A highly efficient and accurate method of detecting and subtyping Influenza A pdm H1N1 and H3N2 viruses with newly emerging mutations in the matrix gene in Eastern Taiwan

Hui-Hua Yang; I-Tsong Huang; Ren-Chieh Wu; Li-Kuang Chen

doi:10.1371/journal.pone.0283074

Peer Review History

Original SubmissionApril 9, 2022
23 Aug 2022 Decision Letter - Maria Elisabeth Johanna Zalm, Editor PONE-D-22-10495A highly efficient and accurate method for detecting and subtyping Influenza A pdm H1N1 and H3N2 viruses with newly emerging mutations in the matrix gene in Eastern TaiwanPLOS ONE Dear Dr. Chen, 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. Your manuscript has been reviewed by two peer-reviewers and their reports are appended below. The reviewers comment that aspects of the study need further explanation, clarification, or review in the terminology used. In addition, the reviewers comment that some of the methodology requires a bit more detail, and that the study does not present the required statistics to assess and compare the sensitivity of the primer sets and probes used in this study. Can you please address the comments raised by the reviewers? Please submit your revised manuscript by Oct 07 2022 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 rebuttal 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. We look forward to receiving your revised manuscript. Kind regards, Maria Elisabeth Johanna Zalm, Ph.D Editorial Office 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 and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. Please provide additional details regarding participant consent. In the ethics statement in the Methods and online submission information, please ensure that you have specified (1) whether consent was informed and (2) what type you obtained (for instance, written or verbal, and if verbal, how it was documented and witnessed). If your study included minors, state whether you obtained consent from parents or guardians. If the need for consent was waived by the ethics committee, please include this information. If you are reporting a retrospective study of medical records or archived samples, please ensure that you have discussed whether all data were fully anonymized before you accessed them and/or whether the IRB or ethics committee waived the requirement for informed consent. If patients provided informed written consent to have data from their medical records used in research, please include this information. 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 in the Acknowledgments Section of your manuscript: This work was supported by Grants from the Centers for Disease Control, R.O.C (Taiwan). (HZ099103, HZ100102, HZ101060, CH102055, CW103035). We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments 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 and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows: The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Please include your amended statements within your cover letter; we will change the online submission form on your behalf. [Note: HTML markup is below. Please do not edit.] 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: Partly Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: No 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. Reviewer #1: Yes Reviewer #2: Yes ****** 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: No Reviewer #2: Yes ****** 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 entitled “ A highly efficient and accurate method for detecting and subtyping Influenza A pdm H1N1 and H3N2 viruses with newly emerging mutations in the matrix gene in Eastern Taiwan" developed an updated and faster method for detection and subtyping of influenza viruses from clinical samples using real-time PCR. The manuscript highlight the importance of regular checking and update of the sequence of primers and probes used for routine diagnosis of influenza from clinical samples however there are a few aspects that require attention before it is suitable for publication : 1- The manuscript has to be revised for English language , there is several words used which is giving another meaning and impression like : line 56 : Influenza A virus strains can "Stably" infect humans... : Stable is not the right word Line 91: Primers and Probes failing to "Adhere" : Bind is much better to use here Line 319 : Influenza virus genome segment "Develops" , "evolve" would be the correct word. and so on , the mistakes with the English Language is repeated in several sections of the MS so i would recommend professional English editing before publication. 