the anti-equine IL-4 antibody, clone 25 (RRID: AB_2737308) as previously described (65). anti-equine IL-4detected: (Dr. Bettina Wagner - Cornell University Cat# IL4 25, RRID:AB_2737308)The assay was detected using a biotinylated mouse anti-goat IgG (H+L) (RRID: AB_2339061, Jackson Immunoresearch Laboratories, West Grove, PA) cross-reactive with deer immunoglobulin. mouse anti-goat IgG H+Ldetected: (Jackson ImmunoResearch Labs Cat# 205-065-108, RRID:AB_2339061)Experimental Models: Cell Lines Sentences Resources The SARS-CoV-2 isolate TGR/NY/20 obtained from a Malayan tiger naturally infected with SARS-CoV-2 and presenting with respiratory disease compatible with SARS-CoV-2 infection (22) was propagated in Vero CCL-81 cells. Vero CCL-81suggested: NoneTo assess the kinetics of replication of SARS-CoV-2 isolate TGR/NY/20, Vero E6, Vero E6/TMPRSS2 and DL cells were cultured in 12-well plates, inoculated with SARS-Cov-2 isolate TGR/NY/20 (MOI = 0.1 and MOI = 1 and harvested at various time points post-inoculation (12, 24, 36, 48 and 72 h pi). Vero E6suggested: RRID:CVCL_XD71)Cell cultures with no CPE were frozen, thawed, and subjected to two additional blind passages/inoculations in Vero E6/TMPRSS2 cell cultures. Vero E6/TMPRSS2suggested: NoneSARS-CoV-2 RNA (strain Hu-WA-1) was derived from Vero cells infected with the virus and cDNA was synthesized using the SuperScript III Reverse Transcriptase (Life Technologies) and oligo dT and six hexamer random primers. Verosuggested: CLS Cat# 605372/p622_VERO, RRID:CVCL_0059)CHO-K1 cells were transiently transfected with the recombinant plasmid constructs. CHO-K1suggested: CLS Cat# 603480/p693_CHO-K1, RRID:CVCL_0214)Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- No conflict of interest statement was detected. If there are no conflicts, we encourage authors to explicit state so.
- No funding statement was detected.
- No protocol registration statement was detected.
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Our take
This study, available as a preprint and thus not yet peer reviewed, demonstrated that white-tailed deer are highly susceptible to SARS-CoV-2 infection, and are capable of transmitting the virus to naïve animals without physical contact, presumably via droplets or aerosols. Farms containing deer and properties with large deer herds in close contact with humans should consider monitoring populations for the presence of SARS-CoV-2 to rapidly contain spread in the case of an outbreak.
Study design
other
Study population and setting
To assess the susceptibility of white-tailed deer (Odocoileus virginianus) to SARS-CoV-2 infection, the authors performed experimental infections of deer cells and live animals. Entry of SARS-CoV-2 into cultured deer lung cells was assessed using an immunofluorescence …
Our take
This study, available as a preprint and thus not yet peer reviewed, demonstrated that white-tailed deer are highly susceptible to SARS-CoV-2 infection, and are capable of transmitting the virus to naïve animals without physical contact, presumably via droplets or aerosols. Farms containing deer and properties with large deer herds in close contact with humans should consider monitoring populations for the presence of SARS-CoV-2 to rapidly contain spread in the case of an outbreak.
Study design
other
Study population and setting
To assess the susceptibility of white-tailed deer (Odocoileus virginianus) to SARS-CoV-2 infection, the authors performed experimental infections of deer cells and live animals. Entry of SARS-CoV-2 into cultured deer lung cells was assessed using an immunofluorescence assay targeting the SARS-CoV-2 nucleoprotein. In vivo infection of deer was evaluated using six-week-old fawns (n = 6) from a breeding herd in Ames, Iowa. Animals were raised in captivity and allowed to acclimate to the biosafety facility for two weeks prior to the start of the experiment. Four animals were inoculated intranasally with 10^6.3 50% tissue culture infective dose of SARS-CoV-2. The two remaining fawns were used as naïve controls housed in the same room to test the potential for indirect viral transmission between deer. Animals were monitored for clinical signs and changes in body temperature. Nasal and rectal swabs were collected between DPI 0 and 21; blood serum and buffy coat samples were collected at DPI 0, 7, 14, and 21. All samples were tested for viral RNA via real-time reverse transcription PCR and for infectious virus via culture on Vero E6 cells. Serum samples were tested reactive and neutralizing antibodies using Luminex and virus neutralization assays, respectively, targeting antibodies against the SARS-CoV-2 nucleoprotein and spike receptor binding domain.
Summary of main findings
Deer lung cells supported SARS-CoV-2 virus entry 24 hours post-inoculation and virus replication for up to 3 days. None of the animals showed any clinical signs of infection, though 3 out of 4 inoculated animals had slightly elevated body temperatures between DPI 1 and 3; body temperatures remained within the normal range in indirect contact animals. Viral RNA was detected in consistently in nasal secretions of inoculated and indirect contact animals between DPI 2 and 21; detection was inconsistent in rectal swabs. No viremia was detected in blood serum or buffy coat samples. Infectious virus was detected in nasal swabs between DPI 2 and 5 from inoculated animals and between DPI 2 and 7 from indirect contact animals. Infectious virus was only detected in fecal swabs from inoculated animals at 1 DPI. Inoculated and indirect contact animals produced reactive and neutralizing antibodies against SARS-CoV-2 N and spike RBD as early as 7 DPI, with increasing antibody titers at 14 and 21 DPI. A single inoculated animal died at DPI 8 from intestinal perforation unrelated to the experiment; the remaining 5 animals were sacrificed at 21 DPI. No gross lesions were observed in any necropsied tissues. SARS-CoV-2 RNA was detected in necropsied multiple upper respiratory tissues, spleen, and lymph nodes via PCR and in situ hybridization, but no infectious virus was detectable.
