SARS-CoV-2 infection in free-ranging white-tailed deer

High infection rate of SARS-CoV-2

We sampled 360 free-ranging white-tailed deer across from nine locations (Figure 1A) in northeast Ohio (USA) between January-March 2021. Across all sites, SARS-CoV-2 was detected by rRT-PCR in 35.8% of nasal swabs from white-tailed deer (129/360, 95% CI 30.9% - 41.0%, Supplementary Table 1). Each site was sampled 1-3 times during the study period, for a total of 18 collection dates (Extended Data Table 1). At least 1 rRT-PCR-positive sample was identified from 17/18 collection dates with the majority of positive sample CT values less than 30 (Figure 1B). Prevalence estimates varied from 13.5% to 70% across the nine sites (Figure 1C). The highest prevalence estimates of SARS-CoV-2 were observed in four sites (2, 5, 7, and 9) situated in the northern section of the sampled area, adjacent to urban areas with higher human population densities (Figures 1A and 1C). Male deer (Chi2 = 25.45, p-value < 0.0005) and heavier deer (Wilcoxon-Mann-Whitney p-value = 0.0056) were significantly more likely to test positive for SARS-CoV-2 (Extended Data Table 2).

Three SARS-CoV-2 lineages identified

We sequenced the complete genome of 14 viruses collected from six of the nine sites, collected at seven time points spanning from 1/26/2021 to 2/25/2021 (Supplementary Table 1). The deer samples were collected approximately six weeks after the peak of Ohio’s 2020-2021 winter epidemic of SARS-CoV-2 in humans, which was dominated by B.1.2 viruses (>50% of human viruses, Figure 2A, Extended Data Table 3). B.1.2 viruses genetically similar to human viruses were detected in deer at four sites: 4, 7, 8, and 9 (Figure 2B). B.1.596, a minor lineage (~11% of human viruses), was identified in seven deer samples at site 1, spanning two collection times (2/2/2021 and 2/25/2021). A rarer lineage, B.1.582 (~1% of human samples), was identified in two deer samples at site 6. No sequences belonging to the alpha (B.1.1.7) or delta (B.1.617.2) lineages were identified in the deer samples, as these variants became widespread in the human population only after February 2021.

Six human-to-deer transmission events

Although B.1.2 was identified in deer at four sites, our phylogenetic analysis found no evidence of B.1.2 viruses transmitting in deer across sites. Rather, each site experienced a separate human-to-deer transmission event of a slightly genetically different B.1.2 virus positioned in a different section of the B.1.2 clade on the phylogenetic tree (Figure 2C). In total, six human-to-deer transmission events were observed: B.1.582 (site 6), B.1.596 (site 1), and B.1.2 (sites 4, 7, 8, and 9). There is a degree of uncertainty about the timing of each viral entry into the deer population, owing to long branch lengths that separate the deer viruses from the ancestral human viruses on the phylogenetic tree. To estimate the timing and location of human-to-deer transmission for the larger cluster of B.1.596 deer viruses, a time-scale Bayesian MCC tree was inferred using a phylogeographic approach (Figure 3A). The MCC tree is consistent with human-to-deer transmission occurring geographically in Ohio (posterior probability = 0.98) and temporally during the winter epidemic when viral loads in humans (and the environment) would be peaking. The MCC tree indicates that 1.596 viruses were introduced into humans in Ohio multiple times from other US states during the fall of 2020 and winter of 2020-2021, forming three co-circulating Ohio clades in humans. The largest Ohio clade then seeded the deer outbreak. Deer viruses in this cluster were collected on February 2 and February 25, 2021, and the MCC tree estimates that human-to-deer transmission occurred several weeks, or possibly months, earlier (Figure 3A). Gaps in sampling in both humans and deer make it difficult to narrow this time estimate further.

Deer-to-deer transmission and evolution

Viable SARS-CoV-2 virus was recovered from two of the deer samples (Extended Data Table 4). Deer-to-deer transmission may have occurred within the three study sites where more than one deer sample was sequenced: site 1 (B.1.596), site 6 (B.1.582), and site 9 (B.1.2). Sites 6 and 9 only had two viruses collected, both from the same sampling date (Figure 2B), limiting what can be inferred about transmission. Instead, our analysis of deer-to-deer transmission and evolution focused on the larger deer cluster of 7 B.1.596 viruses observed in site 1 that spans two collection dates (Figure 3A). A number of uncommon amino acid substitutions were observed in all 7 deer viruses in this clade (i.e. all Site 1 sequences) that were not observed in the most closely related human viruses. Five clade-defining mutations were observed in ORF1ab: a five-residue deletion in nsp1 (Δ82-86), nsp2_T434I, nsp2_P597L, nsp12_A382V, and nsp13_M474I (numbering in Figure 3B refers to ORF1a and ORF1b). A clade-defining deletion (Δ141-144) also was observed in the S1 domain of the spike protein in the 7 deer viruses. All six clade-defining mutations observed in these deer are uncommon among human viruses (< 0.05% frequency globally)²⁶.

Uncommon amino acid changes in the spike protein S1 domain also were observed in singleton deer viruses. A B.1.2 virus from site 7 (hCoV-19/deer/USA/OH-OSU-0212/2021) has a substitution in the spike protein’s N-terminal domain (H245Y). A single B.1.596 virus from site 1 (hCoV-19/deer/USA/OH-OSU-0340/2021) has a substitution in the spike protein receptor binding motif (E484D, Figure 3C). Both mutations are relatively rare in humans, found in < 0.5% of all SARS-CoV-2 viruses sequenced globally. In experimental studies, viruses with the E484D substitution are less sensitive to neutralization by convalescent sera²⁷. The E484D substitution has only been detected in 201 SARS-CoV-2 sequences from humans globally, 71 of which were in the United States, but none of the B.1.596 viruses in humans that were most closely related to the deer virus have this mutation. It is therefore impossible to differentiate if the E484D mutation arose in an unsampled human virus and was transmitted to deer or arose de novo in deer. Additionally, due to low availability of sequence data from deer it is not possible to determine if these spike mutations have transmitted to other deer.