2-Line :152 , Please give more details regarding your positive and negative controls for both real-time PCR and conventional PCR. 3- You used the Term "RNA sequencing" , this is somehow misleading since you only made sequencing of the viral cDNA and not the RNA. 4- No statistics made , Statistical analysis has to be made to address the significance and compare the sensitivity of the 2 sets of primers and probes for better conclusion. Reviewer #2: The manuscript entitled” A highly efficient and accurate method for detecting and subtyping Influenza A pdm H1N1 and H3N2 viruses with newly emerging mutations in the matrix gene in Eastern Taiwan” (Manuscript Number: PONE-D-22-10495) was reviewed, the number of samples were studied in this manuscript are very valuable. These results can provide information on clinically correct medication and vaccine design, but some cases not clear such as: 1) As mentioned in this paper the aim of this project was avoid the false negatives caused using the WHO-recommended primers and probes for detecting influenza A and B viruses, and to shorten total time needed to differentiate influenza A and B. Authors must be clarified this time under 2.5 hours, is include the time of viral RNA extraction? If is include, how many samples? 2) Considering that there are currently rapid one-step commercial kits and some papers, for detecting several respiratory viruses at the same time, authors must be comprised their methods with these commercial kits in the discussion section of the article. such as: A multiplex real-time RT-PCR for simultaneous detection of four most common avian respiratory viruses ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). 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 Privacy Policy. Reviewer #1: No 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.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. Attachments Attachment Submitted filename: PONE-D-22-10495(1).docx https://doi.org/10.1371/journal.pone.0283074.r001
Revision 1
19 Oct 2022 Author Response Responses to Editors and Reviewers (PONE-D-22-10495) We would like to thank the editor and reviewers for their extensive assessment of our manuscript, and for important and helpful comments and suggestions. We have taken all the remarks into account, in a manner that is described in detail below together with our responses to comments. We have responded to all the editor’s and reviewer’s comments in a point-by-point fashion and have revised the manuscript accordingly. The revised portions are indicated by “Track Changes”. An “unmarked” clean version of revised manuscript has also been provided. We think that, following these suggestions, our manuscript has gained in clarity and hope that the changes made will be considered satisfactory. One additional remark is that please update our Funding Statement in the online submission, which should read as: “This work was supported by Grants from the Centers for Disease Control, R.O.C (Taiwan). (HZ099103, HZ100102, HZ101060, CH102055, CW103035).” Responses to Editor: Comment 1: Please include the following items when submitting your revised manuscript. Response: We have provided a rebuttal letter that responds to the academic editor and reviewers in a point-by-point fashion. We have uploaded a marked-up copy of our revised manuscript that highlights changes made to the original version labeled as 'Revised Manuscript with Track Changes'. We have also uploaded an unmarked version without tracked changes labeled as 'Manuscript'. Comment 2: Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. Response: We have ensured that our manuscript meets the style requirements of PLOS ONE. Comment 3: If the need for consent was waived by the ethics committee, please include this information. Response: We have provided a statement to declare that informed consent was waived according to the institutional guidelines (line 126, unmarked version). Comment 4: We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match. Response: We have corrected this mismatching. Comment 5: Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Please include your amended statements within your cover letter; we will change the online submission form on your behalf. Response: We have removed the funding-related text from the manuscript. Also, please update our Funding Statement in the online submission, which should read as: “This work was supported by Grants from the Centers for Disease Control, R.O.C (Taiwan). (HZ099103, HZ100102, HZ101060, CH102055, CW103035).” Responses to Reviewer 1: Comment 1: The manuscript has to be revised for English language, there is several words used which is giving another meaning and impression like: (a) line 56 : Influenza A virus strains can "Stably" infect humans... : Stable is not the right word Response to (a): We have deleted the word “stably” (line 61, unmarked version). (b) Line 91: Primers and Probes failing to "Adhere" : Bind is much better to use here Response to (b): Thank you. We have replaced