key: cord-0899865-jpz7q7dc
authors: Marques, A. D.; Sherrill-Mix, S.; Everett, J.; Adhikari, H.; Reddy, S.; Ellis, J. C.; Zeliff, H.; Greening, S. S.; Cannuscio, C. C.; Strelau, K. M.; Collman, R. G.; Kelly, B. J.; Rodino, K. G.; Bushman, F. D.; Gagne, R. B.; Anis, E.
title: Evolutionary Trajectories of SARS-CoV-2 Alpha and Delta Variants in White-Tailed Deer in Pennsylvania
date: 2022-02-19
journal: nan
DOI: 10.1101/2022.02.17.22270679
sha: 98c8b2b54e6f62cd87dcdd6626c606c621844d0d
doc_id: 899865
cord_uid: jpz7q7dc

The SARS-CoV-2 pandemic likely began by spillover from bats to humans; today multiple animal species are known to be susceptible to infection. White-tailed deer, Odocoileus virginianus are infected in the United States at substantial levels, raising concerns about the formation of a new animal reservoir and potential of spill-back of new variants into humans1. Here we characterize SARS CoV-2 in deer from Pennsylvania (PA) sampled during fall and winter 2021. Of 93 nasal swab samples analyzed by RT-qPCR, 18 (19.3%) were positive for SARS-CoV-2. Seven whole-genome sequences were obtained, which were annotated as alpha and delta variants, the first reported observations of these lineages in deer, documenting multiple new jumps from humans to deer. The alpha lineage persisted in deer after its displacement by delta in humans, and deer-derived alpha variants diverged significantly from those in humans, consistent with a distinctive evolutionary trajectory in deer.

The SARS-CoV-2 pandemic likely began by spillover from bats to humans 1-3 ; today multiple animal species are known to be susceptible to infection [4] [5] [6] [7] [8] . Whitetailed deer, Odocoileus virginianus are infected in the United States at substantial levels [9] [10] [11] , raising concerns about the formation of a new animal reservoir and potential of spill-back of new variants into humans 12 . Here we characterize SARS CoV-2 in deer from Pennsylvania (PA) sampled during fall and winter 2021. Of 93 nasal swab samples analyzed by RT-qPCR, 18 (19.3%) were positive for SARS-CoV-2. Seven whole-genome sequences were obtained, which were annotated as alpha and delta variants, the first reported observations of these lineages in deer, documenting multiple new jumps from humans to deer. The alpha lineage persisted in deer after its displacement by delta in humans, and deer-derived alpha variants diverged significantly from those in humans, consistent with a distinctive evolutionary trajectory in deer.

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The copyright holder for this preprint this version posted February 19, 2022. ;  https://doi.org/10.1101/2022.02. 17.22270679 doi: medRxiv preprint Main text SARS-CoV-2 was detected by RT-qPCR in nasal swabs from 18 of 93 wild white-tailed deer sampled in Pennsylvania during fall and winter 2021 (19. 3%; 95% CI: 12.6, 28.6) (described in Tables S1 and S2). Positive deer were identified in 10 of the 31 Pennsylvania counties surveyed from 10/2/2021 to 12/27/2021 (Figure 1 ). There was no difference in the proportion of positives between volunteer-collected samples and those collected by veterinary technicians (volunteer 11/66 and technician 7/27, p= 0.46). However, deer sampled as road-killed were significantly more likely to be positive than all other sample types (hunter harvested 11/66, road-killed 6/13 and other 1/14, p=0.002).

The prevalence estimate was higher in female deer (35%; 95% CI: 22.13,50.5) than male deer (7.5%; 95% CI: 2.97, 17.9) . No information was available on possible symptoms or disease for the animals sampled. Viral whole-genome sequencing was attempted on the eight samples with the highest viral RNA levels (lowest cycle of threshold values in the RT-qPCR), and high-quality genome sequences were recovered from seven (Table S3 ). To confirm sample provenance, the non-viral sequence reads were analyzed for the deer samples and for human samples that were sequenced in parallel. All deer samples yielded reads mapping to the deer genome, and few or none mapping to the human genome. In contrast, reads from human samples overwhelmingly mapped to the human genome (Figure 2a ), confirming the host species origin of our samples in deer.

. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) Deer genomes annotated as alpha and delta variants, the first reported identification of these lineages in wild white-tailed deer. Previously the alpha variant was shown to be able to infect white-tailed deer that were experimentally inoculated 11 . Figure 2b shows a phylogenetic tree for alpha, and Figure 2c a phylogenetic tree for delta, in each case comparing Pennsylvania (PA) deer to contemporaneous human-derived sequences from across the same Mid-Atlantic region.

The two alpha sequences sampled from deer in adjoining counties in northeastern PA differed by 45 substitutions and were widely separated on the phylogenetic tree. A parsimonious explanation is thus that the two alpha lineages were introduced independently into deer and then diversified during transmission within deer. In addition, these deer-derived alpha genomes appear particularly divergent in contrast to the human alpha genomes sequenced previously in the same region. Only one out of 1240 human genomes were as divergent or more divergent from the inferred alpha common ancestor as were the deer sequence, giving a probability of randomly drawing two such divergent lineages as (1/1240) 2 =6.5X10 -7 .

For the delta variants ( Figure 2C ), two deer genomes were assigned to the same AY.103 clade and differed only by 7 substitutions, making the discrimination between a single or independent introductions for these sequences difficult. Two other deer SARS-CoV-2 genomes differed by over 21 substitutions from the AY.103 sequences and were assigned to the delta AY.88 lineage, which is an uncommon lineage for the PA area with only 0.14% of . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted February 19, 2022. ; https://doi.org/10.1101/2022.02.17.22270679 doi: medRxiv preprint human-derived PA delta genomes being AY.88. The two AY.88 specimens from deer were identical. These likely represent a single introduction into deer given the scarcity of this lineage, the genetic similarity between samples, and the fact that the two isolates were from adjoining counties in northeastern PA and were sampled within a period of 2 days. The last delta sample was assigned to the AY.5 lineage. This genome clustered with the AY.88 specimens and differed by no more than 13 SNPs, making it unclear if it represents an independent introduction. Thus, a simple model would support identification here of at least four independent transmissions of SARS-CoV-2 from humans into deer, two of alpha and two of delta. It is also possible, though less likely, that SARS-CoV-2 entered deer fewer times and subsequently diverged, though this would require independent acquisition in deer of many mutations shared with contemporaneous human lineages.

The alpha and delta SARS-CoV-2 sequences from deer determined here were compared to deer SARS-CoV-2 sequences reported previously from other viral lineages to investigate possible deer-specific sequence polymorphisms. 108 deer genome sequences were downloaded from GISAID (Table S6) This revealed several substitutions that were highly enriched or invariant in deer isolates, but rare or absent in human isolates (Table S4 ). These include three silent mutations in ORF1ab, c7303t, c9430t, and c20259t. Mutation c7303t was found in 86% of these PA deer and 29% of previously published deer, whereas it . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted February 19, 2022. ; https://doi.org/10.1101/2022.02.17.22270679 doi: medRxiv preprint was found in 0.04% of Mid-Atlantic human subjects and 0.09% of global genomes reported by NextStrain. Mutation c9430t was found in 43% of these PA deer and 56% of previously published deer, whereas it was found in 0.35% of Mid-Atlantic human subjects and 0.46% of global genomes reported by NextStrain. Mutation c20259t was found in 43% of these PA deer and 19% of previously published deer, whereas it was absent in Mid-Atlantic human subjects and present in 0.12% of global genomes reported by NextStrain (data from 1/26/2022). The enrichment of these mutations suggests possible functional interaction with deer-specific factors, which could influence RNA synthesis, RNA folding, or protein binding.

