Identification of an optimal exogenous gene insertion site (P–M) and establishment of a reverse genetics system for aMPV/A

Yu Guo; Ting Xue; Bingqing Lu; Jiqing Zheng; Yirong Wang; Xueqiao Xie; Huiting Wang; Furui Cao; Yijun Zhang; Jianhua Wang

doi:10.1371/journal.pone.0347597

Peer Review History

Original SubmissionFebruary 11, 2026
4 Mar 2026 Decision Letter - Haitham Amer, Editor Dear Dr. Guo, 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.. 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.. 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.. 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. 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 . 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 . 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 . 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.... We look forward to receiving your revised manuscript. Kind regards, Haitham Mohamed Amer, PhD Academic Editor PLOS One Journal Requirements: When submitting your revision, we need you to address these additional requirements. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf andandandand https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. Please note that PLOS One has specific guidelines on code sharing for submissions in which author-generated code underpins the findings in the manuscript. In these cases, we expect all author-generated code to be made available without restrictions upon publication of the work. Please review our guidelines at https://journals.plos.org/plosone/s/materials-and-software-sharing#loc-sharing-code and ensure that your code is shared in a way that follows best practice and facilitates reproducibility and reuse. 3. We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match. When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section. 4. Thank you for stating the following financial disclosure: “This work was financially supported by Start-up fund for New Ph.D. Researchers of Suzhou Chien-Shiung Institute of Technology. The Natural Science Foundation of Jiangsu Province (BK20220301). Jiangsu Province high level innovation and entrepreneurship talent introduction plan (JSSCBS20221009). Natural Science Foundation of the Jiangsu Higher Education Institutions (25KJB320013). Science Projects of Taicang City, China (TC2022JC29). Qing Lan Project (Su Teacher [2024]14). Start-up fund for New Ph.D. Researchers of Suzhou Chien-Shiung Institute of Technology.” Please state what role the funders took in the study. If the funders had no role, please state: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." If this statement is not correct you must amend it as needed. Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf. 5. Please note that funding information should not appear in any section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. Please remove any funding-related text from the manuscript. 6. We note that your Data Availability Statement is currently as follows: “If the data are all contained within the manuscript and/or Supporting Information files, enter the following: All relevant data are within the manuscript and its Supporting Information files.” Please confirm at this time whether or not your submission contains all raw data required to replicate the results of your study. Authors must share the “minimal data set” for their submission. PLOS defines the minimal data set to consist of the data required to replicate all study findings reported in the article, as well as related metadata and methods (https://journals.plos.org/plosone/s/data-availability#loc-minimal-data-set-definition). For example, authors should submit the following data: - The values behind the means, standard deviations and other measures reported; - The values used to build graphs; - The points extracted from images for analysis. Authors do not need to submit their entire data set if only a portion of the data was used in the reported study. If your submission does not contain these data, please either upload them as Supporting Information files or deposit them to a stable, public repository and provide us with the relevant URLs, DOIs, or accession numbers. For a list of recommended repositories, please see https://journals.plos.org/plosone/s/recommended-repositories. If there are ethical or legal restrictions on sharing a de-identified data set, please explain them in detail (e.g., data contain potentially sensitive information, data are owned by a third-party organization, etc.) and who has imposed them (e.g., an ethics committee). Please also provide contact information for a data access committee, ethics committee, or other institutional body to which data requests may be sent. If data are owned by a third party, please indicate how others may request data access. 7. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels. In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions. 8. Please upload a new copy of Figures 3 and 4 as the detail is not clear. Please follow the link for more information: https://journals.plos.org/plosone/s/figures 9. 