-->PONE-D-26-16745-->-->Preliminary Molecular Characterization of Foot-and-Mouth Disease Virus SAT-1 Topotype III Associated with Recent Cattle Outbreaks in Syria-->-->PLOS One

Dear Dr. Kassouha,

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Reviewers' comments:

Reviewer's Responses to Questions

-->Comments to the Author

1. 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: Partly

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

Reviewer #1: Yes

Reviewer #2: N/A

Reviewer #3: N/A

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-->3. 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: No

Reviewer #3: Yes

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-->4. 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

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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: 1. Data Availability Statement is incorrect)The authors state: “No data was generated by this study.” This is factually incorrect. Two novel VP1 sequences were generated and submitted to GenBank (accessions PZ124873, PZ124874). The data availability statement must be revised accordingly, e.g.:“The VP1 sequence data generated during this study are available in GenBank under accessions PZ124873 and PZ124874. Other relevant data are within the manuscript.”

2. Sample size and representativeness are very limited. Only six farms were sampled, with sequencing performed on just two isolates. Given the claim of a“complex epidemiological shift,”the authors should acknowledge this as a preliminary observation and avoid overgeneralizing to all of Syria.

3. Missing critical methodological details. PCR annealing temperatures are vaguely reported as “50, 55, or 60°C depending on serotype.”The exact temperature used for SAT-1 must be stated.

4. Phylogenetic tree presentation is poor.(1) Figure 3 is unreadable in the current format (font too small, branches unclear).(2) Bootstrap values are mentioned but not clearly shown on key nodes.(3) The tree should be simplified or split into a main figure + supplementary.

5. Unsupported claims in Discussion.(1) “Virus did not escape from vaccine production facilities”–no data support this. Speculation should be removed or clearly labeled as hypothesis.(2)“Egypt and Jordan are not the source” while plausible, absence of evidence is not evidence of absence. Tone should be more cautious.

6. Ethics statement issues. Verbal consent from farmers is noted but not in line with standard ethical guidelines for animal studies. Written consent or a formal waiver should be documented. The statement should clarify whether any animals were euthanized (apparently none).

6.Writing & Language

(1) Line 2 (page 7): “outbreak”→should be “outbreaks” (plural). Line 5 (page 7): “have raised”→“has raised” (referring to “recent outbreak”). Several sentences are overly long and complex; consider splitting for clarity.

7. Results section. The finding of 8 Syrian O/ME-SA/SA-2018 sequences (page 20) is interesting but feels disconnected from the main SAT-1 focus. This should be briefly integrated into the Discussion, not just reported.

(1)Figure 2 (clinical photos) is useful but lacks scale bars or annotations.

(2) Figure 4 (amino acid alignment) could be merged into a supplementary table.

Reviewer #2: Major Comments

1) Inadequate historical context of FMD in Syria (critical issue)

The manuscript does not provide a comprehensive epidemiological history of FMD in Syria and instead frames the findings as:

“first documented detection of SAT-1 in Syria”

This claim is insufficiently supported and potentially misleading, given that:

* FMD has been historically reported in Syria with multiple serotypes

* SAT-1 circulation has been documented historically in the region

Required revision

* Clarify whether this represents:

* first recent detection

* first molecular characterization

* or first identification of Topotype III

* Include a structured historical overview of FMD circulation in Syria

2) Insufficient description of vaccination practices

The manuscript mentions that vaccines used in Syria include serotypes O, A, and Asia-1, but provides no further detail.

Missing elements:

* vaccine type (imported/local)

* vaccine strains

* antigenic relevance to circulating viruses

This is particularly important given the discussion of vaccine failure.

Required revision

* Provide detailed information on vaccination practices

* Discuss mismatch between circulating SAT-1 and vaccine composition

* Avoid unsupported claims regarding vaccine failure

3) Limited sequencing dataset

Only two isolates were sequenced despite six positive farms.

This limits:

* representativeness

* phylogenetic inference

* epidemiological conclusions

Required revision

* Justify sample selection

* Acknowledge limitations explicitly

* Avoid broad generalizations

4) Overinterpretation of phylogenetic findings

The manuscript proposes:

* transmission routes across countries

* shared outbreak origins

* potential vaccine-related issues

These conclusions are not sufficiently supported by:

* small sample size

* partial VP1 sequencing

Required revision

* Clearly distinguish speculation from evidence

* Reduce strength of causal claims

5) Methodological transparency is insufficient

Important details are missing:

RT-PCR

* controls (positive/negative)

* validation procedures

Sequencing

* platform (e.g., Sanger)

* quality control

* consensus generation

Phylogenetics

* alignment details

* model justification

* reference sequence list

Required revision

Provide sufficient methodological detail to ensure reproducibility.

6) Data availability statement is incorrect

The manuscript states:

“No data was generated by this study”

This is incorrect.

