key: cord-0760898-fhcipjzu
authors: Kuchipudi, Suresh V.; Surendran-Nair, Meera; Ruden, Rachel M.; Yon, Michelle; Nissly, Ruth H.; Nelli, Rahul K.; Li, Lingling; Jayarao, Bhushan M.; Vandegrift, Kurt J.; Maranas, Costas D.; Levine, Nicole; Willgert, Katriina; Conlan, Andrew J. K.; Olsen, Randall J.; Davis, James J.; Musser, James M.; Hudson, Peter J.; Kapur, Vivek
title: Multiple spillovers and onward transmission of SARS-CoV-2 in free-living and captive white-tailed deer
date: 2021-11-06
journal: bioRxiv
DOI: 10.1101/2021.10.31.466677
sha: 282c2b9417722fbea9232bc3d13af8b3b1cd7ec7
doc_id: 760898
cord_uid: fhcipjzu

Many animal species are susceptible to SARS-CoV-2 and could potentially act as reservoirs, yet transmission of the virus in non-human free-living animals has not been documented. White-tailed deer (Odocoileus virginianus), the predominant cervid in North America, are susceptible to SARS-CoV-2 infection, and experimentally infected fawns can transmit the virus. To test the hypothesis that SARS-CoV-2 may be circulating in deer, we tested 283 retropharyngeal lymph node (RPLN) samples collected from 151 free-living and 132 captive deer in Iowa from April 2020 through December of 2020 for the presence of SARS-CoV-2 RNA. Ninety-four of the 283 deer (33.2%; 95% CI: 28, 38.9) samples were positive for SARS-CoV-2 RNA as assessed by RT-PCR. Notably, between November 23, 2020 and January 10, 2021, 80 of 97 (82.5%; 95% CI 73.7, 88.8) RPLN samples had detectable SARS-CoV-2 RNA by RT-PCR. Whole genome sequencing of the 94 positive RPLN samples identified 12 SARS-CoV-2 lineages, with B.1.2 (n = 51; 54.5%), and B.1.311 (n = 19; 20%) accounting for ~75% of all samples. The geographic distribution and nesting of clusters of deer and human lineages strongly suggest multiple zooanthroponotic spillover events and deer-to-deer transmission. The discovery of sylvatic and enzootic SARS-CoV-2 transmission in deer has important implications for the ecology and long-term persistence, as well as the potential for spillover to other animals and spillback into humans. These findings highlight an urgent need for a robust and proactive “One Health” approach to obtaining a better understanding of the ecology and evolution of SARS-CoV-2. One-Sentence Summary SARS-CoV-2 was detected in one-third of sampled white-tailed deer in Iowa between September 2020 and January of 2021 that likely resulted from multiple human-to-deer spillover and deer-to-deer transmission events.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of coronavirus disease 2019 in humans, is a novel coronavirus in the genus Betacoronavirus (subgenus Sarbecovirus) (1). SARS-CoV-2 was first identified in Wuhan, China, toward the end of 2019 (2) , and has caused a pandemic with 247 million COVID-19 cases and over 5 million deaths globally as of November 1 st , 2021 (3) . The virus continues to evolve, with a growing concern for the emergence of new variants. SARS-CoV-2 uses the host angiotensin-converting enzyme 2 (ACE-2) receptor to enter cells (4) . ACE-2 receptors are well conserved across vertebrate species including humans (5) , and computational analyses predict high binding affinities of SARS-CoV-2 to the ACE-2 receptor in multiple animal species, indicating potential susceptibility to infection (5) . Included amongst these are three species of cervids: the Père David's deer (Elaphurus davidianus), reindeer (Rangifer tarandus), and white-tailed deer (Odocoileus virginianus) (6) .

The widespread and global dissemination of SARS-CoV-2 among humans provides opportunities for spillovers into non-human hosts (7) . Indeed, SARS-CoV-2 infections have been documented in dogs, cats, zoo animals (e.g. tigers and lions) and farmed mink (8, 9) . In principle, SARS-CoV-2 infection of a non-human animal host might result in the establishment of a reservoir that can further drive the emergence of novel variants with potential for spillback to humans. This type of transmission cycle has been described among workers on mink farms (10) . However, widespread SARS-CoV-2 transmission in a free-living animal species has not yet been documented.