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. Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? Reviewer #1: Yes Reviewer #2: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? -->?> Reviewer #1: Yes Reviewer #2: N/A ****** 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.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: No Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English??> Reviewer #1: Yes Reviewer #2: No ****** Reviewer #1: 1. Study design and experimental unit (critical) The manuscript does not clearly define: • the true experimental unit • whether the analysis is based on: individual animals/samples or flocks or farms or time points This is essential to avoid pseudoreplication. ➡️ You must explicitly state: • what n represents in each analysis • how many independent units were used • whether repeated measurements were accounted for If measurements come from the same flock over time, a mixed model / repeated-measures approach is required. 2. Sample size justification There is no power calculation or rationale for sample size. you should state: how the sample size was determined or that all available samples were included (for retrospective studies) 3. Insufficient methodological detail (reproducibility issue) Several essential details are missing or unclear: Biological material • source • inclusion/exclusion criteria • health status • management conditions For PCR / molecular work: • primer sequences (or proper citation) • cycling conditions • amplicon size • positive & negative controls • limit of detection 4. Statistical analysis – incomplete and sometimes inappropriate The statistical section needs major improvement: You must report: • software used • test for normality • exact statistical tests for each variable • post-hoc test (if multiple comparisons) • confidence intervals (recommended for PLOS ONE) • definition of significance level 5. Confounding factors not controlled The manuscript does not sufficiently account for: • time effects • management changes • environmental variation • genetic line differences • seasonal effects These must be: • either controlled statistically • or discussed as limitations 6. Overinterpretation of results Several conclusions imply causation, while the design only supports association. Change wording such as: “improved”, “resulted in”, “led to” to “was associated with”, “showed higher”, “suggests” 8. Introduction – needs a sharper knowledge gap Currently too descriptive. Add: • what is unknown • why this study is necessary • the specific hypothesis End the introduction with a clear, testable objective. 9. Results section The Results section sometimes: • repeats methods • includes interpretation Move all interpretation to the Discussion. • Always report exact P values • Provide measures of variation (SD or SEM) 10. Discussion – structure The discussion should follow this order: 1. Main finding 2. Comparison with previous studies 3. Biological explanation 4. Practical relevance 5. Limitations 6. Future directions Currently, the flow is not always logical. Abbreviations Define at first use. Tables & figures • Must be self-explanatory • Include statistical test used in the legend • Define error bars Reviewer #2: PONE-D-26-07403 Optimal Exogenous Gene Insertion Site (P-M) and Reverse Genetics System for aMPV/A Avian metapneumovirus is an important avian pathogen that causes not only respiratory diseases, such as turkey rhinotracheitis and swollen head syndrome, but also reproductive disorders in multiple bird species. Currently, aMPV is classified into four major subtypes. In this study, the authors describe the construction of a reverse genetics system for aMPV/A and systematically evaluate seven intergenic regions for insertion of an exogenous EGFP reporter gene, identifying the P–M junction as the most favorable site based on replication capacity, transgene expression, and genetic stability. The work has potential relevance for aMPV vector development; however, substantial issues in methodology, data presentation, and interpretation remain, and major revision is required before the manuscript can be considered for publication. -The manuscript contains several major scientific concerns that must be addressed. Most critically, inconsistent subtype labeling (aMPV/A vs. rMPV/B and raMPV/C) raises doubts about data validity and must be clarified. The study also fails to report compliance with the paramyxovirus rule of six, genome design details, and key reverse genetics parameters, which could confound replication comparisons. Interpretations regarding the transcription gradient and the designation of the P–M junction as the “optimal” insertion site are plausible but overstated given the absence of supporting mRNA/protein quantification, extended stability testing, and control for alternative explanations. Overall, additional methodological detail, validation, and more cautious interpretation are required to support the