the word by “bind” (line 92, unmarked version). (c) Line 319: Influenza virus genome segment "Develops" , "evolve" would be the correct word. Response to (c): Thank you. We have replaced the word by “evolve” (line 329, unmarked version). (d) the mistakes with the English Language is repeated in several sections of the MS so i would recommend professional English editing before publication. Response to (d): In response to the recommendation from the reviewer, we have asked help to improve the language of our revised manuscript by a professional English editor (KGSupport). The certificate of this English editing service is provided at the end of this document. Comment 2: Line :152 , Please give more details regarding your positive and negative controls for both real-time PCR and conventional PCR. Response: We thank the reviewer for reminding us this important issue. In response to this comment, we have now provided details regarding our positive and negative controls for both real-time PCR and conventional PCR (lines 170-173, unmarked version). The statement reads as: “We used RNA extracted from Influenza A/New Caledonia/20/99(H1N1), Influenza A/Wisconsin/67/05(H3N2) and Influenza B Florida/07/04 as positive controls and sterile water as the negative control in each run.” Comment 3: You used the Term "RNA sequencing" , this is somehow misleading since you only made sequencing of the viral cDNA and not the RNA. Response: We thank the reviewer for this suggestion. The misleading term has now been replaced by “viral cDNA sequencing” (lines 193 and 194, unmarked version). Comment 4: No statistics made, Statistical analysis has to be made to address the significance and compare the sensitivity of the 2 sets of primers and probes for better conclusion. Response: We thank the reviewer for this excellent suggestion. In response to this comment, we have additionally provided data of analytical sensitivity of the conventional singleplex and our multiplex assays analyzed by standard curves of CT values. For comparisons of CT values and Pearson correlation coefficients between the conventional singleplex and our multiplex assays, we have used independent sample t-tests and no significances were detected. Thus, we have made the following revisions: (1) This additional set of data is presented as Table 5 (page 29, unmarked version). (2) The results of these analyses have been reported in the Result section (lines 248-254 , unmarked version). These statements read as:” Table 5 shows the data of the analytical sensitivity of the multiplex assays developed in this study and the traditional singleplex assays. The CT values for each standard concentration obtained from the use of MAF/R, MBF/R, and InfAF/R in the multiplex assays did not significantly differ from those in the singleplex assays. Further analyses of standard curves of CT values revealed that the correlation coefficients, slopes, and amplification efficiencies of these two types of assays had no significant differences.” (3) The results of these analyses have been reported in the abstract (lines 52-53, unmarked version) and the statement reads as: “The analytical sensitivity of this multiplex RT-PCR assay was as good as that of the conventional singleplex assay.” (4) A paragraph of statistical analysis has been added to the revised manuscript (lines 224-230, unmarked version). We sincerely hope that the reviewer could approve our explanations. Responses to Reviewer 2: Comment 1: As mentioned in this paper the aim of this project was avoid the false negatives caused using the WHO-recommended primers and probes for detecting influenza A and B viruses, and to shorten total time needed to differentiate influenza A and B. Authors must be clarified this time under 2.5 hours, is include the time of viral RNA extraction? If is include, how many samples? Response: We thank the reviewer for reminding us this important issue. The time of viral RNA extraction was not included in the test duration of 2.5 hours, which was indicated only for the multiplex RT-PCR assay. In response to the reviewer’s comment, we have added a statement in the revised manuscript to clarify this issue (lines 370-371, unmarked version). The statement reads as; “The total test duration did not include the time of viral RNA extraction.” Comment 2: Considering that there are currently rapid one-step commercial kits and some papers, for detecting several respiratory viruses at the same time, authors must be comprised their methods with these commercial kits in the discussion section of the article. such as: A multiplex real-time RT-PCR for simultaneous detection of four most common avian respiratory viruses Response: We thank the reviewer for providing the valuable reference, which has been added to our reference list (ref. 25). In response to this comment, we