https://orcid.org/0000-0003-1372-7818
Figures
Abstract
Natural infections of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been documented in over 60 animal species, some distantly related. Several marine mammals have been predicted as highly susceptible to SARS-CoV-2 infection based on the homology of their ACE2 receptors to those of humans. To assess potential exposure of marine mammals to SARS-CoV-2, we conducted an opportunistic survey from 2020 to 2025 and tested 1,808 swabs and 378 serum samples from 21 marine mammal species in the United States. All the swabs tested by RT-qPCR were negative, indicating the absence of active infection. A low level of SARS-CoV-2 neutralizing antibodies was detected in three pinniped species, including Phoca vitulina (harbor seal, 13.6%, 95% CI: 5.2–27.4%), Zalophus californianus (California sea lion, 7%, 95% CI: 1.9–17.0%), and Halichoerus grypus (grey seal, 3.7%, 95% CI: 0.5–12.7%). These findings represent the first serological evidence of SARS-CoV-2 exposure in marine mammals in the United States, highlighting the need for continued monitoring of these populations and further research on SARS-CoV-2 transmission dynamics in wildlife.
Citation: Sanogo IN, Puryear WB, Simulynas AF, DiGiovanni R, Goertz CE, Hunter N, et al. (2026) Serological evidence of SARS-CoV-2 exposure in marine mammals in the United States between 2020 and 2025. PLoS One 21(7): e0351734. https://doi.org/10.1371/journal.pone.0351734
Editor: Mohamed Samy Abousenna, Central Laboratory for Evaluation of Veterinary Biologics, Agricultural Research Center, EGYPT
Received: January 17, 2026; Accepted: June 1, 2026; Published: July 9, 2026
This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
Data Availability: All relevant data are within the paper and its supporting information files.
Funding: This study was supported by the U.S. Department of Agriculture under contract AP23OA000000C022 (awarded to Jonathan Runstadler, Tufts University); the National Institutes of Health - Centers of Excellence for Influenza Research and Response (CEIRR) under contract 75N93021C00014 (awarded to Jonathan Runstadler, Tufts University); the National Institutes of Health Biocontainment Laboratory Operation Cooperative Agreement UC7AI180310 (New England Regional Biosafety Laboratory, Tufts University); and the National Marine Fisheries Service John H. Prescott Marine Mammal Rescue Assistance Grant Program under awards NA22NMF4390247 and NA23NMF4390303 (awarded to Kathy Burek Huntington, AVPS). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the virus responsible for the COVID-19 pandemic, is known for its remarkable ability to infect a wide range of distantly related animal species. Natural infections of SARS-CoV-2 have been documented in over 60 animal species, including many free-ranging wildlife species [1,2]. Given the high transmissibility of SARS-CoV-2 and its endemic prevalence in humans, there is increasing concern regarding its potential spillover to new animal species, including marine mammals [3,4].
Several studies have confirmed the presence of SARS-CoV-2 genome fragments in domestic untreated wastewater and marine environments [5–8]. Poor wastewater management or natural disasters may lead to the discharge of untreated wastewater, potentially carrying infectious materials into natural water systems. Consequently, marine mammal species inhabiting coastal areas with contaminated natural water may be exposed to a variety of new pathogens [4,9]. However, to date, infectious SARS-CoV-2 has not been found in untreated or treated wastewater or aquatic environments [10,11]. Marine mammals can also be exposed to human viruses in various other ways, including activities involving direct contact with humans, such as during rehabilitation and field research, or in stochastic ways associated with fishing or recreational activities. The rehabilitation of stranded marine mammals requires close interaction between humans and animals, which could expose marine mammals to human viruses and potentially increase the risk of reverse zoonosis [12]. Rehabilitated animals could introduce a novel pathogen acquired during rehabilitation into marine environments, leading to potential consequences for naïve populations [13].
Numerous marine mammal species, including most pinniped and cetacean species, particularly P. vitulina, was predicted to be highly susceptible to SARS-CoV-2 infection based on in silico modeling of SARS-CoV-2 spike protein binding affinity to their ACE2 receptors [4,14,15]. To date, natural SARS-CoV-2 infection in marine mammal species has not been reported, and the potential effect of SARS-CoV-2 spillover to marine mammals remains unknown. However, only a limited number of coronaviruses have been described in marine mammals, primarily alphacoronaviruses and gammacoronaviruses identified in seals and cetaceans, including one associated with epizootic pneumonia and mortality in Pacific harbor seals (Phoca vitulina richardii) [16,17]. Importantly, no betacoronaviruses closely related to SARS-CoV-2 have been reported in these hosts.
