A recent study posted to bioRxiv* conducted surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in wildlife in Vermont, United States (US).
Study: Surveillance of Vermont wildlife in 2021-2022 reveals no detected SARS-CoV-2 viral RNA. Image Credit: TomReichner/Shutterstock.com
*Important notice: bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.
Background
Natural SARS-CoV-2 infections in animals have been reported. Animals that facilitate virus transmission within species become viral reservoirs, leading to evolutionary changes that pose a risk if reintroduced in humans.
This scenario has been documented in mink farms. The Netherlands registered five outbreaks in 2020, leading to SARS-CoV-2 infections in over half of the mink farms.
Sequencing data indicated multiple spillover and spillback events between minks and humans. Recent studies demonstrated the susceptibility of North American Cervidae family members to SARS-CoV-2.
While studies have reported wildlife infection with SARS-CoV-2 in several states in the US and Canada, no information is available for Vermont.
The study and findings
In the present study, researchers conducted SARS-CoV-2 surveillance in animals in Vermont. The surveillance was conducted during the hunting and trapping seasons in 2021 and the hunting season in 2022 throughout the state. They sampled grey/red foxes, fishers, coyotes, bobcats, white-tailed deer, black bears, and otters.
Deer were prioritized for sampling; most sampling (in deer) occurred in 2022 at 470 samples compared to 17 in 2021.
At the end of the 2021 season, RNA was extracted from samples, and reverse-transcription quantitative polymerase chain reaction (RT-qPCR) was performed to detect SARS-CoV-2 RNA using N1 and N2 primer sets.
No viral RNA was detectable in any of the 2021 samples. Of the 472 samples in 2022, 133 were positive for both primers. The mean cycle threshold (CT) was 36.6 and 38 for N1 and N2, respectively. Additionally, multiple samples were positive for one of the primers; 28 tested positive for N1, and 56 were positive for N2.
This sudden high positivity in 2022 samples, with high mean CT values and the absence of samples with CT < 30 and CT < 33 for N2, pointed to the possibility of contamination. The laboratory at the University of Vermont initiated a separate in vitro project involving the expression of SARS-CoV-2 nucleocapsid.
DNA constructs with N1/N2 primer-recognizable sequences were present in the laboratory. Therefore, the team evaluated whether the positive test results were authentic or due to contamination. The researchers obtained environmental swabs from common items/surfaces in the laboratory.
All swabs tested positive for N1 and N2, with CT values reaching 23.6. No negative control was amplified in the reaction. Next, they performed qPCR to determine whether the contamination was viral DNA or RNA. On average, positive controls (clinical SARS-CoV-2 specimens) in qPCR had 5.4 cycles higher CT for N1 than in RT-qPCR.
Two positive control specimens with the N2 primer were undetectable in qPCR, whereas CT was 1.8 cycles higher for one positive control positive for N2.
The CT values of all laboratory specimens were consistent between qPCR and RT-qPCR tests, suggesting viral DNA contamination. Further, the team compared RT-qPCR and qPCR reactions on select positive samples from deer.
SARS-CoV-2 nucleic acids were detectable, with consistent CT values between tests. This suggested that the initial positive results for animal samples were likely DNA contamination.
Therefore, the team repeated RT-qPCR tests using an envelope (E) gene primer set since the DNA constructs used in the laboratory lacked E gene sequences. All 2022 samples were undetectable by this primer set, suggesting that wildlife in Vermont did not contain SARS-CoV-2 RNA.
Conclusions
In summary, the study did not find SARS-CoV-2 in wildlife in Vermont. This was surprising because prior studies have reported positivity rates of > 30% and seropositivity rates of > 40% in white-tailed deer.
The sparse population and relatively low coronavirus disease 2019 (COVID-19) rate in Vermont might have reduced the risk of SARS-CoV-2 transmission from humans to deer.
While the findings are reassuring, it is unlikely to continue indefinitely, especially as cases are increasingly reported in wildlife in neighboring regions. Surveillance should be conducted throughout North America to detect viral transmission and adaptation in wildlife.
*Important notice: bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.
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