Our study was prompted by a recent report that 40% of free-living white-tailed deer in the USA had antibodies against SARS-CoV-2 (11) . More recent studies have provided evidence of SARS-CoV-2 transmission among experimentally infected deer under controlled conditions (6) . To test the hypothesis that infection and subsequent transmission of SARS-CoV-2 of deer occurs in nature, we assayed 283 retropharyngeal lymph node (RPLN) samples collected from free-living and captive deer in Iowa from April 2020 through January 2021. We discovered that one-third of the deer sampled over the course of the study had SARS-CoV-2 nucleic acid in their RPLN. We then sequenced the SARS-CoV-2 genomes present in all positive samples and found that the genomes represented multiple lineages that corresponded to viral genotypes circulating contemporaneously in humans. In the aggregate, our results are consistent with a model of multiple

We next explored regional differences in observed SARS-CoV-2 positivity among deer at the county level across the State. Figure 2A shows the widespread distribution of positive samples recovered from deer throughout Iowa and illustrates a strong temporal trend in SARS-CoV-2 positivity as the year progressed. The study identified 10 counties with at least one positive sample (Table 1 ). The largest number of RPLN samples represented in the collection were from a single game preserve (Preserve 2; Fig 2B) in Southeastern Iowa. Overall, 23 of the 112 deer RPLN samples from this preserve were found to be positive for SARS-CoV-2 RNA, with the first positive in September and the second in October 2020, and 11 of 38 deer sampled in November and all 10 deer sampled in December 2020, suggesting a rapidly increasing herd-level prevalence.

Seven counties had at least 10 samples collected, with all 11 specimens from Allamakee county being found to be SARS-CoV-2 positive, as were 21 of the 28 samples collected from Appanoose county (Table 1; Supplementary Table 1 ). In contrast, none of the 9 samples collected from Black

Hawk county were positive, nor were the 6 RPLN samples from Henry county. While the exact reasons for this heterogeneity in PCR positive response rates are unknown, the timing of collection in relation to the SARS-CoV-2 spread in deer may play a role. For instance, the samples from Henry county were collected during April and May of 2020 during the early of the pandemic and well before the first positive sample was identified in deer. Similarly, all 9 RPLN samples tested from Black Hawk county were collected prior to the mid-November peak of reported SARS-CoV-2 cases in humans in Iowa. Together, these results suggest the widespread presence of SARS-CoV-2 RNA in deer across the State of Iowa, with strong evidence of temporal clustering.

To begin to understand the genomic diversity of SARS-CoV-2 associated with free-living and captive deer, we characterized the complete SARS-CoV-2 genomes from all 94 deer RPLN positive for the presence of viral RNA. A high-level of sequencing coverage was obtained, and Pangolin version 3.1.11 (https://github.com/cov-lineages/pangoLEARN, last accessed October 27, 2021) was used to identify SARS-CoV-2 lineages using previously described genome sequence analysis pipelines (13) (14) (15) . Next, we used an automated vSNP pipeline (https://github.com/USDA-VS/vSNP) to identify SNPs and construct phylogenetic trees in the context of 84 additional publicly available animal origin SARS-CoV-2 isolates as well as from 372 SARS-CoV-2 isolates identified from humans in Iowa during this same period (Supplementary Table 2 Finally, to better visualize phylogenetic relationships amongst circulating SARS-CoV-2 originating in free-living and captive deer, we generated an SNP-based maximum likelihood tree including available human and animal lineage isolates (Fig. 3 ). As evident from the branching patterns of the phylogram, the results highlight the presence of multiple independent but closely related SARS-CoV-2 lineages circulating amongst deer in Iowa, as well as provide strong evidence for transmission within deer as many of the genomes from individual deer shared complete genomic identity (no SNPs) or differed by between 1 and 5 SNPs. The results also highlight several branches with shared human and deer origin SARS-CoV-2 isolates circulating in Iowa that are related to but distinct from isolates previously identified from outbreaks from animals such as farmed mink or otters or other domesticated animal species. Hence, taken together, the results provide strong evidence of multiple spillover events of SARS-CoV-2 and the subsequent circulation of these strains within free-living and captive deer.

