USDA Announcement

Wildlife exposure to SARS-CoV-2 across a human use gradient
BioRxiv preprint 7Nov2022

Abstract
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 18 different wildlife species in the Eastern U.S. and detected widespread exposure to
SARS-CoV-2 across wildlife species. Using quantitative reverse transcription polymerase chain
reaction and whole genome sequencing, we conclusively detected SARS-CoV-2 in the Virginia
opossum and had equivocal detections in six additional species. Species considered human
commensals like squirrels, and raccoons had high seroprevalence, ranging between 62%-71%,
and sites with high human use had three times higher seroprevalence than low human-use areas.
SARS-CoV-2 genomic data from an infected opossum and molecular modeling exposed
previously uncharacterized changes to amino acid residues observed in the 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 isolation. These mutations were not identified in human samples 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 in fueling de novo
mutations that may eventually appear in humans.

Selected parts of the Discussion
Our combined results suggest that a high diversity of species are exposed to SARS-CoV2 in the wild. Prevalence of active infections among wildlife was generally low and ranged from
1.6-20.0%. We found that 7.8% of opossums, a marsupial, were positive (including equivocal
detections), similar to white-tailed deer (10.0%).

The Eastern gray squirrel also had detections on both RT-qPCR (1.6%) and using
serological tests (71.4%). Several other species that were examined for antibodies to SARSCoV-2 suggested high levels of previous exposure, including raccoons (64%) and skunks (67%).

SARS-CoV-2 is being transmitted from humans to wildlife; there was greater rates of transmission in locations where there are more humans. Omicron variants of SARS-CoV-2 were detected in an opossum before those variants were detected in the local human population. Either the mutations developed in the opossum, or the opossum was infected from humans with this variant (and then sampled) before human sampling detected the new variant. Inadequate variant sampling of humans or human to opossum transmission followed by mutation within the opossum and then transmission back to humans.