-->PONE-D-26-02880-->-->Serological Evidence of SARS-CoV-2 Exposure in Marine Mammals in the United States between 2020 and 2025-->-->PLOS One

Dear Dr. Sanogo,

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-->Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

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Reviewer #1: Partly

Reviewer #2: Partly

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-->2. Has the statistical analysis been performed appropriately and rigorously? -->

Reviewer #1: No

Reviewer #2: N/A

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The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.-->

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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-->5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)-->

Reviewer #1: The manuscript “Serological Evidence of SARS-CoV-2 Exposure in Marine Mammals in the United States between 2020 and 2025” is an interesting manuscript that reports evidence of SARS-CoV-2 exposure in marine mammals. The information presented by the authors is interesting and important to understand the ecology of SARS-CoV-2 and is clear that was a really big endeavor to achieve. However, despite the importance of this report, there are many technical issues that must be solved before being considered to be published. Also lab results interpretation is limited and as a consequence discussion is limited and for moments contradictory.

The main methodology issue is the performance of the ELISA. Authors do not describe in detail how this was performed. The reference that they present to validate the use of this test is for human test, not for a panmammalian as is claimed in the manuscript.

To properly evaluate the results detailed methodology is needed to detect possible pitfalls in the laboratory analyses. One of the most important details is the secondary antibody or protein used, as well as, all the controls used.

About the interpretation of the lab results, it is necessary to declare the limitation of the test used. For example, how specific are the primers used for the rt-pcr? If the primers are so specific and the strain is little different, the rt-pcr can lose the diagnostic, otherwise, a more degenerated primer can detect even different variants of the virus, making the result much more trustful.

Some data of the animals is missing. This is especially important for Zalophus, where the animals tested positive where actively captured. The site of capture is vital to understand how the animals get in contact with the virus.

Discussion should be rewrite and reinforce, in its actual form is just a superficial comparison. One important issue to be discussed is the lab results (based on the methodology used) and its limitations. The discussion is lack of analysis of the geographic distribution, difference of the species, antibody titer, among others.

Here some specific comments that could help to improve the manuscript:

Line 84-85: this sentence does not have enough sustain. The similitude does not explain why could be potentially infected at "low viral concentration", is it because it is similar to humans and humans can infect a low viral concentration?

Line 124: viral transport mediums need specification to know how long the virus or at least the virus RNA can last.

Line 135: declare the primers used and the range of detection for SARS-CoV-2. How specific are the primers just a specific strain of any SARS-CoV-2 strain.

Line 143: the 23 reference does not include a generic protocol for mammals, this article is about tests for humans.

Line 144-145: Authors omitted the most important part of the protocol, since the previous reference is for testing in humans.

Clearly and highly detailed protocol must be included, with emphasis in secondary antibody. The secondary antibody (or protein) must be capable of recognizing FC from immunoglobulins of mammals. This part is crucial to understand the effectiveness of the serological test.

If there is not a previous use of this methodology, authors must include the standardization phase as a supplementary material.

Line 147: what the authors used has negative controls?

Line 149: What does “healthy” mean for the authors? How could the authors rule-out a subclinical or previous exposition?

Line 149: why the authors selected this species and no others?

Line 156: If a validation (or more precisely comparison) is attempted, a statistical test must be run, such as a concordance test such as kappa. And in this way, positive and negative samples must be included, not just positive.

Line 163: Controls for the neutralization test must be declared. How the authors can test the true inactivation of the serum. Some carnivore serums are still killing cells after heat (56oC) inactivation.

Line 165: a data analyses section is necessary

Line 172: authors can not ensure if the virus is active just by pcr. But you can say that at least there is some RNA of the virus in the sample.

Line 174: even when authors already mention how many samples analyzed, it would be helpful to put 18 out of how many, and of course a prevalence or frequency along with confidence intervals.

Line 175: since CA sea lions come from active capture, it is important to declare and to discuss where the animals were captured. Especially since this species is more likely to have a natural infection. Is this site close to human settlements?

Line 178: Same 12 samples of the confirmatory ELISA?

Line 180: the first sentence is confusing, it looks like authors test both condition stranded and active capture. However, just samples from grey came from both sources, active and stranded, so this declaration implies that you evaluate both.

Line 187: this raises some questions. Samples were analyzed separately? This is increasing the number of positive samples. Authors must clean the data base specially of the resampling individuals where there is not so much time difference.

Line 191: is not 3/7 since one was not tested, must be 3/6

Line 195-197: some of the table legend was already in the methodology, not necessary here.

Line 199: is necessary to include confidence interval

Line 201: Is important to mention that you are considering a positive animal (or sample) when it was positive to ELISA and SN.

Line 202-205: Authors have data to compare prevalence between species, maybe sex and age, why the authors did not? A simple x-square test can solve this. It was a big effort to lose this information.