Most viruses causing disease in humans have originated in animals and many are capable of transmitting between multiple host species (16, 17) . The ability to infect a range of host species is a risk factor for disease emergence (18, 19) . Despite this knowledge, reservoir host(s) are rarely identified and studied. Indeed, the wild animal reservoir(s) of SARS-1, SARS-CoV-2 and MERS-CoV are still not known. There have been numerous cases of isolated human-to-animal transmission of SARS-CoV-2 involving companion, farmed, and zoo animals since the COVID-19 pandemic began (8, 9, 20, 21) . Our study is the first to provide evidence of widespread dissemination of SARS-CoV-2 into any free-living species, in this instance, the white-tailed deer.

While the precise route(s) of transmission of SARS-Cov-2 from humans to deer are unknown, there are several ways in which deer may be exposed to the virus from humans, including through feeding in backyards or even when a susceptible deer may come in contact with potentially infectious material saliva, urine, etc.) from an infected human in forested areas or exurban environments. Deer may also become exposed to SARS-CoV-2 through contact with wastewater discharges, infected fomites, or other infected animals. Regardless of the route of transmission from humans, our results suggest that deer have the potential to emerge as a major reservoir host for SARS-CoV-2, a finding that has important implications for the future trajectory of the Predicting how the utilization of a new host species by a virus can affect virulence in the primary host is not simple. In theory, pathogen evolution to an optimum in a single-host system is determined by a trade off with transmission, but this becomes more complicated in multiple-host systems (28). With the infection spreading so quickly through the deer population, as seen in our study, this could potentially result in fade out with insufficient susceptible deer recruits to sustain the infection within the deer population alone. Alternatively, with sizeable annual birth cohorts or invasion into areas where deer have not previously been infected, the virus may continue to spread among susceptible deer or circulate with the deer population. However, even while the dynamics in these multi-host systems can be complex, they often result in more stable dynamics with multiple reservoir hosts. The pathogens that utilize many hosts can be at a selective advantage since they are not lost during periods soon after the fade out.

Finally, the white-tailed deer is the most abundant wild cervid species in the United States, with an estimated 25 million individuals. Deer hunting is the most popular form of hunting in the United

States, contributing over $20 billion to the US GDP and supporting more than 300,000 jobs in 2016 (29). Given the social relevance and economic importance of deer to the US economy, even though experimental evidence suggests that SARS-CoV-2 infected deer remain largely asymptomatic, the clinical outcomes and health implications of SARS-CoV-2 infection in freeliving deer are unknown, and warrant further investigation. For these reasons, the discovery of sylvatic and enzootic transmission in a substantial fraction of free-living deer has important implications for the natural ecology and long-term persistence of the SARS-CoV-2, including through spillover to other free-living or captive animals and potential for spillback to humans.

The study has several limitations: The RPLN samples tested were from only one State in the USA, and the sampling was not uniform within the State. However, while the generalizability of our findings remains to be tested, we see no reason why this scenario has also not already played out in other regions with large deer populations with opportunities for contact with humans. Another limitation of our study was that RPLN samples tested were all from 2020 and early 2021, representing the early part of the pandemic before the global dissemination of the highly successful Alpha and Delta variants. Hence, surveillance efforts with robust longitudinal sampling approaches are urgently needed to determine whether deer will become long-term reservoirs for SARS-CoV-2 and potentially assume a role as generators of novel variant viruses that may repeatedly re-emerge in humans or spillover to other animal hosts.

To help predict or prevent the emergence of the next pandemic and control infectious diseases with pandemic and panzootic potential, a better understanding of the human-animal molecular and ecological interface and its relevance to infection transmission dynamics is essential (9) . Thus, we call for an urgent need to implement a more proactive and robust "One Health" approach to better understand the ecology and evolution of SARS-CoV-2 in deer and other free-living species.

