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

Human-to-animal spillover of SARS-CoV-2 virus has occurred in a wide range of animals, but thus far, the establishment of a new natural animal reservoir has not been detected. Here, we detected SARS-CoV-2 virus using rRT-PCR in 129 out of 360 (35.8%) free-ranging white-tailed deer (Odocoileus virginianus) from northeast Ohio (USA) sampled between January-March 2021. Deer in 6 locations were infected with at least 3 lineages of SARS-CoV-2 (B.1.2, B.1.596, B.1.582). The B.1.2 viruses, dominant in Ohio at the time, spilled over multiple times into deer populations in different locations. Deer-to-deer transmission may have occurred in three locations. The establishment of a natural reservoir of SARS-CoV-2 in white-tailed deer could facilitate divergent evolutionary trajectories and future spillback to humans, further complicating long-term COVID-19 control strategies.

One-Sentence Summary: A significant proportion of SARS-CoV-2 infection in free-ranging US white-tailed deer reveals a potential new reservoir.
Main Text: To date, SARS-CoV-2, the virus responsible for coronavirus disease 2019 , has caused over 4 million deaths globally (1). The zoonotic origins of SARS-CoV-2 are not 5 fully resolved (2), leading to active surveillance for susceptible host species and potential new reservoirs. Natural infections of SARS-CoV-2 linked to human exposure have been reported in both domestic animals (e.g. cats, dogs, ferrets) and in wildlife under human care (e.g. several species of big cats, Asian small-clawed otters, western lowland gorillas, and mink) (3). Experimental infections have identified additional animal species susceptible to SARS-CoV-2 10 including hamsters, North American raccoons, striped skunks, white-tailed deer, raccoon dogs, fruit bats, deer mice, domestic European rabbits, bushy-tailed woodrats, tree shrews, and multiple non-human primate species (4)(5)(6)(7)(8)(9)(10)(11)(12)(13). Moreover, several naturally or experimentally infected species were capable of intra-species SARS-CoV-2 transmission (cats, ferrets, fruit bats, hamsters, raccoon dogs, deer mice, white-tailed deer) (6-8, 10, 14-16). Vertical transmission has 15 also been suggested to occur in experimentally infected white-tailed deer (16). An in silico study modeling ACE2 receptor binding residues across host species predicted cetaceans, rodents, primates, and cervids are at highest risk for infection due to predicted binding between the ACE2 receptor and the SARS-CoV-2 spike protein (17). 20 Limited evidence for SARS-CoV-2 infection or exposure (antibodies) in free-ranging animal populations has also begun to emerge. In Utah (USA), free-ranging mink presumed to be escapees from a mink farm tested positive for SARS-CoV-2 antibodies, and a subset tested positive via rRT-PCR for SARS-CoV-2 (18). In Spain, two free-ranging mink also tested positive for SARS-CoV-2 (19). Additionally, antibodies for SARS-CoV-2 have been identified 25 in 152 free-ranging white-tailed deer (seroprevalence 40%) sampled across Michigan, Pennsylvania, Illinois, and New York (USA) (20). The presence of SARS-CoV-2 in free-ranging animals raises critical concerns about the potential establishment of a new reservoir, future spillback to humans and spillover to other species including livestock based on prior studies of experimental transmission of other betacoronaviruses from cervids to cattle (21). 30 White-tailed deer are of particular interest because their ACE2 receptor binding residues are predicted to have high affinity for SARS-CoV-2 (17); they demonstrate experimental susceptibility to and transmission of SARS-CoV-2 (7,16); and, SARS-CoV-2 antibodies have been reported in free-ranging deer (20). In this study, we report the presence of SARS-CoV-2 in 35 129 out of 360 (35.8%) free-ranging white tailed deer (Odocoileus virginianus) from northeast Ohio tested via rRT-PCR between January-March 2021. Genetic sequence data was used to estimate the number of human-to-deer transmission events, characterize the genetic diversity of the virus in deer, and identify phylogenetic clades of deer-only viruses consistent with deer-todeer transmission in certain locations.
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 5, 2021. ; https://doi.org/10.1101/2021.11.04.467308 doi: bioRxiv preprint At least 1 rRT-PCR-positive sample was identified from 17/18 collection dates. Fewer male deer (n=149) were sampled than female deer (n=211), but males (Chi2 = 25.45, p-value < 0.0005) and heavier deer (Wilcoxon-Mann-Whitney p-value = 0.0056) were more likely to test positive for SARS-CoV-2 (Table S3). Other potential predictors including deer age and harvest method (bait site versus opportunistically) were not associated with SARS-CoV-2 detection (Table S3). The 5 highest prevalence estimates of SARS-CoV-2 were observed in four sites that are more urban and surrounded by higher human population densities ( Figure 1).
We obtained 14 whole-genome sequences from six of the nine sites representing seven different collection dates (from 1/26/2021 to 2/25/2021, Table S1). The genetic composition of the deer 10 sequences reflected the dominant lineages that were circulating in humans in Ohio during January-February 2021 ( Figure 2, Table S4