Line 229: the specific clinical history of the positive animals, specially from those stranded, are really important to evaluate the odds of get infected in the rehabilitation center. Antibodies usually need at least 21 to get formed, in this way if the sample was get before that time, seroconversion should be before the contact to humans.

Line 250-252: Make clear what is the relevance of this for a SARS-CoV-2 study, particularly the final part of this paragraph “the clinical significance of the HSCoV remains unclear due to incomplete sampling during the outbreak”

Line 255-257: No agree with this, new variants are more specific to humans, original ones had more potential to cross transition.

Line 274: “using contemporary SARS-CoV-2 strains in the VNT could have increased its sensitivity” indeed but also if the antibodies of your study came from cross reaction, new strain could detect even lower prevalence.

Figures: quality must be improved.

I recommend to the authors to explore to the natural dynamics of immunoglobulin production and decay. Also, difference between neutralizing antibodies and those antibodies that have not neutralizing activity. To considered exposure to the virus, this difference is important. In this way, almost all antibodies need exposure to the antigen to be produce, but not necessary all produce neutralizing activity (i.e. protection to the pathogen).

It is important to know if your secondary antibody allows you to identify the isotype of immunoglobulin that you found in the ELISA test. Is not the same interpretation if you find IgG or IgM.

Finally, the description of other potential coronaviruses present in marine mammals is important and even more, how probable is the cross reaction with SARS-CoV-2. This is a key point since there are no other complementary techniques.

Reviewer #2: Overall, this article is well written and presents interesting findings. However, several methodological and interpretive points require clarification to strengthen confidence in the results.

General Comments

# The authors do not mention whether any pan coronavirus or consensus coronavirus PCR assays were performed on the swabs. Given the possibility of cross reactivity in serological assays, this information is important for interpreting the results.

# Because serological assays can yield false positives, the study design and conclusions should be interpreted cautiously. The authors should report the sensitivity of the ELISA RBD assay particularly in any relevant mammalian species to help readers assess the likelihood of missed positives.

# Virus neutralization testing (VNT) was not performed on all serum samples. The authors should clarify how they calculated the percentage of neutralizing antibodies and whether these analyses are appropriate given that VNT was only conducted on indirect ELISA RBD–positive samples.

# Including a larger number of pre pandemic serum samples would strengthen the study by helping determine whether the assays produce any unexpected positives and by increasing confidence in the VNT results.

# The absence of evidence for active infection makes it difficult to interpret the low to moderate VNT titers observed in a few animals. Additionally, different VNT formats can vary in false positive and false negative rates, which should be acknowledged.

Line Specific Comments

Lines 142–144: What is the sensitivity of the ELISA RBD assay, particularly for any relevant mammalian species? Including this information would help readers assess the likelihood of false negatives. If sensitivity is low, some positive animals may have been missed; if high, it strengthens confidence in the findings.

Lines 149–150: Only one pre pandemic serum sample was used as a negative control. Is it possible to include additional pre pandemic samples? This would help determine whether any of the assays produce unexpected positives and would strengthen the overall conclusions.

Line 156: If I understand correctly, all serum samples were first screened using the ELISA RBD assay, and only RBD positive samples were subsequently tested by ELISA Spike and VNT. If this is accurate, please clarify the reference to “20 positive samples (n = 20)” here. In the Results section, you report 18 ELISA RBD positive samples. Reconciling these numbers would improve clarity.

Lines 166–168: Please specify in the results section the number of animals tested by PCR and by ELISA. Although the table provides totals, including these figures directly in the text would improve readability. For example, briefly state the total number of animals tested, how many underwent PCR, how many were tested serologically, and how many received both.

Lines 198–201: As I understand it, VNT was performed only on ELISA-RBD positive samples. If so, analyses comparing VNT results across ELISA RBD negative animals may not be appropriate unless VNT was performed on all samples or the ELISA RBD assay is known to be 100% sensitive.

Lines 204–205: The same concern applies here: analyses involving VNT results should account for the fact that VNT was not conducted on ELISA RBD negative samples.

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Reviewer #1: No

Reviewer #2: No

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-->PONE-D-26-02880R1-->-->Serological Evidence of SARS-CoV-2 Exposure in Marine Mammals in the United States between 2020 and 2025-->-->PLOS One

Dear Dr. Sanogo,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Jun 18 2026 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:-->

• A letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

-->

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

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As the corresponding author, your ORCID iD is verified in the submission system and will appear in the published article. PLOS supports the use of ORCID, and we encourage all coauthors to register for an ORCID iD and use it as well. Please encourage your coauthors to verify their ORCID iD within the submission system before final acceptance, as unverified ORCID iDs will not appear in the published article. Only  the individual author can complete the verification step; PLOS staff cannot  verify ORCID iDs on behalf of authors.

We look forward to receiving your revised manuscript.

Kind regards,

Stephen Raverty

Academic Editor

PLOS One

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Additional Editor Comments:

There are minor edits which should addressed prior to publication of this manuscript. Thank you for your detailed responses to the reviewers comments.