Exposure of marine mammals to SARS-CoV-2 could have serious consequences, particularly for highly social species such as seals and sea lions, which interact in large groups [18]. Such exposure could facilitate their role as a new reservoir and pose a future zoonotic threat. Consequently, investigating the epidemiology of SARS-CoV-2 in marine mammal hosts and assessing potential spillover events among these species will provide valuable information for public health. Several studies have investigated the epidemiology of SARS-CoV-2 in terrestrial mammals [2,19–21], but research on its circulation in marine mammals remains limited. In this study, we conducted an opportunistic survey of SARS-CoV-2 at the human–animal interface in diverse marine mammal species, including animals admitted to rehabilitation facilities and those sampled during active capture-release studies along the U.S. east and west coasts and Alaska, to assess potential exposure in these populations.
Methods
Ethics statement
This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The study was approved by Tufts University Institutional Animal Care and Use Committee (Number: G2023-02). Active capture-release studies on the West Coast were conducted under NMFS Permit #22678. Active capture-release studies of Steller sea lions (Eumetopias jubatus) were conducted under NMFS Permit #22289 and NMFS Alaska IACUC approval NWAK 18−03, and work in the Alaska Maritime National Refuge under permit #74500-15-005. Active capture-release studies of H. grypus were conducted under NMFS Permit #21719 and #26939, and U.S. Fish and Wildlife Service, Eastern Massachusetts NWR Complex Special Use Permits #53514-FY2019-01 and #53510-FY2022-01. Sea otter samples were collected under USFWS LOA-PER0051451. Additional approvals included NOAA Permit 24359, SA-AKR-2020-04, SA-AKR-2023-02, and USFWS Permit MAPER0032559 (AVPS). Samples provided by the Alaska SeaLife Center were obtained under NOAA SA-AKR-2025-05 for non-ESA–listed species. NOAA MMPA/ESA Permit 24359 applied to all samples from ESA-listed species.
Sample origin and collection
A total of 1,808 nasal, oral, and rectal swab samples from 21 marine mammal species and 378 serum samples from 6 pinniped, two cetacean and one mustelid species were collected between 2020 and 2025 (Table 1) through a network of collaborators across the U.S. Samples were collected opportunistically from both live and dead stranded animals under each responding organizations federal (with the National Oceanic and Atmospheric Administration’s National Marine Fisheries Service) and regional stranding agreement. A subset of species was also sampled as part of active capture-release studies for health or stock assessments under the Marine Mammal Protection Act (MMPA), permitting, and following established procedures for marine mammal sampling. No animals were anesthetized or euthanized for the purpose of this study. All animal handling was performed by trained marine mammal rehabilitation personnel following standard animal welfare protocols. As determined by each facility or researchers’ authorizations or permitting followed by feasibility of collection, an individual animal may have had multiple swabs collected and sera, only swab(s), or only sera collected (S1 Table). Individual swabs were immediately placed into vials containing viral transport medium (VTM) consisting of Medium 199 supplemented with bovine serum albumin, gentamicin, benzylpenicillin, nystatin, and sulfamethoxazole [22] and stored at −20 °C or below at each collecting facility until shipment to the laboratory. Whole blood samples were centrifuged, and serum was collected in cryovials and stored at −20°C.
RNA extraction and SARS-CoV-2 RT-PCR testing
RNA was extracted from swab samples using the Mag-Bind Viral DNA/RNA Extraction Kit (Omega Bio-Tek, Norcross, GA, USA) on a KingFisher Flex platform, following the manufacturer’s protocol. All samples were tested for SARS-CoV-2 viral RNA using a real-time RT-PCR assay targeting the open reading frame 1b non-structural protein 14 (ORF1b-nsp14) region (HKU-ORF1 primers: Forward 5′-TGGGGYTTTACRGGTAACCT-3′, Reverse 5′-AACRCGCTTAACAAAGCACTC-3′, Probe 5′-FAM-TAGTTGTGATGCWATCATGACTAG-TAMRA-3′) [23]. Samples with a cycle threshold (Ct) value < 40 were considered putatively positive for SARS-CoV-2 RNA.
Each batch of samples included a positive control (genomic RNA from SARS-CoV-2 isolate USA-WA1/2020, BEI resources NR-52281) and a negative control (VTM). All samples were further tested for evidence of viable RNA by detection of endogenously expressed β-actin (Ct < 40) as previously described [24]. Animals with a minimum of one swab sample with detectable β-actin were considered valid for further interpretation regarding the presence or absence of SARS-CoV-2 RNA detection.
Detection of SARS‐CoV‐2 antibodies by ELISA-RBD
SARS-CoV-2 antibodies from serum were detected using an indirect pan-mammalian ELISA specifically targeting the SARS-CoV-2 receptor binding domain (RBD), following a previously described protocol [24]. The ELISA assay was initially validated in-house for detecting total immunoglobulins in serum samples across multiple mammalian orders, including marine mammals (S1 Fig).