Discussion

Our finding that white-tailed deer are frequently infected with SARS-CoV-2 viruses raises profound questions about the future trajectory of SARS-CoV-2. The potential establishment of a new reservoir of SARS-CoV-2 viruses in white-tailed deer could open new pathways for evolution, transmission to other wildlife species, and potential spillback of novel variants to humans that our immune system has not seen before. SARS-CoV-2 viruses have a high capacity for adaptive evolution when infection rates are high in a community or population. It is therefore alarming that over one-third of deer in our study were positive by PCR, suggesting an active or recent infection during last winter’s major wave. A number of mutations were observed in white-tailed deer that are very low frequency in humans, including a mutation in the receptor binding motif. Such mutations could potentially be amplified in a new reservoir host with high infection rates and different constraints on evolution. There is an urgent need to expand monitoring of SARS-CoV-2 viruses in potential wildlife hosts to document the breadth of the problem in white-tailed deer nationally, understand the ecology of transmission, and track evolutionary trajectories going forward, including in other potential host species.

The impact of urban sprawl on disease ecology is well documented for Lyme disease and other multihost zoonotic systems that include white-tailed deer, rodents, and other species that have become ubiquitous and well adapted in expanding US urban and semi-urban environments, creating opportunities for pathogen exchange. Approximately 30 million free-ranging white-tailed deer are distributed broadly across urban, suburban, and rural environments in the United States, and can live at densities of greater than 45 deer per square mile in some areas²⁸. Ohio is home to >700,000 free-ranging white-tailed deer²⁹ and another 440 commercial deer farms³⁰. Estimates of deer density in and around our sites range from approximately 8/km² to upwards of 30/km². There are no deer farms in the study area and public feeding of deer is prohibited. There is ample forage available around urban and suburban residences in gardens and plantings, drawing deer into close proximity with humans and their companion animals. Therefore, is unsurprising that deer in urban sites were at higher risk for infection in our study. Urban settings provide ample opportunities for deer to have direct and indirect contact with human-contaminated sources (e.g. trash, backyard feeders, bait stations, wildlife hospitals) that could serve as a pathway for viral spillover into wildlife. Additionally, urban and suburban environments include waterways that could be contaminated by multiple sources^18,31. Viable SARS-CoV-2 is shed in human stool. SARS-CoV-2 RNA and has also been detected in wastewater^32,33 and urban runoff ³⁴; although, infectivity of SARS-CoV-2 from these sources is undetermined. The recent detection of genetically distinct SARS-CoV-2 virus fragments in New York City wastewater introduces an intriguing hypothesis that SARS-CoV-2 could be transmitting cryptically in rodents³⁵. However, although sensitive techniques for detecting viral RNA in wastewater have vastly improved, providing a potentially useful tool for early detection of outbreaks, isolating or whole-genome sequencing viruses to characterize their genetic diversity remains challenging.

A major outstanding question is how the virus transmits between deer. Deer are social animals that live in small herds and frequently touch noses. It is unclear if baiting the deer prior to harvest contributed the increased frequency of SARS-CoV-2 in this study but concentrating deer with bait could have potentially facilitated pathogen transmission through a population. However, baiting is regularly used in deer management programs and the practice is commonly employed by deer hunters, which makes understanding the effect of baiting on SARS-CoV-2 transmission in free-ranging deer paramount for future studies. The increased rate of infection in males in this study could reflect sex-linked differences in behavior that increase disease transmission. The higher prevalence of chronic wasting disease and tuberculosis in male white-tailed deer is 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 size³⁶. Deer may experience high levels of viremia and shedding that may be conducive to environmental or aerosol transmission. Another question is whether deer experience clinical disease and whether clinical signs such as sneezing or nasal discharge increase the risk of transmission. Two previous experimental studies reported only subclinical infections in white-tailed deer challenged with SARS-CoV-2, but these are very small sample sizes^14,23.

While extensive measures were taken to prevent cross-contamination during sample collection and testing, the nature field work makes it impossible to completely exclude the possibility. Cross-contamination during sample collection would not invalidate the detection of SARS-CoV-2 in white-tailed deer but would artificially inflate prevalence estimates. However, the extent of genomic diversity among the sequences recovered on a single sampling day (e.g. site 1, sampling 2) indicates cross-contamination during sample collection was likely minimal.

Although our study was limited to northeastern Ohio, these findings have implications for other US states, including Michigan, Pennsylvania, New York, and Illinois where high rates of exposure to SARS-CoV-2 in white-tailed deer was reported earlier based on serology²⁴. Serological assays are notoriously difficult to interpret and many animal health experts hoped the results were an artifact. Moreover, the detection of antibodies does not prove active infection. Unfortunately, our study suggests that the antibodies observed in deer in other states may have arisen from active infection, and this is just the tip of the iceberg.

Moreover, it is worth noting that white-tailed deer are a relatively convenient surveillance target because of their abundance and accessibility. The detection of SARS-CoV-2 in free-ranging white-tailed deer naturally raises the question whether less accessible species are also being infected through viral spillover from humans, which calls for broader surveillance efforts.

Methods:

Extended Data

Supplementary Material

Data Availability:

For all 14 SARS-CoV-2 viruses from white-tailed deer sequenced in this study, sequencing data are available on NCBI Sequence Read Archive (SRA) and assembled genome sequences are on GISAID. SRA and GISAID accession numbers are provided in Extended Data Table 4.