Samples from deer were compared longitudinally to samples from the mid-Atlantic region recovered from infected human subjects ( Figure 2D ). The alpha samples from deer were recovered in November of 2021, months after the alpha variant was displaced by delta in humans. In contrast, the delta isolates from deer were contemporaneous with the delta wave in humans. This suggests possible persistence of the alpha wave selectively in deer. Consistent with this, the deer alpha lineages appear relatively more diverged from human alpha isolates than do the deer delta lineages from human delta isolates (p=0.00056 for t-test comparing branch lengths of deer alpha versus delta isolates).

In summary, a survey of SARS-CoV-2 in 93 deer in Pennsylvania over the fall-winter of 2021 showed 19% of the deer sampled to be positive. Prior surveys carried out over the fall and winter of 2020 showed similarly high point prevalence of infection in Iowa (33.2%) 9 and Ohio (35.8%) 10 . We report the first . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 

Samples were collected from hunter harvested deer by state employee volunteers as well as by veterinary technicians responding to calls about sick or injured deer that needed to be euthanized (e.g., deer with neurologic symptoms, hit by cars). Nasal swabs were taken within hours of death, placed in phosphate buffered saline (PBS) and stored in commercial refrigerators in field offices.

Samples were shipped to the University of Pennsylvania within one week of . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 

Sequence data were processed as previously described 18 . BWA aligner tool (v0.7.17) was used to align subject sequences to the Wuhan reference sequence (NC_045512.2) 19 . Samtools package (v1.10) was used to filter alignments 20 . Variants were called using Pangolin lineage software (3.1.17 with the PangoLEARN 2021-12-06 release) 21, 22 .

The proportion of host sequences was inferred using a sampling of raw reads from all samples on any sequencing batch performed with deer specimen.

. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted February 19, 2022 Human samples were sequenced as described for deer samples.

Deer and human sequences are available at GENBANK and GISAID (Table S3 and Table S7 ). NextClade was used to align sequences to the Wuhan reference 26 . IQ-TREE (v1.6.12) was used to infer a tree using maximumlikelihood methods using 1,000 bootstrap replicates 27 . Visualization of the inferred tree was performed using FigTree (v.1.4.4) 28 .

Sequence accession numbers for deer-derived SARS-CoV-2 genomes can be found in Table S3 (OM570187-OM570193). Accession numbers for human SARS-CoV-2 genomes can be found in Table S7 . Computer code is available at https://doi.org/10.5281/zenodo.4046252.

We are grateful to hunters and wildlife personnel who provided specimens, and to Laurie Zimmerman for artwork and help with manuscript preparation. We acknowledge help from staff of the Philadelphia Department of Public Health. This work was supported in part by the Penn University Research Foundation. SG is supported by the Robert J. Kleberg, Jr. and Helen C. Kleberg

Foundation. Funding was provided by a contract award from the Centers for . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 

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. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 

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(Delta) AY.25 (Delta) AY.44 (Delta) AY.103 (Delta) AY.119 (Delta) Other AY.# (Delta) B

1 (Gamma)

Analysis of SARS-CoV-2 whole genome sequences from white-tailed deer in PA and comparison to local human-derived sequences. A) Phylogeny of contemporaneous human alpha lineage sequences from PA, collected from 1/6/2021 to 11/16/2021, with deer sequences marked (1240 human and 2 deer sequences). B) Phylogeny of human delta lineage sequences from PA, collected from 10/14/2021 to 11/28/2021, with deer sequences marked (440 human and 5 deer sequences). C) Longitudinal comparison of deer variants and contemporary human variants. The bar plots show the progression of SARS-CoV-2 variants detected by genome sequencing in humans in eastern PA from 1/31/2021 to1/3/2021. The variants are color-coded according to the key to the right. The variants from white-tailed deer sequences are shown at the top of the figure