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Reviewers' comments:

Reviewer's Responses to Questions

-->Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.-->

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

-->2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. -->

Reviewer #1: Yes

Reviewer #2: Yes

**********

-->3. Has the statistical analysis been performed appropriately and rigorously? -->

Reviewer #1: Yes

Reviewer #2: Yes

**********

-->4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.-->

Reviewer #1: Yes

Reviewer #2: Yes

**********

-->5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.-->

Reviewer #1: Yes

Reviewer #2: Yes

**********

-->6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)-->

Reviewer #1: Authors solve all queries. Just a few minor comments.

It will be desirable to include representative figures of the controls, positives and negatives samples of the seroneutralization test.

Protein A/G binds to the FC of any immunoglobulin, is not specific for IgG, therefore authors are reporting total Ig.

Inganäs, M. (1981). Comparison of Mechanisms of Interaction between Protein A from Staphylococcus aureus and Human Monoclonal IgG, IgA and IgM in Relation to the Classical Fcγ and the Alternative F (ab') 2γ Protein A Interactions. Scandinavian journal of immunology, 13(4), 343-352.

Scientific names should be used more than common names, once both names were mentioned.

Reviewer #2: (No Response)

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Reviewer #1: No

Reviewer #2: No

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-->

Serological Evidence of SARS-CoV-2 Exposure in Marine Mammals in the United States between 2020 and 2025

PONE-D-26-02880R2

Dear Dr. Sanogo,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Mohamed Samy Abousenna, Ph.D

Academic Editor

PLOS One

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

-->Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.-->

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

Reviewer #4: All comments have been addressed

**********

-->2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. -->

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

**********

-->3. Has the statistical analysis been performed appropriately and rigorously? -->

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

**********

-->4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.-->

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

**********

-->5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.-->

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

**********

-->6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)-->

Reviewer #1: (No Response)

Reviewer #2: (No Response)

Reviewer #3: Overall, the revised manuscript is substantially improved, and most of the previous comments have been adequately addressed. I have one minor suggestion regarding the concluding paragraph. The discussion around the possible establishment of endemic infection and subsequent viral evolution in marine mammal hosts may imply a stronger conclusion than is directly supported by the current data, given the absence of PCR-positive animals and the limited serological evidence presented. Therefore, the authors may wish to consider slightly softening this wording so that the conclusions remain closely aligned with the finding

Reviewer #4: Reviewer comment #1

1. Abstract and Introduction

The Introduction is well written and provides a good background on SARS-CoV-2 spillover and marine mammal susceptibility. However, it does not fully justify the study design, hypothesis, significance of serology, or the interpretation of findings reported in the abstract as follows:

1.1. Aim and hypothesis: To make the study scientifically stronger, the author should sufficiently define the objectives and hypothesis and emphasize that this study is the first to provide serological evidence of SARS-CoV-2 exposure in marine mammals in the United States.

1.2. There is a lack of a clear rationale for combining PCR and serology. As the abstract reports both RT-qPCR and serological testing, the Introduction mainly discusses infection risk. It does not clearly explain why antibody surveillance is essential in addition to PCR testing.

1.3. The abstract emphasizes that this is the “first serological evidence” of SARS-CoV-2 exposure in marine mammals in the United States. However, the Introduction does not adequately explain why serological detection alone is epidemiologically important.

Could the authors explain the significance of detecting neutralizing antibodies in marine mammals despite the absence of active infection by RT-qPCR?

1.4. Additionally, given the gap, the authors should note which surveillance studies have already been conducted globally and which gaps remain.

Reviewer comment #2

2. Methodology

The methodology is strong and suitable for its objectives, with notable strengths in laboratory workflow and confirmatory testing. The study demonstrates excellent adherence to ethical standards. It includes a large, diverse sample and a rigorous serological testing strategy.

Below are some improvements that could be clarified to enhance the study's rigour:

2.1. Have the authors recorded any demographic data for the sampled animals, such as sex, age, and clinical condition, since environmental factors may influence serological findings? Please add demographic and environmental variables.

2.2. According to Table 1, the geographic and temporal distributions of the samples collected between 2020 and 2025 were not included in the table. Could the authors add additional information regarding the sampling locations and yearly distribution of the collected samples, if applicable?

Reviewer comment #3

3. Results

The results are clearly displayed and well structured. There are no major methodological errors.

Reviewer comment #4

4. Discussion

The discussion is well written and has an excellent start emphasizing that this study is the first in investigating SARS-CoV-2 in several marine mammal species on both the East and West coasts of the U.S.

Reviewer comment #5

5. Figures

Figures 1 and 2 need to be prepared in Illustrator to ensure the highest resolution. Alternatively, they can be exported in high-quality vector formats, such as PDF or EPS, which are preferred for publication.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Reviewer #4: Yes: Ruba Abdulrahman Ashy

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Attachments

Attachment

Submitted filename: Comments-PLOs One-PONE-D-26-02880_R2. Reviewer 2.pdf