have added a paragraph (lines 378-385, unmarked version) to discuss the methods of that work (ref. 25). The paragraph reads as: “A recent work [25] developed a multiplex real-time RT-PCR for the simultaneous detection of the four most common avian respiratory viruses: avian influenza virus, infectious bronchitis virus, Newcastle disease virus, and infectious laryngotracheitis virus. They used various primers and probes for these viruses, which were encompassed into one tube for simultaneous detections. Their approach was similar to ours except that our multiplex assay was designed for the simultaneous detection of Influenza B virus or pdm H1N1 and H3N2, the two most prevalent strains of Influenza A.” We have also added statements (lines 385-388, unmarked version) to discuss the methods of certain commercial kits and an additional reference (ref. 26) was added to the list. These statements read as: “Certain commercial kits such as FilmArray multiplex PCR [26] apply nucleic acid amplification test-based methods to detect multiple respiratory pathogens in a single test. However, unlike the results of our multiplex assay reporting CT values, the results of these commercial kits show only positive or negative diagnosis.” We sincerely hope that the reviewer could approve our explanations. Attachments Attachment Submitted filename: Responses to Comments from Editor and Reviewer.pdf https://doi.org/10.1371/journal.pone.0283074.r002
13 Dec 2022 Decision Letter - Baochuan Lin, Editor PONE-D-22-10495R1A highly efficient and accurate method of detecting and subtyping Influenza A pdm H1N1 and H3N2 viruses with newly emerging mutations in the matrix gene in Eastern TaiwanPLOS ONE Dear Dr. Chen, 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. While the reviewer agreed that the revised version showed improvement, there are still several issues that needs to be addressed. The authors established a multiplex RT-PCR assay for rapid detection and distinguishing pdm H1N1 and H3N2 in eastern Taiwan. The authors combined WHO and Taiwan CDC suggested primers/probes in the multiplex PCR which mitigated the low detection efficiency of M-mutant H3N2 viruses (evolved and appeared after 2011) using WHO suggested primers/probes set. The assay was verified using 5709 clinical specimens. While not mentioned in the abstract and introduction, the authors did phylogenetic analysis of 94 samples (44 pdm H1N1 and 50 H3N2). I am at a lost on the intention of the phylogenetic analysis, which needs to be mentioned in abstracts and introduction to strengthen the rationale of the study. In addition, the quality of the language still needs to be improved. We suggest you thoroughly copyedit your manuscript for language usage, spelling, and grammar. If you do not know anyone who can help you do this, you may wish to consider employing a professional scientific editing service. Whilst you may use any professional scientific editing service of your choice, PLOS has partnered with both American Journal Experts (AJE) and Editage to provide discounted services to PLOS authors. Both organizations have experience helping authors meet PLOS guidelines and can provide language editing, translation, manuscript formatting, and figure formatting to ensure your manuscript meets our submission guidelines. To take advantage of our partnership with AJE, visit the AJE website (http://learn.aje.com/plos/) for a 15% discount off AJE services. To take advantage of our partnership with Editage, visit the Editage website (www.editage.com) and enter referral code PLOSEDIT for a 15% discount off Editage services. If the PLOS editorial team finds any language issues in text that either AJE or Editage has edited, the service provider will re-edit the text for free." Specific comments: Abstract, line 45 – 46: This sentence is not relevant. Suggest deletion. Materials and method, line 209: BLAST please reference “J Mol Biol. 1990 Oct 5;215(3):403-10. doi: 10.1016/S0022-2836(05)80360-2.Basic local alignment search tool S F Altschul 1, W Gish, W Miller, E W Myers, D J Lipman” Results, line 247 – 248: Suggest moving tables 3 & 4 to supplemental materials. The purpose of the 44 pdm H1N1 and 50 H3N2 samples for phylogenetic analysis. Discussion, line 341 – 344: This sentence is not relevant. Suggest deletion. Discussion, line 357 – 377: Suggest moving this paragraph to the beginning of the discussion. Discussion, line 366 – 368: Please explain how the delayed C_T values help distinguishing pdm H1N1 and H3N2. Where is Table 1A? Please submit your revised manuscript by Jan 27 2023 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 rebuttal 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. We look forward to receiving your revised manuscript. Kind regards, Baochuan Lin, Ph.D. Academic Editor PLOS ONE [Note: HTML markup is below. Please do not edit.] 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 ******** 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 ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: 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 ****** 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 ****** 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) ****** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). 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 Privacy Policy. Reviewer #1: 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.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0283074.r003