Immulon 4 HBX plates (part # 3855) were coated with purified SARS-CoV-2 RBD protein (NR-52366, BEI Resources) at a concentration of 2 µg/ml and incubated at 4 °C for 24 hours. After incubation, plates were washed three times with phosphate-buffered saline containing 0.1% Tween-20 (PBS-T) and blocked with Pierce Protein-Free Blocking Buffer (Thermo Fisher, catalog no. PI37573) for 1 hour at room temperature. Serum samples were initially diluted 1:5 in PBS in separate plates, then transferred to the ELISA plates to achieve a final dilution of 1:50 in PBS-T supplemented with 1% milk. Plates were incubated for 2 hours at room temperature. Following incubation, plates were washed three times with PBS-T, and 50 μL of the secondary antibody, Pierce Recombinant Peroxidase-Conjugated Protein A/G (Thermo Fisher, catalog no. 32490), diluted 1:10,000 in PBS-T containing 1% milk, was added to each well. Plates were incubated for 1 hour at room temperature. Plates were then washed and developed with SigmaFast OPD tablets (Sigma-Aldrich catalog no. P9187).
Absorbance values were measured at 490 nm on a BioTek Synergy 4 Multidetection Plate Reader, with the positive cutoff defined as the mean plus three standard deviations (μ + 3σ) of the negative controls (n = 8). All samples were run in duplicate, and each plate included serum from spike (S) protein-immunized alpacas as a positive control and serum from uninfected H. grypus obtained before the COVID-19 pandemic in 2018 as a negative control (S2 Fig). Positive samples from the initial ELISA-RBD screening were validated by a confirmatory ELISA using 3-fold serial dilutions starting at 1:100 against the full-length SARS-CoV-2 spike protein (BEI Resources, NR-52308). Positives were defined as samples showing a signal greater than the mean plus three standard deviations (μ + 3σ) of the negative controls in at least the first two consecutive dilutions [25].
SARS‐CoV‐2 neutralization assay
A virus neutralization test (VNT) to validate the ELISA-positive samples (n = 18) was performed under BSL-3 conditions at the New England Regional Biosafety Laboratory (NERBL). Heat-inactivated (56 °C, 30 min) serum samples were serially diluted two-fold, starting at 1:4, in duplicate. Sera dilutions were incubated with 200 TCID50 of SARS-CoV-2 isolate USA-WA1/2020 (BEI resources, NR-52281) for 1 h at room temperature. The mixtures were subsequently added to confluent Vero E6 cells (ATCC-CRL-1586) in 96-well plates and maintained at 37 °C with 5% CO₂. Each plate included a virus-only control, a cell-only control, and a serum-only control. Cytopathic effect (CPE) was independently examined under a light microscope by two operators at 5 days post-infection, and the neutralization titer was recorded as the highest serum dilution that provided 100% neutralization of the reference virus and completely prevented CPE in both duplicate wells. Neutralizing titers equal to or greater than 16 were considered seropositive. An animal was considered seropositive only if the corresponding sample tested positive by both ELISA and VNT.
Statistical analyses: SARS-CoV-2 seropositivity rate was calculated as the proportion of seropositive animals among the total number of individuals tested for each species. Exact 95% confidence intervals were estimated using the binomial distribution. Differences in seropositivity rates among species were assessed using Fisher’s exact test. All statistical analyses were performed in R (version 2025.05.1; R Foundation for Statistical Computing, Vienna, Austria), and statistical significance was defined as p < 0.05.
Results
A total of 1,017 individual animals were tested. Among these, 639 were tested only for SARS-CoV-2 RNA, 163 were tested only for antibodies, and 215 were tested for both viral RNA and antibodies. Of the 1,808 total swabs tested, 1,207 swabs tested positive for β-actin by RT-qPCR. This represents at least one viable sample from 656 of the 854 individual animals tested, confirming the presence of amplifiable RNA in 76.8% of the overall animals tested (S2 Table). Detection of endogenous β-actin was successful in the majority of pinniped species tested (9/12), with Z. californianus, E. jubatus, and M. angustirostris as the exceptions. The detection of endogenous β-actin was also successful in the majority of cetacean species (7/9) and in E. lutris kenyoni (S2 Table). All swabs were negative for SARS-CoV-2 RNA (S1 Table), indicating the absence of detectable SARS-CoV-2 RNA in marine mammal samples collected from 2020 to 2025.
In contrast, 18 out of 378 (4.8%; 95% CI: 2.6–6.9%) serum samples tested positive for binding antibodies against the SARS-CoV-2 RBD, including seven P. vitulina, three H. grypus, one P. groenlandicus, and seven Z. californianus (Fig 1). Of those, 12 also tested positive in a separate confirmatory ELISA against SARS-CoV-2 full spike protein, four tested negative, and two were untested due to limited sample volume. Neutralizing antibodies against SARS-CoV-2 WA1/2020 were detected in nine of the 12 ELISA spike-positive samples, as well as in three additional samples, two not tested by Spike ELISA and one Spike ELISA-negative sample, with neutralization titers ranging from 1:16–1:128 (Table 2).