Required revision

Replace with a compliant statement including GenBank accession numbers.

7) Vaccine update recommendation requires stronger evidence and broader surveillance (critical point)

The manuscript recommends updating vaccination strategies to include SAT-1/III strains.

While this is a reasonable consideration, the recommendation is premature and insufficiently supported.

Key concerns:

* Conclusions are based on only two sequenced isolates

* It is unclear whether:

* SAT-1 Topotype III is the only circulating lineage

* other SAT-1 topotypes (e.g., Topotype I) may also be present

* No antigenic matching data are provided

Required revision

The authors should adopt a more evidence-based and cautious interpretation:

* Vaccine strain selection in FMD should rely on:

* large-scale molecular surveillance across regions and hosts

* continuous sequencing of circulating strains

* antigenic matching studies (e.g., virus neutralization tests, r1-values)

The recommendation should be revised along the following lines:

“While the detection of SAT-1/III highlights a potential gap in current vaccination strategies, expanded nationwide surveillance and antigenic characterization are required to determine the diversity of SAT-1 strains circulating in Syria and to inform evidence-based vaccine strain selection.” According to Abousenna, M.S., Khafagy, H.A., Mohamed, A.A. et al. Emergency response for recently

isolated Foot and Mouth Disease virus type A Africa in Egypt 2022. Scientific Reports , 15, 4475

(2025). https://doi.org/10.1038/s41598-025-88906-4

Abousenna M.S. Emergency evaluation for existing vaccine against recently isolated Foot and

Mouth Disease Virus type SAT2 in Egypt 2018. VacciMonitor , 31(1):9-14 (2022).

This will align the manuscript with established FAO/WRLFMD principles and avoid premature policy recommendations.

Reviewer #3: Dear Authors;

The study contains extremely important findings. Firstly, This is one of the few reports from Syria for a long time. The second but not the least important finding is the circulating virus which is very closely related to a vaccine strain. A very recent study showed that this strain was also responsible from the outbreaks in Iran. You may add this reference before publication. This study had some flaws such as very limited number of samples and lack of serological data that might help to understand the complete picture. Also, vaccines used in that country may help to understand possible source of the outbreaks. Full genome sequencing may also help to date the outbreaks.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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-->PONE-D-26-16745R1-->-->Preliminary Molecular Characterization of Foot-and-Mouth Disease Virus SAT-1 Topotype III Associated with Recent Cattle Outbreaks in Syria-->-->PLOS One

Dear Dr. Kassouha,

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 Jul 27 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.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

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,

Mohamed Samy Abousenna, Ph.D

Academic Editor

PLOS One

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1. If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise.

2. Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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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 #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 #2: Yes

**********

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

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 #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 #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 #2: The authors have made substantial efforts to address the reviewers’ comments, and the manuscript has improved considerably compared with the original submission. The revisions have strengthened the methodological description, clarified the molecular analyses, improved the discussion, and added important information regarding sequencing procedures, study limitations, and data availability.

The study provides valuable information on the molecular characterization of FMDV SAT-1 Topotype III detected in Syria and represents an important contribution to regional Foot-and-Mouth Disease surveillance and epidemiology.

Most of the previous comments have been adequately addressed. However, a few minor issues should be corrected before acceptance:

1. Statements referring to the “first detection” of SAT-1 in Syria should be revised to “first molecularly confirmed detection” or “first molecular characterization” to avoid overstatement.

2. Conclusions regarding vaccine failure should be moderated, as no vaccine efficacy data were generated in the present study. The discussion should instead refer to a potential vaccine mismatch or insufficient antigenic coverage.

3. Some epidemiological interpretations regarding virus origin and transmission routes remain speculative and should be expressed more cautiously.

4. Information based on personal communications should be replaced with citable references where possible or removed.

5. The reference list requires careful verification. In particular, Reference 22 contains an apparent error in the first author’s initials (“Abousenna MJV”), which should be corrected to “Abousenna MS.” The authors are encouraged to check the entire bibliography for similar citation errors.

6. Minor English-language editing is still recommended to improve clarity and readability.

Overall, the manuscript is scientifically sound, addresses an important topic, and is close to being suitable for publication. I recommend acceptance after minor revision.

**********

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Reviewer #2: No

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-->

Preliminary Molecular Characterization of Foot-and-Mouth Disease Virus SAT-1 Topotype III Associated with Recent Cattle Outbreaks in Syria

PONE-D-26-16745R2

Dear Dr. Kassouha,

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):

The reviewer comments have been adequately addressed; however, please perform a final careful editorial revision to improve the English language, grammar, consistency, and clarity throughout the manuscript before acceptance.

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 #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 #2: Yes

**********

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

Reviewer #2: N/A

**********

-->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 #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 #2: No

**********

-->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 #2: (No Response)

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Reviewer #2: No

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