Revision 2
26 Dec 2022 Author Response Responses to Editor (PONE-D-22-10495_R2) We would like to thank the editor for their extensive assessment of our manuscript, and for important and helpful comments and suggestions. We have taken all the remarks into account, in a manner that is described in detail below together with our responses to comments. We have responded to all the editor’s comments in a point-by-point fashion and have revised the manuscript accordingly. The revised portions are indicated by “Track Changes”. An “unmarked” clean version of revised manuscript has also been provided. We think that, following these suggestions, our manuscript has gained in clarity and hope that the changes made will be considered satisfactory. Responses to Editor’s Comments: Major Comments: Comment 1: While not mentioned in the abstract and introduction, the authors did phylogenetic analysis of 94 samples (44 pdm H1N1 and 50 H3N2). I am at a lost on the intention of the phylogenetic analysis, which needs to be mentioned in abstracts and introduction to strengthen the rationale of the study. Response: We thank the editor for this excellent suggestion. In response to the suggestion, we have added statements in the abstract and introduction to address this issue. The statements in the abstract read as:” Further, the phylogenetic analyses of our samples revealed that the characteristics of these viruses were different from those reported previously using samples collected during 2012–2013” (lines 54-56). The statements in the introduction section read as:” In this study, we also report the results of the phylogenetic analyses of Influenza A pdm H1N1 and H3N2 viruses associated with the pandemic that occurred during 2012–2013 in Taiwan [14]. Since then, the changes in phylogenetic characteristics of these viruses have not yet been investigated. Therefore, in this study, we further performed phylogenetic analyses for the Influenza A pdm H1N1 and H3N2 viruses that caused the 2011–2015 pandemic” (lines 117-122). Comment 2: In addition, the quality of the language still needs to be improved. We suggest you thoroughly copyedit your manuscript for language usage, spelling, and grammar. If you do not know anyone who can help you do this, you may wish to consider employing a professional scientific editing service. Response: In response to the suggestion, our revised manuscript (R2 version) has been professionally edited by Editage for improvements of language; this company is recommended by the journal. The certificate of this English editing service has been uploaded alongside the revised manuscript. Specific comments: Comment 1: Abstract, line 45 – 46: This sentence is not relevant. Suggest deletion. Response: In response to the suggestion, the sentence has been deleted (at the position of line 45). Comment 2: Materials and method, line 209: BLAST please reference “J Mol Biol. 1990 Oct 5;215(3):403-10. doi: 10.1016/S0022-2836(05)80360-2. Basic local alignment search tool S F Altschul 1, W Gish, W Miller, E W Myers, D J Lipman” Response: We thank the editor for providing this important reference, which has been added to the reference list and cited (Materials and method, line 233). The numbers of other references in the text have been revised accordingly. Comment 3: Results, line 247 – 248: Suggest moving tables 3 & 4 to supplemental materials. Response: In response to the suggestion, these two tables have been moved to supplemental materials (S1 and S2 Tables) (line 273). The number of Table 5 in the text have been changed to Table 3 accordingly (line 274 and line 290). Comment 4: The purpose of the 44 pdm H1N1 and 50 H3N2 samples for phylogenetic analysis. Response: In response to this suggestion, we have added statements in the abstract section to address this issue.” Further, the phylogenetic analyses of our samples revealed that the characteristics of these viruses were different from those reported previously using samples collected during 2012–2013” (lines 54-56). The statements in the introduction section read as:” In this study, we also report the results of the phylogenetic analyses of Influenza A pdm H1N1 and H3N2 viruses associated with the