Seropositive samples were detected in P. vitulina, H. grypus, P. groenlandicus, and Z. californianus. In H. grypus, no neutralizing antibodies were detected in samples collected during capture-release studies. In all three seal species, one of the seropositive samples failed to neutralize SARS-CoV-2 WA1/2020, leaving 6/7 P. vitulina, 2/3 H. grypus, and 0/1 P. groenlandicus with sera that both bound to SARS-CoV-2 antigens on ELISA, and also neutralized whole live virus (Table 2). Nearly all of these detections were from the northeastern United States in 2023, with a single neutralizing antibody-positive P. vitulina detected in the Pacific Northwest in October 2023 (Fig 2). The earliest detection of a positive neutralizing antibody sample occurred in a single P. vitulina in the mid-Atlantic in November 2021. P. vitulina SSC23–081 had sera collected on 2 occasions, 8 days apart. Both samples from that animal were seropositive with comparable values on all serology assays.
Color intensity indicates the relative number of samples collected per state. SARS-CoV-2 neutralizing antibodies were detected in three marine mammal species, including Phoca vitulina (blue), Halichoerus grypus (purple), and Zalophus californianus (Gray). State boundaries were derived from public domain U.S. Census Bureau TIGER/Line shapefiles [26].
For Z. californianus, seropositive samples were detected against the SARS-CoV-2 RBD antigen in 7 animals that were part of active capture-release efforts on San Miguel Island, CA, during the springs of 2023 and 2024. 3/6 seropositive samples also bound to SARS-CoV-2 spike protein in a separate analysis, and 4/7 neutralized whole SARS-CoV-2 WA1/2020 (Table 2).
Overall, the highest percentage of animals with neutralizing antibodies against SARS-CoV-2 WA1/2020 was observed in stranded P. vitulina (13.6%, 95% CI: 5.2–27.4%, N = 44), followed by actively captured and released Z. californianus (7%, 95% CI: 1.9–17.0%, N = 57), and stranded H. grypus (3.7%, 95% CI: 0.5–12.7%, N = 54). No statistically significant differences in seropositivity were detected among the three species.
No SARS-CoV-2 neutralizing antibodies were detected in serum samples collected from active capture-release studies for E. jubatus (n = 34), or C. ursinus (n = 20), nor during sampling of stranded P. groenlandicus (n = 11), D. delphis (n = 2), and E. lutris kenyoni (n = 2). The overall percentage of SARS-CoV-2 neutralizing antibodies in all pinniped samples tested across species, geographies, and years was 3.2% (95% CI: 1.7–5.5% N = 378).
Species are clustered by stranded sampling (black silhouettes) and active capture-release sampling (grey silhouettes). Bars show the total number of sera samples tested, with one sample per animal. Seronegative samples are shown in blue, SARS-CoV-2 RBD ELISA positive samples are shown in orange, and SARS-CoV-2 WA1/2020 neutralizing antibody positive samples are shown in red. The percentage of neutralizing antibody positives is shown in the box.
Discussion
To date, this study provides the first investigation of SARS-CoV-2 in several marine mammal species on both the East and West coasts of the U.S. We tested samples from a total of 1,018 individual animals representing 21 marine mammal species, sampled from live and dead stranded animals and during active capture-release studies. We found no evidence of SARS-CoV-2 viral RNA in any of the swab samples tested, indicating the absence of active infection in these species at the time of sampling. Consistent with our findings, SARS-CoV-2 surveillance studies of cetaceans stranded along the Italian coast (2020–2021) and pinnipeds stranded in Brazil also reported no evidence of viral RNA detection [14,27]. Interestingly, although no active infection was detected in any of the marine mammals tested, we found neutralizing antibodies to SARS-CoV-2 at a low seroprevalence (3.2% overall) in three marine mammal species, including the Z. californianus, H. grypus, and P. vitulina, sampled from different locations, indicating their exposure to the virus.
It remains unclear how these animals came into contact with the virus. However, SARS-CoV-2 transmission from humans to wildlife, particularly white-tailed deer, has been reported in multiple instances, highlighting the capacity of the virus for reverse zoonosis [2,28,29]. Cross-species transmission of pathogens from humans to marine mammals has been documented [30,31]. For example, the human influenza A(H1N1)pdm09 virus was detected in free-ranging northern elephant seals, supporting the possibility that human respiratory viruses can spill over into marine mammals [31,32]. It has been suggested that these seals may have been exposed to the H1N1 virus through feces discharged from sewage-dumping ships. A similar mechanism could be hypothesized for SARS-CoV-2 exposure in marine mammals, as the virus is shed in the feces of infected humans [33] and cruise ship traffic has increased in recent years in the areas we sampled. Nevertheless, the rapid loss of SARS-CoV-2 infectivity in feces suggests that contaminated human waste is unlikely to represent a major transmission route [34]. In contrast, direct contact between humans and marine mammals could provide opportunities for cross-species transmission. The samples tested in this study were primarily collected from animals in rehabilitation facilities, where close human-animal interactions are common [13]. One of the seropositive P. vitulina in this study, with a high neutralizing antibody titer (animal ID - NA25012Pv), was a juvenile male admitted to a rehabilitation facility after stranding in Delaware with severe injuries and ingested fishing gear requiring surgical intervention. Although the animal ultimately died following complications from surgery, its rehabilitation history involved prolonged care and close contact with animal care personnel, which may have provided opportunities for human-to-animal exposure to SARS-CoV-2. Therefore, we cannot rule out the possibility that seropositive animals were exposed to infected humans during rehabilitation, as serum samples were not routinely collected before admission to the rehabilitation centers, and asymptomatic human individuals may have gone undetected.