conclusions. Title -The title can be changed as follows: Identification of an Optimal Exogenous Gene Insertion Site (P–M) and Establishment of a Reverse Genetics System for aMPV/A abstract -The claim that vector vaccine development is hindered solely by the lack of a reverse genetics system and unclear insertion sites is inaccurate as it is not the only reason! - Please include key quantitative results for example fold differences in EGFP expression, viral titer comparisons, and passage stability, etc -Also revise the sentence to: This study establishes a stable and efficient reverse genetics system for aMPV/A… Introduction -Line 50-51 aMPV poses a significant threat to the global poultry industry, causing substantial50 economic losses[7]. can be moved to begingin of introduction -Line 48 revise as follow Subtypes A and B are the most widely distributed aMPV lineages globally, although their regional prevalence varies and other subtypes may predominate in specific geographic areas -The novelty is not clearly defined given prior insertion-site studies; What gap specifically exists for subtype A? Methods 2.1 Cell Line, Virus, Plasmids, and Nucleic Acid This subtitle does not make sense. Change it to "Cells" and explain the cell line after mentioning which viruses are used. Move the plasmid details to 2.2. Revise, please, to: BSR T7/5 and Vero cells were maintained in our laboratory. containing 10% fetal bovine serum (FBS) and antibiotics -The methods section lacks critical detail for reproducibility, like genome design or transfection conditions, I mean exact genome length after insertion, gene start/stop signals, intergenic sequences used, cloning strategy details and rescue efficiency -Statistical analysis is also insufficiently described. Results -Virus growth curve analysis revealed that…Do you mean Growth-curve analysis demonstrated that? For most paramyxoviruses like metapneumovirusesthe genome length must be a multiple of six nucleotides Otherwise rescue efficiency and replication are impaired! So not he question is whether the rule of six was maintained final genome lengths and whether EGFP insertions were adjusted accordingly? -The figures need more detailed captions -English language editing is strongly recommended. ****** what does this mean?). If published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our For information about this choice, including consent withdrawal, please see our For information about this choice, including consent withdrawal, please see our For information about this choice, including consent withdrawal, please see our Privacy Policy..--> Reviewer #1: Yes: Aalaa S. A. SaadAalaa S. A. SaadAalaa S. A. SaadAalaa S. A. Saad Reviewer #2: No ******** https://doi.org/10.1371/journal.pone.0347597.r001
Revision 1
8 Mar 2026 Author Response Response to Reviewers Response to academic editor： 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. Reply：The manuscript has been revised according to the format of PLOS ONE. All the modifications have been marked as the highlighted yellow parts in the text. 2. Please note that PLOS One has specific guidelines on code sharing for submissions in which author-generated code underpins the findings in the manuscript. Reply：There is no information about code in my research. 3. We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match. Reply：I have revised the Financial Disclosure based on the content of Funding Information. 4. Please state what role the funders took in the study. If the funders had no role, please state: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." Reply：Funder statement：The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. 5. Please note that funding information should not appear in any section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. Please remove any funding-related text from the manuscript. Reply：The funding information has been deleted. 6. Please confirm at this time whether or not your submission contains all raw data required to replicate the results of your study. Reply：All the original data have been supplemented and uploaded to the supplementary file. 7. In your cover letter, please notewhether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Reply：All the original data have been supplemented and uploaded to the supplementary file. 8. Please upload a new copy of Figures 3 and 4 as the detail is not clear. Please follow the link for more information: Reply：I can assure you that the uploaded images are high-definition ones in accordance with the journal's requirements. However, after the PDF was generated, the images became very blurry. I have tried many methods but the situation remains the same. I'm very sorry. However, I have re-uploaded the pictures, hoping this will be helpful. 9. 