Study monitoring Vermont wildlife finds no SARS-CoV-2

A recent study published on bioRxiv* conducted surveillance for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in wildlife in Vermont, United States (USA).

Study: Vermont wildlife surveillance in 2021-2022 reveals no SARS-CoV-2 viral RNA detected. Image Credit: TomReichner/Shutterstock.com

*Important Notice: bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be considered conclusive, guide clinical practice/health-related behaviors, or treated as established information.

Background

Natural SARS-CoV-2 infections in animals have been reported. Animals that facilitate transmission of the virus within species become viral reservoirs, driving evolutionary changes that pose a risk if reintroduced into humans.

This scenario has been documented on mink farms. The Netherlands recorded five outbreaks in 2020, leading to SARS-CoV-2 infections in more than half of mink farms.

Sequencing data indicated multiple spillover and return events between mink and humans. Recent studies have demonstrated the susceptibility of members of the North American deer family to SARS-CoV-2.

Although studies have reported wildlife infection with SARS-CoV-2 in several states in the United States and Canada, no information is available for Vermont.

The study and the conclusions

In the current study, researchers conducted surveillance for SARS-CoV-2 in Vermont animals. Monitoring was conducted during hunting and trapping seasons in 2021 and hunting season in 2022 statewide. They sampled grey/red foxes, fishers, coyotes, bobcats, white-tailed deer, black bears and otters.

Deer were prioritized for sampling; most sampling (in deer) took place in 2022 at 470 samples compared to 17 in 2021.

At the end of the 2021 season, RNA was extracted from the samples and quantitative reverse-transcription polymerase chain reaction (RT-qPCR) was performed to detect SARS-CoV-2 RNA using of primer sets N1 and N2.

No viral RNA was detectable in any of the 2021 samples. Of the 472 samples in 2022, 133 were positive for both primers. The average cycle threshold (C_J) was 36.6 and 38 for N1 and N2, respectively. Additionally, several 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 a high average C_J values ​​and absence of samples with C_J < 30 and C_J < 33 for N2, indicates the possibility of contamination. The University of Vermont lab launched a separate study in vitro project involving the expression of the SARS-CoV-2 nucleocapsid.

DNA constructs with sequences recognizable by the N1/N2 primers were present in the laboratory. Therefore, the team assessed whether the positive test results were genuine or due to contamination. Researchers obtained environmental swabs from common laboratory objects/surfaces.

All swabs tested positive for N1 and N2, with C_J values ​​reaching 23.6. No negative control was amplified in the reaction. Then they performed qPCR to determine if the contamination was viral DNA or RNA. On average, positive controls (SARS-CoV-2 clinical specimens) in qPCR had 5.4 cycles of higher C_J for N1 only in RT-qPCR.

Two positive control samples with the N2 primer were undetectable in qPCR, while C_J was 1.8 cycles higher for a positive control positive for N2.

The C_J values ​​from all laboratory samples were consistent between qPCR and RT-qPCR assays, suggesting viral DNA contamination. Additionally, the team compared RT-qPCR and qPCR reactions on selected positive samples from deer.

SARS-CoV-2 nucleic acids were detectable, with constant C_J values ​​between tests. This suggests that the initial positive results for the animal samples were likely DNA contamination.

Therefore, the team repeated the RT-qPCR tests using a set of envelope (E) gene primers because the DNA constructs used in the lab lacked E gene sequences. All 2022 samples were undetectable by this set of primers, suggesting that Vermont wildlife did not contain SARS-CoV-2 RNA.

conclusion

In summary, the study did not find SARS-CoV-2 in Vermont wildlife. This was surprising as previous studies reported positivity rates >30% and seropositivity rates >40% in white-tailed deer.

The sparse population and relatively low rate of coronavirus disease 2019 (COVID-19) in Vermont may have reduced the risk of transmission of SARS-CoV-2 from humans to deer.

While the results are reassuring, this is unlikely to continue indefinitely, especially as cases are increasingly being reported in wildlife from 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 considered conclusive, guide clinical practice/health-related behaviors, or treated as established information.