In this study, we found the highest SARS-CoV-2 seropositivity rate (13.6%) in P. vitulina. Notably, P. vitulina was predicted to be highly susceptible to SARS-CoV-2 compared to other pinnipeds based on the homology of their ACE2 viral receptors with human ACE2 receptors, suggesting their potential risk of infection by SARS-CoV-2 if exposed to the virus [4]. Moreover, an epizootic pneumonia outbreak and the associated die-off of P. vitulina were linked to infection with another coronavirus (harbor seal coronavirus – HSCoV) detected in archived tissues during a retrospective study [17]. However, the clinical significance of the HSCoV remains unclear due to incomplete sampling during the outbreak. Nevertheless, this finding demonstrates that coronaviruses can infect marine mammals and may be associated with disease outbreaks.
Unexpectedly, SARS-CoV-2 neutralizing antibodies were detected in Z. californianus sampled during active capture-release studies, albeit at low titers, despite this species being predicted to have very low susceptibility to infection based on ACE2 binding models [4]. However, while in silico predictions provide useful insight into receptor compatibility, SARS‑CoV‑2 has demonstrated its remarkable ability to infect a broad range of mammalian hosts, indicating that factors beyond ACE2 binding affinity contribute to host susceptibility [35]. Moreover, the Z. californianus sampled in this study were captured and released in San Miguel Island, where they may interact with humans through activities such as ecotourism and research operations. These human-animal interfaces may increase opportunities for exposure to SARS-CoV-2 through spillover from infected individuals [36,37].
Although SARS-CoV-2 neutralizing antibodies have been detected in stranded H. grypus, no evidence of exposure has been observed in actively captured and released individuals. Notably, H. grypus sampled during active capture-release studies are typically healthy, newly weaned pups, which may have limited opportunities for exposure to the virus.
The majority of seropositive animals were identified on the East Coast; however, this pattern likely reflects the geographic distribution and nature of our sampling rather than a true regional difference in exposure. Most samples were collected from the East Coast, and the majority of West Coast samples were obtained from actively captured animals, which may have had a lower likelihood of exposure compared with stranded or rehabilitated individuals.
While our study offers valuable insights into the prevalence of SARS-CoV-2 in marine mammals, it is essential to consider some limitations. First, the majority of samples tested were collected opportunistically and consisted primarily of swab specimens for the detection of active SARS-CoV-2 infection. Serum samples, which are more likely to provide evidence of exposure to SARS-CoV-2, were collected from only six of the 21 species from which swabs were collected. Additionally, the sensitivity of RT-PCR assays relies on primer binding to conserved genomic regions, and mutations in SARS-CoV-2 primer target sites can reduce assay sensitivity and potentially result in false negatives. However, the ORF1b region (encoding non-structural protein 14) targeted in our assay is among the most conserved regions of the SARS-CoV-2 genome, which reduces the likelihood that sequence variation substantially affected detection sensitivity [38]. Importantly, in this study, no pan coronavirus or consensus coronavirus PCR assays were performed; therefore, the presence of other coronaviruses in the sampled animals cannot be excluded. However, to date, only a limited number of coronaviruses have been described in marine mammals, primarily alphacoronaviruses and gammacoronaviruses identified in seals and cetaceans [16,17]. Second, sampling for certain species was limited to fewer than ten individuals. Therefore, we may lack information on potential exposure to the virus for many marine mammal species. Third, most serum samples exhibited low to moderate neutralizing titers (≤1:32) despite showing high optical density values in the ELISA, and three confirmatory ELISA-positive samples collected between 2023 and 2024 lacked detectable neutralizing activity. ELISA detects binding antibodies against viral antigens, indicating prior exposure to the virus. However, not all binding antibodies possess neutralizing activity. Therefore, the presence of binding antibodies in the absence of detectable neutralization may reflect exposure that elicited predominantly non-neutralizing antibodies [39]. Although we cannot completely rule out potential cross-reactivity with other coronaviruses, current evidence indicates that ELISA assays targeting the SARS-CoV-2 receptor-binding domain (RBD) are highly specific [40]. In addition, ELISA-positive samples were confirmed using virus neutralization tests (VNT), which further reduces the likelihood of cross-reactivity. However, VNT was performed using only a single SARS-CoV-2 strain (Wuhan-like strain USA/WA1/2020). Given that the sampling period spanned 2020–2025, the inclusion of multiple SARS-CoV-2 strains in the neutralization assay could have increased its sensitivity, particularly for detecting antibodies against antigenically divergent variants. Finally, although the secondary antibody used in the ELISA recognizes immunoglobulins across a broad range of mammalian species and was validated in-house for multiple taxa, including marine mammals (S1 Fig), the possibility of false-negative results, particularly in animals with low antibody titers, cannot be entirely excluded
In conclusion, this survey found no evidence of active SARS-CoV-2 transmission among marine mammals in the United States from 2020 to 2025. Nonetheless, the establishment of endemic infection in a mammalian host with substantial human contact could facilitate viral evolution and spillover as a newly emerging human variant. Although no active infections were found, indirect evidence of past SARS-CoV-2 exposure highlights the importance of future studies on the ecological and molecular mechanisms of SARS-CoV-2 transmission to marine mammals and their responses. It also emphasizes the need for ongoing surveillance of populations that could be at risk.
