The spillover of SARS-CoV-2 into humans has caused one of the most devastating pandemics in recorded history. Human-animal interactions have led to transmission events of SARS-CoV-2 from humans to wild and captive animals. However, many questions remain about how extensive SARS-CoV-2 exposure is in wildlife, the factors that influence wildlife transmission risk, and whether sylvatic cycles can generate novel variants with increased infectivity and virulence. We sampled 22 different wildlife species in Virginia, U.S.A. We detected widespread exposure to SARS-CoV-2 across six wildlife species. Using quantitative reverse transcription polymerase chain reaction, we detected SARS-CoV-2 RNA in the Virginia opossum and had equivocal detections in six additional species. Furthermore, we used whole genome sequencing to confirm the presence of SARS-CoV-2 and compare mutations present to known circulating strains. Species that exhibit peridomestic tendencies had high seroprevalence, ranging between 62%-71%, and sites with high human presence had three times higher seroprevalence than low human-use areas across all species combined. SARS-CoV-2 genomic data from an opossum and molecular modeling exposed one previously uncharacterized change to an amino acid residue in the Spike receptor binding domain (RBD), which predicts improved binding between the Spike protein and human angiotensin-converting enzyme (ACE2) compared to the dominant variant circulating at the time of collection. Overall, our results highlight widespread exposure to SARS-CoV-2 in wildlife and suggest that areas with high human activity may serve as important points of contact for cross-species transmission. Furthermore, this work highlights the potential role of wildlife as reservoirs for SARS-CoV-2.

The emergence of SARS-CoV-2 has resulted in unprecedented consequences for humans across the globe. Transmission of SARS-CoV-2 among species has the potential to generate new and more virulent variants, posing a threat to both public health and animal populations. However, the ability of SARS-CoV-2 to infect wildlife other than white tailed deer and mustelids in nature remains unknown. We examined exposure to SARS-CoV-2 in 22 wildlife species, which are commonly found across the Eastern U.S. We found widespread SARS-CoV-2 exposure in six common wildlife species, which was elevated in areas with high human activity. Our results highlight the capacity of SARS-CoV-2 to spread through wildlife communities.