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. Reply：No similar problems occurred. Response to Reviewer 1： 1. Study design and experimental unit (critical) Reply：In the "2. MATERIALS AND METHODS" section, a new subsection "2.8 Experimental unit and replication design" has been added. The research design and experimental units were elaborately described. Line:203-210 2. Sample size justification Reply：In the "2. MATERIALS AND METHODS" section, a new subsection "2.8 Experimental unit and replication design" has been added. It indicates that n is greater than or equal to 3, which means each experiment is replicated more than three times. Line:203-210 3. Insufficient methodological detail (reproducibility issue) Reply：The primer sequences used in the study have been uploaded as supplementary files. 4. Statistical analysis – incomplete and sometimes inappropriate Reply：The section on 2.7 Statistical Analysis has been revised again. 5. Confounding factors not controlled Reply：In Section 2.1, the standardized control of experimental conditions is supplemented, and the aforementioned confounding factors are explicitly excluded: All cell cultures and virus experiments are conducted in a controlled laboratory environment. Line:196-202 6. Overinterpretation of results Reply：Vague expressions such as “were associated with” and “showed higher” have been replaced with precise alternatives: “suggests”, “improved”, “resulted in” and “led to”. 7. Introduction – needs a sharper knowledge gap Currently too descriptive. Reply：The introduction section was supplemented and revised as per the requirements, and a clear research objective was added at the end. Line:115-122 8. Results section：repeats methods， includes interpretation Reply：Make the necessary revisions based on the opinions. Remove the repetitive descriptions of the methods in the results section and place all the explanatory content in the discussion section. 9. Discussion – structure Reply：The discussion section has been revised in accordance with the comments, and the consistency of abbreviations, as well as the presentation and formatting of figures and tables, have been carefully reviewed and verified throughout the manuscript. Response to Reviewer 2： 1. Title Reply：It has been revised to: Identification of an Optimal Exogenous Gene Insertion Site (P–M) and Establishment of a Reverse Genetics System for aMPV/A Line:1-2 2. Abstract: The claim that vector vaccine development is hindered solely by the lack of a reverse geneticssystem and unclear insertion sites is inaccurate as it is not the only reason! Reply：It has been revised to: The development of highly efficient viral vector vaccines is constrained by multiple factors, among which the lack of a mature reverse genetics system and the uncertainty regarding the optimal insertion site for foreign genes represent one of the core technical bottlenecks. Line:21-24 3. Abstract: Please include key quantitative results for example fold differences in EGFP expression, viral titer comparisons, and passage stability, etc. Reply：Based on the suggestions, revisions were made as follows: The results demonstrated that the expression level at the P M locus after EGFP insertion was approximately 1.2 fold higher than that of other recombinant viruses. In addition, there was no significant difference in viral titer, and good genetic stability was observed over 20 generations. Line:27-33 4. Also revise the sentence to: This study establishes a stable and efficient reverse genetics system for aMPV/A… Reply：Based on the suggestions, revisions were made as follows: This study establishes a stable and efficient reverse genetics system for aMPV/A, which provides a critical technical platform for aMPV/A molecular mechanism research and novel recombinant vector vaccine development, with important practical value for global aMPV prevention and control. 5. Line 50-51 aMPV poses a significant threat to the global poultry industry, causing substantial50 economic losses[7]. can be moved to begingin of introduction Reply：Put in the first sentence of the first paragraph of the introduction. Line:39-40 6. Line 48 revise as follow Subtypes A and B are the most widely distributed aMPV lineages globally, although their regional prevalence varies and other subtypes may predominate in specific geographic areas Reply：Based on the suggestions, revisions were made as follows: Subtypes A and B are the most widely distributed aMPV lineages globally, although their regional prevalence varies and other subtypes may predominate in specific geographic areas. Line:49-51 7. The novelty is not clearly defined given prior insertion-site studies; What gap specifically exists for subtype A? Reply：Based on the suggestions, revisions were made as follows: Prior studies have identified optimal exogenous gene insertion sites and established reverse genetics systems for aMPV/C subtypes[17,18,28], whereas no systematic investigation has been conducted on insertion site selection for aMPV/A—a subtype that has recently re-emerged in North America (USA and Mexico) and caused large-scale outbreaks in the poultry industry[8,9,10]. To address this gap, the present study constructed a pBR322-T7 RNA polymerase dual-based aMPV/A reverse genetics system and systematically evaluated seven intergenic insertion sites, representing the first report on the optimal exogenous gene insertion locus for aMPV/A to date. Line:116-123 8. 