Widespread exposure to SARS-CoV-2 in wildlife communities

Pervasive SARS-CoV-2 infections in humans have led to multiple transmission events to animals. While SARS-CoV-2 has a potential broad wildlife host range, most documented infections have been in captive animals and a single wildlife species, the white-tailed deer. The full extent of SARS-CoV-2 exposure among wildlife communities and the factors that influence wildlife transmission risk remain unknown. We sampled 23 species of wildlife for SARS-CoV-2 and examined the effects of urbanization and human use on seropositivity. Here, we document positive detections of SARS-CoV-2 RNA in six species, including the deer mouse, Virginia opossum, raccoon, groundhog, Eastern cottontail, and Eastern red bat between May 2022–September 2023 across Virginia and Washington, D.C., USA. In addition, we found that sites with high human activity had three times higher seroprevalence than low human-use areas. We obtained SARS-CoV-2 genomic sequences from nine individuals of six species which were assigned to seven Pango lineages of the Omicron variant. The close match to variants circulating in humans at the time suggests at least seven recent human-to-animal transmission events. Our data support that exposure to SARS-CoV-2 has been widespread in wildlife communities and suggests that areas with high human activity may serve as points of contact for cross-species transmission.

about two miles down the road.However, the lands owned by the research station, where we trapped, were off-limits to public use.Pandapas Pond (PP) is located in Montgomery county and is a high use area for outdoor recreation including trail running, hiking, mountain biking, and fishing.We placed traps along a forest service road.The area is dominated by trees and leaf debris on the ground.Caldwell fields (CF) is located also in Montgomery county.The area is lightly used for general outdoor recreation including hunting.2. Comparative analysis of SARS-CoV-2 sequences from wild animals isolated in southwest Virginia.Consensus sequences for SARS-CoV-2 isolated from wild animal specimen were analyzed for sequence coverage and percent identity compared to the SARS-CoV-2 reference genome (NC_045512), Pango lineage, closely related SARS-CoV-2 sequence isolated from a human, and unique amino acid substitutions, which are not found in their closely related sequences.See Table S5   *Low-high depending on bat species 7,9,15 Groundhog 7% -*Low (Alpine marmot, Yellowbellied marmot) 16 , medium (Alpine marmot)

Figure 1 .Supplementary Figure 13 .
Examination of SARS-CoV-2 exposure relationship between human residence (U.S. 2020 census data) and seroprevalence collected from 5 different sites in VA, USA.Black line indicates the model fit and the grey ribbons (shadowed area) represent 95% confidence intervals (intercept = -1.277,β = 0.607, P = 0.059).Color of the circle indicates the species sampled and each circle represents and individual (n=67).Supplementary Figure 12.Equipment modified for animal processing.A modified plastic container (a & b).We used a vaporizer with a small O2 tank to supply isoflurane into the chambers (b).We made masks from the top part of a plastic bottle (c).We used a modified bucket chamber for anesthetizing animals trapped in larger cages (d).Distribution of SARS-CoV-2 variants in circulation during the 2022 sampling period.(a) Map of counties where sequence data was obtained.(b) Summary of weekly distribution of SARS-CoV-2 variants circulating in human communities in Southwest Virginia between May and September, 2022 as determined by RT-qPCR, RMA, and WGS sequencing.

Table 1 .
Summary of species tested including both RT-qPCR and serology (60% cut-off) results for SARS-CoV-2 after the virus had arrived in the United States.
2Species sampled at one of the 8 study sites 13 Supplementary Table

Table 3 .
for unresolved sequences.Summary of SARS-CoV-2 amplicon sequences obtained from wild animals.Sequences were analyzed for their length in bases and their position and percent identity relative to the reference genome (NC_045512.2).

Table 4 .
Number of seropositive samples under 4 different percent neutralization cutoff values from the 49 samples collected in summer 2022 in Virginia, U.S.A from 6 species.The four different percent neutralization cutoff values we evaluated include: 40% (Pos 40), 50% (Pos 50), 60% (Pos 60; the value we used for Fig 2c), and 70% (Pos 70).Additionally, we include the seropositivity values for each species under the 4 different cutoff values.

Table 5 .
Generalized linear mixed model results evaluating the relationship between urbanization and seroprevalence of mammals with species as a random effect.We evaluated this relationship using 5 different % neutralization cutoff values.All reported P values are two-tailed and the model consisted a single predictor variable and multiple comparisons were not made.

Table 7 .
Comparison of results from this study, experimental infection studies and predicted susceptibility based on modeling of the ACE2 receptor.RT-qPCR and seroprevalence data from our study are combined with data from previously published research on whether species were capable of being infected in the lab and their predicted susceptibility based on modeling of the ACE2 receptor.For species that do not have an exact match we included closely related species, which are indicated after the semicolon.* Indicates instances where a species has not been evaluated but information for a closely related species is available.