Supporting information
S1 Table. Metadata for each animal sampled in this study.
https://doi.org/10.1371/journal.pone.0351734.s001
(XLSX)
S2 Table. β-actin detection in swabs collected from marine mammals.
Numbers report individual animals for each species as negative (N) or positive (P) for endogenous β-actin detection, Ct < 40. Animals were considered to have viable RNA on swab samples if a minimum of 1 swab sample was positive. Percentage shows the number of animals for each species with viable RNA.
https://doi.org/10.1371/journal.pone.0351734.s002
(DOCX)
S1 Fig. Species and sample breadth results for ELISA SARS-CoV-2 RBD ELISA.
Phylogenetic tree shows IgG1 CH2-CH3 orthologs for representative mammalian orders. Circles denote sample types tested per species: red (serum), blue (oral/nasal), brown (fecal). Circles with an X were tested but failed at the internal control. No circle means a sample could not be collected from that species.
https://doi.org/10.1371/journal.pone.0351734.s003
(TIF)
S2 Fig. Representative ELISA-RBD plate layout.
96-well Immulon 4 HBX microtiter plate showing the experimental setup and distribution of controls and samples. Black boxes indicate blank wells used for background correction. Red boxes indicate positive controls, and green boxes indicate negative controls. The remaining wells correspond to samples.
https://doi.org/10.1371/journal.pone.0351734.s004
(TIF)
Acknowledgments
We express our deep gratitude to the stranding and marine mammal rehabilitation organizations that contributed to the collection of samples in this study. We thank Lauren Brandkamp and Caroline Genther of Whale and Dolphin Conservation, Mandy Keogh from NMFS Alaska PRD, Barbie Halaska from the Marine Mammal Center, and the many additional staff and volunteers for their assistance in providing biological samples. We thank the Prescott Grant Program for supporting the work of many of the organizations that provided samples. We thank Dr. Florian Krammer and Dr. Charles Shoemaker for generously providing purified SARS-CoV-2 proteins and serum from spike (S) protein-immunized alpacas, respectively. We thank Dr. Kaitlin Sawatzki for the early development of the assays used in this study.
The following reagent was deposited by the Centers for Disease Control and Prevention and obtained through BEI Resources, NIAID, NIH: SARS-Related Coronavirus 2, Isolate hCoV-19/USA-WA1/2020, NR-52281. The following reagents were produced under HHSN272201400008C and obtained through BEI Resources, NIAID, NIH: Spike Glycoprotein Receptor Binding Domain (RBD) from SARS-Related Coronavirus 2, Wuhan-Hu-1 with C-Terminal Histidine Tag, Recombinant from HEK293F Cells, NR-52366; Spike Glycoprotein (Stabilized) from SARS-Related Coronavirus 2, Wuhan-Hu-1 with C-Terminal Histidine Tag, Recombinant from Baculovirus, NR-52308
This is a contribution of the New England Regional Biosafety Laboratory. This manuscript serves as Sea Research Foundation publication #393.
The scientific results and conclusions and any views or opinions expressed herein are those of the authors and do not necessarily reflect the views or policies of the included agencies or organizations. Reference to trade names does not imply endorsement by the National Marine Fisheries Service, NOAA.