2.1 Cell Line, Virus, Plasmids, and Nucleic Acid This subtitle does not makesense. Change it to "Cells" and explain the cell line after mentioning which viruses are used. Move the plasmid details to 2.2. Reply：According to the suggestions, the section "2.2 Construction of recombinant virus and auxiliary plasmids" has been supplemented with "Virus and nucleic acid information", "Genome design and compliance with the Rule of Six", "Details of gene start/stop and intergenic sequences", "Cloning strategy details" and "Supplement on plasmid construction". Line:136-159 9. -Statistical analysis is also insufficiently described. Reply：Based on the revision suggestions, I have rewritten 2.7 Statistical Analysis and added 2.8 Experimental Unit and Replication Design. Line:197-211 10. Results-Virus growth curve analysis revealed that…Do you mean Growth-curve analysis demonstrated that? Reply：The original sentence “Virus growth curve analysis revealed that…” was revised to: Growth-curve analysis demonstrated that. Line:214 11. So not he question is whether the rule of six was maintained final genomelengths and whether EGFP insertions were adjusted accordingly? Reply：The 6-base principle has been added to the "Genome design and Rule of Six compliance" section in the "2.2 Construction of recombinant virus and auxiliary plasmids" subsection. Line:141-146 12. The figures need more detailed captions Reply：Modify and supplement all the legends. Line:160-167;219-226;233-243;250-260 13. English language editing is strongly recommended. Reply：Revise the entire text in English to make it smooth and fluent, following English expression conventions. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0347597.r002
23 Mar 2026 Decision Letter - Haitham Amer, Editor Dear Dr. Guo, 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 May 07 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.. 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.. 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.. 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. 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 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 . 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 . 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 . 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.... We look forward to receiving your revised manuscript. Kind regards, Haitham Mohamed Amer, PhD Academic Editor PLOS One Journal Requirements: 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. Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 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??> Reviewer #1: Yes Reviewer #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? -->?> Reviewer #1: Yes Reviewer #2: I Don't Know ****** 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.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 Response) ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English??> Reviewer #1: Yes Reviewer #2: No ****** Reviewer #1: 1. Inconsistent virus nomenclature in Results section 3.1: The text states "rMPV/B-NP-EGFP" and "rMPV/B-GL-EGFP" (page 46, lines 214-217), but the manuscript is focused on aMPV/A. This appears to be a typographical error; it should likely read "raMPV/A-NP-EGFP" and "raMPV/A-GL-EGFP" to be consistent with the rest of the document and Figure 2. 2. Incorrect section numbering: In the Results section, the subheadings for exogenous gene expression (page 46, line 233) and genetic stability (page 47, line 244) are numbered 2.2 and 2.3. These should be 3.2 and 3.3 to align with the main section numbering. 3. Gene stop signal inconsistency: In the methods (page 43, line 151), the gene stop signal is listed as 3'- tattagaaaaaa- 3', which appears to be a correction from the earlier version. However, the abstract and results still refer to the P-M locus. The authors should double-check that the correct gene start/stop sequences are consistently used throughout the manuscript and supplementary files. 4. Figure 2 y-axis label: The label currently reads "logio TCIDso/mL" (page 46, line 226). This should be corrected to "log₁₀ TCID₅₀/mL". Reviewer #2: The comments have been addressed and are now ready for publication. However, some language editing and minor technical formatting remain, which can be addressed after acceptance. ****** what does this mean?). If published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our For information about this choice, including consent withdrawal, please see our For information about this choice, including consent withdrawal, please see our For information about this choice, including consent withdrawal, please see our Privacy Policy..--> Reviewer #1: Yes: Aalaa S. A. SaadAalaa S. A. SaadAalaa S. A. SaadAalaa S. A. Saad Reviewer #2: No ******** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] To ensure your figures meet our technical requirements, please review our figure guidelines: https://journals.plos.org/plosone/s/figures You may also use PLOS’s free figure tool, NAAS, to help you prepare publication quality figures: https://journals.plos.org/plosone/s/figures#loc-tools-for-figure-preparation. NAAS will assess whether your figures meet our technical requirements by comparing each figure against our figure specifications. Attachments Attachment Submitted filename: R2.docx https://doi.org/10.1371/journal.pone.0347597.r003