The Iowa Department of Natural Resources (DNR) routinely collects medial retropharyngeal lymph nodes (RPLNs) from white-tailed deer across Iowa for its statewide Chronic Wasting Disease (CWD) surveillance program. Tissue samples were collected by trained field staff. Paired

RPLNs were then removed and placed into separate Whirl-Paks with corresponding sample identification numbers and frozen at -20°F in a standard chest or standing freezer. A total of 283 RPLN samples collected between April 2020 to January 2021 were studied (Supplementary Table   1 ). An additional 60 RPLN archived samples from the 2019 deer hunting season were included as process negative control samples (Supplementary Table 1 ).

RPLN tissues were processed by adding 3ml UTM (Copan) to a whirl-pak bag containing the tissue and placing the bag in the stomacher on a high setting for 120 seconds. Liquid volume was recovered and centrifuged at 3,000 rpm for 5 minutes to pellet cellular debris. 400 μL of the RPLN tissue homogenate supernatant was used for viral RNA extraction with a KingFisher Flex machine (ThermoFisher Scientific) with the MagMAX Viral/Pathogen extraction kit (ThermoFisher Scientific) following the manufacturer's instructions.

The presence of SARS-CoV-2 nucleic acid was assessed by a real-time reverse transcriptionpolymerase chain reaction (RT-PCR) assay using the OPTI Medical SARS-CoV-2 RT-PCR kit following the manufacturer's instructions on an ABI 7500 Fast instrument (ThermoFisher Scientific). The OPTI Medical SARS-CoV-2 RT-PCR assay detects two different targets in the gene encoding viral nucleocapsid (N) protein coding region (12, 30) . The assay is highly sensitive with a limit of detection of 0.36 copies/µl. The internal control RNase P (RP) was used to rule out human contamination. We generated a standard curve using SARS-CoV-2 RNA with a known copy number. Using the standard curve, viral RNA copies per milliliter of tissue homogenate were calculated.

To ensure assay specificity, a subset of 25 positive and 25 negative samples were additionally tested with the TaqPath kit (ThermoFisher Scientific) targeting the SARS-CoV-2 ORF1ab, N gene, and S gene (30, 31) . The results were concordant with both assays. Further, to ensure samples were not inadvertently contaminated with human origin tissue or fluids during harvesting or

processing, all samples were tested and found negative for the presence of human RNaseP. As a final check of assay specificity, none of the 60 RPLN samples collected in 2019 prior to the first reported case in humans in the United States were found positive for the presence of SARS-CoV-2 RNA.

Total RNAs extracted from RPLN samples was used for sequencing the whole genomes of SARS-CoV-2 as previously described (13) (14) (15) 

Viral genomes were assembled with the BV-BRC SARS-Cov2 assembly service (The Bacterial 

Authors declare that they have no competing interests.

All SARS-CoV-2 consensus genomes are deposited in GISAID and raw reads submitted to NCBI's Table 2 ). The sequences were screened for quality thresholds, and SNP positions called against the SARS-CoV-2 reference were determined together with SNP alignments and used to assemble a maximum-likelihood phylogenetic trees using RAxML. The results show several genetically distinct clusters of animal and human SARS-CoV-2 lineages circulating within the Iowa deer herd, suggesting multiple likely spillover events from humans to deer. Several branches with shared human and deer origin SARS-CoV-2 isolates circulating in Iowa were observed. The sequences from deer were genetically distinct from isolates from previous outbreaks in farmed mink and otters but showed close clustering with SASRS-CoV-2 genomes recovered humans in Iowa. 

As the positivity proportion among the collected samples increased over the months of collection depicted on X axis, the viral copy numbers (y-axis) increased in a range of 268 to 5.4 x 10 8 copies/ml with a median of 106,000 viral copies/ml. 

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We thank Abhinay Gontu, Shubhada Chothe, Padmaja Jakka and Abirami Ravichnadran from