References
- 1. Fang R, Yang X, Guo Y, Peng B, Dong R, Li S, et al. SARS-CoV-2 infection in animals: Patterns, transmission routes, and drivers. Eco Environ Health. 2023;3(1):45–54. pmid:38169914
- 2. Goldberg AR, Langwig KE, Brown KL, Marano JM, Rai P, King KM, et al. Widespread exposure to SARS-CoV-2 in wildlife communities. Nat Commun. 2024;15(1):6210. pmid:39075057
- 3. Audino T, Grattarola C, Centelleghe C, Peletto S, Giorda F, Florio CL, et al. SARS-CoV-2, a Threat to Marine Mammals? A Study from Italian Seawaters. Animals (Basel). 2021;11(6):1663. pmid:34204885
- 4. Mathavarajah S, Stoddart AK, Gagnon GA, Dellaire G. Pandemic danger to the deep: The risk of marine mammals contracting SARS-CoV-2 from wastewater. Sci Total Environ. 2021;760:143346. pmid:33160659
- 5. González-Aravena M, Galbán-Malagón C, Castro-Nallar E, Barriga GP, Neira V, Krüger L, et al. Detection of SARS-CoV-2 in Wastewater Associated with Scientific Stations in Antarctica and Possible Risk for Wildlife. Microorganisms. 2024;12(4):743. pmid:38674687
- 6. La Rosa G, Mancini P, Iaconelli M, Veneri C, Bonanno Ferraro G, Del Giudice C, et al. Tracing the footprints of SARS-CoV-2 in oceanic waters. Sci Total Environ. 2024;906:167343. pmid:37751837
- 7. Polo D, Lois M, Fernández-Núñez MT, Romalde JL. Detection of SARS-CoV-2 RNA in bivalve mollusks and marine sediments. Sci Total Environ. 2021;786:147534. pmid:33984699
- 8. Yousif M, Rachida S, Taukobong S, Ndlovu N, Iwu-Jaja C, Howard W, et al. SARS-CoV-2 genomic surveillance in wastewater as a model for monitoring evolution of endemic viruses. Nat Commun. 2023;14(1):6325. pmid:37816740
- 9. Miller MA, Byrne BA, Jang SS, Dodd EM, Dorfmeier E, Harris MD, et al. Enteric bacterial pathogen detection in southern sea otters (Enhydra lutris nereis) is associated with coastal urbanization and freshwater runoff. Vet Res. 2010;41(1):1. pmid:19720009
- 10. Sherchan S, Thakali O, Ikner LA, Gerba CP. Survival of SARS-CoV-2 in wastewater. Sci Total Environ. 2023;882:163049. pmid:36990233
- 11. Atoui A, Cordevant C, Chesnot T, Gassilloud B. SARS-CoV-2 in the environment: Contamination routes, detection methods, persistence and removal in wastewater treatment plants. Sci Total Environ. 2023;881:163453. pmid:37059142
- 12. Lopes De Melo F, Bezerra B, De Oliveira Luna F, Navarrete Barragan A, Moraes R, Arcoverde L, et al. Investigation of Coronavirus (SARS-CoV-2) in Antillean Manatees (Trichechus Manatus Manatus) in Northeast Brazil. Sci World J Cancer Sci Ther. 2019;1.
- 13. Moore M, Early G, Touhey K, Barco S, Gulland F, Wells R. Rehabilitation and release of marine mammals in the United States: risks and benefits. Mar Mamm Sci. 2007;23:731–50.
- 14. Audino T, Berrone E, Grattarola C, Giorda F, Mattioda V, Martelli W, et al. Potential SARS-CoV-2 Susceptibility of Cetaceans Stranded along the Italian Coastline. Pathogens. 2022;11(10):1096. pmid:36297153
- 15. Li S, Yang R, Zhang D, Han P, Xu Z, Chen Q, et al. Cross-species recognition and molecular basis of SARS-CoV-2 and SARS-CoV binding to ACE2s of marine animals. Natl Sci Rev. 2022;9(9):nwac122. pmid:36187898
- 16. Legnardi M, Franzo G, Cecchinato M, Si H, Baston R, Mazzariol S, et al. First Detection of Gammacoronavirus in a Striped Dolphin (Stenella coeruleoalba) from the Adriatic Sea. Animals (Basel). 2024;14(18):2725. pmid:39335313
- 17. Nollens HH, Wellehan JFX, Archer L, Lowenstine LJ, Gulland FMD. Detection of a respiratory coronavirus from tissues archived during a pneumonia epizootic in free-ranging Pacific harbor seals Phoca vitulina richardsii. Dis Aquat Organ. 2010;90(2):113–20. pmid:20662367
- 18. Liwanag HEM, Oraze J, Costa DP, Williams TM. Thermal benefits of aggregation in a large marine endotherm: huddling in California sea lions. J Zool. 2014;293:152–9.
- 19. Castañeda D, Isaac EJ, Schnieders BP, Kautz T, Romanski MC, Moore SA, et al. Absence of SARS-CoV-2 in wildlife of northeastern Minnesota and Isle Royale National Park. Zoonoses Public Health. 2024;71(6):744–7. pmid:38853397
- 20. Yaglom HD, Van Pelt L, Howard AL, Jansen B, Smith P, Sorensen R, et al. Convenience sampling yields no evidence of SARS-CoV-2 infection in free-ranging mammalian wildlife in Arizona, USA, 2021–23. J Wildl Dis. 2024;60:1016–20.
- 21. Ehrlich M, Madden C, McBride DS, Nolting JM, Huey D, Kenney S, et al. Lack of SARS-CoV-2 Viral RNA Detection among a Convenience Sampling of Ohio Wildlife, Companion, and Agricultural Animals, 2020–2021. Animals. 2023;13:2554.
- 22.