Revision 2
24 Mar 2026 Author Response Response to Reviewers Response to Reviewer#1： 1. Inconsistent virus nomenclature in Results section 3.1: The text states "rMPV/B-NP-EGFP" and "rMPV/B-GL-EGFP" (page 46, lines 214-217), but the manuscript is focused on aMPV/A. This appears to be a typographical error; it should likely read "raMPV/A-NP-EGFP" and "raMPV/A-GL-EGFP" to be consistent with the rest of the document and Figure 2. Reply：Thank you for your feedback. The text has been updated to read "raMPV/A-NP-EGFP" and "raMPV/A-GL-EGFP" on lines 214-217. 2. Incorrect section numbering: In the Results section, the subheadings for exogenous gene expression (page 46, line 233) and genetic stability (page 47, line 244) are numbered 2.2 and 2.3. These should be 3.2 and 3.3 to align with the main section numbering. Reply：Thank you for your feedback. The sequence numbers of the subheadings have been changed to 3.2 and 3.3. (This change has been made on lines 233 and 244.) 3. Gene stop signal inconsistency: In the methods (page 43, line 151), the gene stop signal is listed as 3'- tattagaaaaaa- 3', which appears to be a correction from the earlier version. However, the abstract and results still refer to the P-M locus. The authors should double-check that the correct gene start/stopsequences are consistently used throughout the manuscript and supplementary files. Reply：Thank you for the correction. The "GS: 5'- ggacaagtcaaa-3' " and "gene stop (GS: 5'-ttatgaaaaaaa-3')" signals have been changed to lines 148-149. 4. Figure 2 y-axis label: The label currently reads "logio TCIDso/mL" (page 46, line 226). This should be corrected to "log₁₀ TCID₅₀/mL". Reply：Thank you for the correction. The y-axis of FIG2 has been modified to log₁₀ TCID₅₀/mL. Attachments Attachment Submitted filename: Response to Reviewers2.docx https://doi.org/10.1371/journal.pone.0347597.r004
5 Apr 2026 Decision Letter - Haitham Amer, Editor Identification of an Optimal Exogenous Gene Insertion Site (P–M) and Establishment of a Reverse Genetics System for aMPV/A PONE-D-26-07403R2 Dear Dr. Guo, 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. An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. For questions related to billing, please contact and clicking the ‘Update My Information' link at the top of the page. For questions related to billing, please contact and clicking the ‘Update My Information' link at the top of the page. For questions related to billing, please contact and clicking the ‘Update My Information' link at the top of the page. For questions related to billing, please contact billing support.... 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, Haitham Mohamed Amer, PhD Academic Editor PLOS One https://doi.org/10.1371/journal.pone.0347597.r005
Formally Accepted
Acceptance Letter - Haitham Amer, Editor PONE-D-26-07403R2 PLOS One Dear Dr. Guo, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS One. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset You will receive further instructions from the production team, including instructions on how to review your proof when it is ready. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few days to review your paper and let you know the next and final steps. Lastly, if your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. 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. You will receive an invoice from PLOS for your publication fee after your manuscript has reached the completed accept phase. If you receive an email requesting payment before acceptance or for any other service, this may be a phishing scheme. Learn how to identify phishing emails and protect your accounts at https://explore.plos.org/phishing. If we can help with anything else, please email us at customercare@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Haitham Mohamed Amer Academic Editor PLOS One https://doi.org/10.1371/journal.pone.0347597.r006

Open letter on the publication of peer review reports

PLOS recognizes the benefits of transparency in the peer review process. Therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. Reviewers remain anonymous, unless they choose to reveal their names.

We encourage other journals to join us in this initiative. We hope that our action inspires the community, including researchers, research funders, and research institutions, to recognize the benefits of published peer review reports for all parts of the research system.

Learn more at ASAPbio .