Study strengths
The experimental design tested the potential for indirect transmission between deer using a plexiglass barrier to prevent nose-to-nose contact between inoculated and naïve animals.
Limitations
The study used a very small number of individuals, and the animals were all of uniform age and from the same source population. Thus, the results may not be generalizable to all white-tailed deer populations. The animals were also not tested for concurrent or past infection with other coronaviruses that might affect susceptibility to infection or serological tests. The authors were unable to detect infectious virus in any of the tissues sampled from necropsied animals at 8 and 21 DPI. Testing for infectious virus in tissues at additional timepoints would be necessary to find the active sites of virus replication.
Value added
Previous studies have shown that the ACE2 receptors of white-tailed deer and other deer and ruminants are highly similar to the human version (see https://doi.org/10.1073/pnas.2010146117), so it was assumed that deer would be susceptible to SARS-CoV-2 infection. White-tailed deer frequently live nearby humans in suburban areas in North America, are frequently hunted, and can be farmed, though not extensively. Due to this interface with humans, there was concern that transmission of SARS-CoV-2 from humans could establish virus circulation in wild or farmed deer populations. This is the first study to show that SARS-CoV-2 can infect and be transmitted by a deer species.
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SciScore for 10.1101/2021.01.13.426628: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement Animals, inoculation and sampling All animals were handled in accordance with the Animal Welfare Act Amendments (7 U.S. Code §2131 to §2156) and all study procedures were reviewed and approved by the Institutional Animal Care and Use Committee at the National Animal Disease Center (IACUC approval number ARS-2020-861). Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources At 24 h post-inoculation, cells were fixed with 3.7% formaldehyde for 30 min at room temperature, permeabilized with 0.2% Triton X-100 (in Phosphate … SciScore for 10.1101/2021.01.13.426628: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement Animals, inoculation and sampling All animals were handled in accordance with the Animal Welfare Act Amendments (7 U.S. Code §2131 to §2156) and all study procedures were reviewed and approved by the Institutional Animal Care and Use Committee at the National Animal Disease Center (IACUC approval number ARS-2020-861). Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources At 24 h post-inoculation, cells were fixed with 3.7% formaldehyde for 30 min at room temperature, permeabilized with 0.2% Triton X-100 (in Phosphate Buffered Saline [PBS]) and subjected to an immunofluorescence assay (IFA) using a monoclonal antibody (MAb) antiACE2 (Sigma-Aldrich), and then incubated with a goat anti-rabbit IgG (goat anti-rabbit IgG, Alexa Fluor 488®), and using a monoclonal antibody (MAb) anti-SARS-CoV-2 nucleoprotein (N) (clone B6G11) produced and characterized in Dr. antiACE2suggested: Noneanti-rabbit IgGsuggested: Noneanti-SARS-CoV-2 nucleoprotein (Nsuggested: NoneDiel’s laboratory, and then incubated with a goat anti-mouse IgG secondary antibody (goat anti-mouse IgG, Alexa Fluor® 594), and Nuclear counterstain was performed with DAPI, and visualized under a fluorescence microscope. anti-mouse IgGsuggested: NoneFluorescent beads were coupled with the anti-equine IL-4 antibody, clone 25 (RRID: AB_2737308) as previously described (65). anti-equine IL-4detected: (Dr. Bettina Wagner - Cornell University Cat# IL4 25, RRID:AB_2737308)The assay was detected using a biotinylated mouse anti-goat IgG (H+L) (RRID: AB_2339061, Jackson Immunoresearch Laboratories, West Grove, PA) cross-reactive with deer immunoglobulin. mouse anti-goat IgG H+Ldetected: (Jackson ImmunoResearch Labs Cat# 205-065-108, RRID:AB_2339061)Experimental Models: Cell Lines Sentences Resources The SARS-CoV-2 isolate TGR/NY/20 obtained from a Malayan tiger naturally infected with SARS-CoV-2 and presenting with respiratory disease compatible with SARS-CoV-2 infection (22) was propagated in Vero CCL-81 cells. Vero CCL-81suggested: NoneCell susceptibility and growth curves The susceptibility and kinetics of replication of the SARS-CoV-2 in DL cells was assessed in vitro and compared to virus replication in Vero E6 and Vero E6/TMPRSS2. Vero E6suggested: RRID:CVCL_XD71)Positive samples were subjected to end point titrations by limiting dilution using the Vero E6/TMPRSS2 cells and virus titers were determined using the Spearman and Karber’s method and expressed as TCID50.ml-1. Vero E6/TMPRSS2suggested: NoneFollowing incubation of serum and virus, 50 µl of a cell suspension of Vero cells was added to each well of a 96-well plate and incubated for 48 h at 37 °C with 5% CO2. Verosuggested: NoneResults from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.
Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).
Results from JetFighter: We did not find any issues relating to colormaps.
About SciScore
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