WHO. Collecting, preserving and shipping specimens for the diagnosis of avian influenza A(H5N1)virus infection: guide for field operations. 2006. Available: https://iris.who.int/handle/10665/69392
- 23. Chu DKW, Pan Y, Cheng SMS, Hui KPY, Krishnan P, Liu Y, et al. Molecular Diagnosis of a Novel Coronavirus (2019-nCoV) Causing an Outbreak of Pneumonia. Clin Chem. 2020;66(4):549–55. pmid:32031583
- 24. Sawatzki K, Hill NJ, Puryear WB, Foss AD, Stone JJ, Runstadler JA. Host barriers to SARS-CoV-2 demonstrated by ferrets in a high-exposure domestic setting. Proc Natl Acad Sci U S A. 2021;118(18):e2025601118. pmid:33858941
- 25. Stadlbauer D, Amanat F, Chromikova V, Jiang K, Strohmeier S, Arunkumar GA, et al. SARS-CoV-2 Seroconversion in Humans: A Detailed Protocol for a Serological Assay, Antigen Production, and Test Setup. Curr Protoc Microbiol. 2020;57(1):e100. pmid:32302069
- 26.
US Census Bureau. TIGER/Line Shapefiles, Current, State and Equivalent. 2025 [cited 11 Jun 2025]. Available: https://www.census.gov/geographies/mapping-files/time-series/geo/tiger-line-file.html
- 27. Costa-Silva S, Ewbank AC, Duarte-Benvenuto A, Sacristán C, Soares RM, Sánchez-Sarmiento AM, et al. Novel paramyxovirus in wild pinnipeds, Brazil. Vet Res Commun. 2025;49(4):242. pmid:40591094
- 28. Chandler JC, Bevins SN, Ellis JW, Linder TJ, Tell RM, Jenkins-Moore M, et al. SARS-CoV-2 exposure in wild white-tailed deer (Odocoileus virginianus). Proc Natl Acad Sci U S A. 2021;118(47):e2114828118. pmid:34732584
- 29. Hale VL, Dennis PM, McBride DS, Nolting JM, Madden C, Huey D, et al. SARS-CoV-2 infection in free-ranging white-tailed deer. Nature. 2022;602(7897):481–6. pmid:34942632
- 30. Faires MC, Gehring E, Mergl J, Weese JS. Methicillin-resistant Staphylococcus aureus in marine mammals. Emerg Infect Dis. 2009;15(12):2071–2. pmid:19961710
- 31. Plancarte M, Kovalenko G, Baldassano J, Ramírez AL, Carrillo S, Duignan PJ, et al. Human influenza A virus H1N1 in marine mammals in California, 2019. PLoS One. 2023;18:e0283049.
- 32. Goldstein T, Mena I, Anthony SJ, Medina R, Robinson PW, Greig DJ, et al. Pandemic H1N1 influenza isolated from free-ranging Northern Elephant Seals in 2010 off the central California coast. PLoS One. 2013;8(5):e62259. pmid:23690933
- 33. Jones DL, Baluja MQ, Graham DW, Corbishley A, McDonald JE, Malham SK, et al. Shedding of SARS-CoV-2 in feces and urine and its potential role in person-to-person transmission and the environment-based spread of COVID-19. Sci Total Environ. 2020;749:141364. pmid:32836117
- 34. Wurtzer S, Lacote S, Murri S, Marianneau P, Monchatre-Leroy E, Boni M, et al. Reduction in SARS-CoV-2 Virus Infectivity in Human and Hamster Feces. Viruses. 2022;14(8):1777. pmid:36016399
- 35. Dunowska M. Cross-species transmission of coronaviruses with a focus on severe acute respiratory syndrome coronavirus 2 infection in animals: a review for the veterinary practitioner. N Z Vet J. 2023;71(4):159–71. pmid:36927253
- 36. Holcomb K, Young JK, Gerber LR. The influence of human disturbance on California sea lions during the breeding season. Anim Conserv. 2009;12:592–8.
- 37. Laake JL, Lowry MS, DeLong RL, Melin SR, Carretta JV. Population growth and status of california sea lions. J Wildl Manag. 2018;82(3):583–95.
- 38. Takada K, Ueda MT, Shichinohe S, Kida Y, Ono C, Matsuura Y, et al. Genomic diversity of SARS-CoV-2 can be accelerated by mutations in the nsp14 gene. iScience. 2023;26(3):106210. pmid:36811085
- 39. Ding S, Laumaea A, Benlarbi M, Beaudoin-Bussières G, Gasser R, Medjahed H, et al. Antibody Binding to SARS-CoV-2 S Glycoprotein Correlates with but Does Not Predict Neutralization. Viruses. 2020;12(11):1214. pmid:33114742
- 40.
Peterhoff D, Glück V, Vogel M, Schuster P, Schütz A, Neubert P, et al. A highly specific and sensitive serological assay detects SARS-CoV-2 antibody levels in COVID-19 patients that correlate with neutralization.