Discussion
Free-ranging white-tailed deer are distributed broadly across urban, suburban, and rural 35 environments in the United States, and can live at densities of greater than 45 deer per square mile in some areas (22). Ohio is home to >600,000 free-ranging white-tailed deer (23) and another 440 commercial deer farms (24). Direct or indirect human-deer interaction occurs in parks and backyards, on livestock and cervid farms, in wildlife hospitals, and through hunting. The prevalence, distribution, and genetic variation of SARS-CoV-2 strains identified in free-40 ranging deer across Ohio raises a series of questions. How is the virus directly or indirectly transmitted from humans to deer? How extensively and efficiently are free-ranging deer transmitting the virus within or between species, in Ohio or other US states? Could viral adaptions occur in deer as has been observed in farmed mink? Could white-tailed deer serve as a reservoir for future spillovers of SARS-CoV-2 to humans or to other susceptible species? 45 The results from this study indicate that there have been multiple independent introductions of SARS-CoV-2 into deer occurring at different times and different locations around northeast . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 5, 2021. ; https://doi.org/10.1101/2021.11.04.467308 doi: bioRxiv preprint Ohio. How these deer acquired SARS-CoV-2 from humans remains unclear. Our results are consistent with at least six human-to-deer transmission events occurring during Ohio's major pandemic wave last winter, which peaked from November 2020 -January 2021. Although it is difficult to date precisely when these human-to-deer transmissions occurred, human-to-deer transmissions may be most likely during the weeks of highest virus circulation in humans. A 5 similar temporal association was observed previously during the 2009 H1N1 influenza pandemic when the vast majority of transmission events from humans to swine and other species occurred during times of peak pandemic activity in humans. While high quality sequence coverage across the entire genome proved challenging for these post-mortem samples, it is likely that the presence of inhibitors within samples and/or sample degradation between collection and testing 10 may have occurred. A comprehensive surveillance program in the future could provide additional data to help resolve remaining questions. Additionally, the recent wave of Delta variant infections in the human population likely represents another opportunity for SARS-CoV-2 to spillover into white-tailed deer. Sequencing of samples from the fall of 2021 will also determine whether the lineages seeded in deer last winter have continued to circulate. 15 It may be unsurprising that deer in urban sites were at higher risk for infection in our study. While it remains unknown how human-to-deer transmission occurs mechanistically, urban settings provide greater opportunities for direct and indirect contact with human-contaminated sources (e.g. trash, backyard feeders, bait stations), as well as contact with waterways that could 20 be contaminated by multiple sources (11,25). Viable SARS-CoV-2 is shed in human stool and has been demonstrated in wastewater (26, 27). Viable SARS-CoV-2 can be isolated from tap water and wastewater under experimental conditions (26). SARS-CoV-2 RNA has been identified in urban runoff in Ohio, which is useful for early detection systems. Virus isolation was not attempted and it is unknown whether SARS-CoV-2 was viable in these samples and 25 capable of transmitting to other species (28). The recent detection of genetically disticnt SARS-CoV-2 virus fragments in New York City wastewater introduces the possibility that SARS-CoV-2 is transmitting cryptically in rodents (29). Contaminated waterways were also suggested as potential sources of infection for the free-ranging mink that tested positive for SARS-CoV-2 in Spain (19). Another question is how the virus transmits mechanistically between deer, whether 30 by aerosols, direct contact, or environmentally. Prospective sequencing of samples from these deer populations will help determine whether the virus transmits long-term or only transiently in deer.
As SARS-CoV-2 continues to evolve it will be important to determine whether particular virus 35 lineages are more associated with infection, transmission, disease, and persistence in deer. Although free-ranging white-tailed deer are highly susceptible to SARS-CoV-2 infection, it is unknown whether this leads to clinical disease. Two previous experimental studies reported subclinical infections in white-tailed deer challenged with SARS-CoV-2 (7,16). The increased rate of infection in males found in this study could reflect differential susceptibility between 40 sexes, but could also be explained by sex-linked differences in behavior that influence disease transmission. Male white-tailed deer also have a higher prevalence of chronic wasting disease and tuberculosis, which has been attributed to larger male home ranges, increased movement and contact with other deer during breeding season (fall/winter), and dynamic male social group composition and size (30). Monitoring SARS-CoV-2 evolution in wildlife populations, including 45 white-tailed deer, mustelids, rodents, and other susceptible species will be critical to future COVID-19 management and mitigation efforts.
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Tables S1 -S5 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 5, 2021. ; https://doi.org/10.1101/2021.11.04.467308 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 5, 2021. ; https://doi.org/10.1101/2021.11.04.467308 doi: bioRxiv preprint Figure 2. Timing of SARS-CoV-2 waves in Ohio, USA. The number of weekly COVID-19 cases in humans in Ohio is presented from April 2020 -September 2021 (y-axis). The proportion of viruses sequenced each week in Ohio for routine surveillance that are assigned to one of five Pango lineages (or "Other") is indicated by shading. Genomic data prior to December 2020 was 5 too sparse to reliably estimate lineage frequency. The shading and placement of each circle indicates the timing and lineage of SARS-CoV-2 viruses found in white-tailed deer at seven time points in this study.
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 5, 2021. ; https://doi.org/10.1101/2021.11.04.467308 doi: bioRxiv preprint  . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 5, 2021. ; https://doi.org/10.1101/2021.11.04.467308 doi: bioRxiv preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 5, 2021. ; https://doi.org/10.1101/2021.11.04.467308 doi: bioRxiv preprint