pandemic that occurred during 2012–2013 in Taiwan [14]. Since then, the changes in phylogenetic characteristics of these viruses have not yet been investigated. Therefore, in this study, we further performed phylogenetic analyses for the Influenza A pdm H1N1 and H3N2 viruses that caused the 2011–2015 pandemic” (lines 117-122). Comment 5: Discussion, line 341 – 344: This sentence is not relevant. Suggest deletion. Response: In response to the suggestion, the sentence has been deleted (line 408). Comment 6: Discussion, line 357 – 377: Suggest moving this paragraph to the beginning of the discussion. Response: We thank the editor for the suggestion. This paragraph has been moved to the first paragraph of the discussion (lines 365-394). Comment 7: Discussion, line 366 – 368: Please explain how the delayed CT values help distinguishing pdm H1N1 and H3N2. Where is Table 1A? Response: Table 1A was a typo and should be Table 4 in our last R1 version. The Table 1A has been corrected to S2 Table (supplementary materials) in this R2 version. We apologize for the error we made. We have also added statements to explain how the delayed CT values help distinguishing pdm H1N1 and H3N2. These statements read as:” In this study, the delayed CT values obtained when the MAF and MAR primers and MA probe were used (S2 Table) could be used to distinguish Influenza A pdm H1N1 and H3N2 serotypes. Specifically, Influenza A pdm H1N1 virus could be identified when the CT values obtained based on the use of the MAF and MAR primers and MA probe were very similar to those obtained when the InfAF and InfAR primers and InfA probe were used (S1 Table). Conversely, Influenza A H3N2 could only be identified when the CT values obtained based on the use of MAF and MAR primers and the MA probe were much greater than those detected based on the use of InfAF and InfAR primers and InfA probe or were even undetectable (S2 Table)” (lines 375-383). Attachments Attachment Submitted filename: Responses to Comments from Editor_R2.pdf https://doi.org/10.1371/journal.pone.0283074.r004
6 Jan 2023 Decision Letter - Baochuan Lin, Editor PONE-D-22-10495R2 A highly efficient and accurate method of detecting and subtyping Influenza A pdm H1N1 and H3N2 viruses with newly emerging mutations in the matrix gene in Eastern Taiwan PLOS ONE Dear Dr. Chen, 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. The revised manuscript showed significant improvement over previous version. However, there are a few issues that still need to be addressed. 1. Line 83, suggest changing "Therefore, presently,..." to "Presently..." 2. Line 171, delete "of" 3. Line 268, suggest changing "Further, the..." to "The..." 4. Line 268 - 269, It seems that table S1 and S2 only listed representative samples of pdmH1N1, and H3N2, not all the test samples. Please clarify. 5. Line 271, suggest deleting "data" 6. Line 309 - 310, need to rephrase this sentence for clarity. Does the second set of primers and probe remedy the situation mentioned in the previous sentences? 7. Line 369, suggest changing "...sometimes causes..." to "...caused by..." 8. Line 370, suggest deleting "simultaneously applied" 9. Line 371-372, suggest deleting "...with early CT values..." 10. Line 419, suggest deleting "...previous..." Please submit your revised manuscript by Feb 20 2023 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 rebuttal 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. We look forward to receiving your revised manuscript. Kind regards, Baochuan Lin, Ph.D. Academic Editor PLOS ONE Journal Requirements: 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. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: [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.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0283074.r005
Revision 3
16 Jan 2023 Author Response Responses to Editor (PONE-D-22-10495_R3) We would like to thank the editor for their extensive assessment of our manuscript, and for important and helpful comments and suggestions. We have taken all the remarks into account, in a manner that is described in detail below together with our responses to comments. We have responded to all the editor’s comments in a point-by-point fashion and have revised the manuscript accordingly. The revised portions are indicated by “Track Changes”. An “unmarked” clean version of revised manuscript has also been provided. We think that, following these suggestions, our manuscript has gained in clarity and hope that the changes made will be considered satisfactory. Responses to Editor’s Comments: General comment: The revised manuscript showed significant improvement over previous version. However, there are a few issues that still need to be addressed. Response: We thank the editor for his/her positive feedback and for valuable suggestions to enhance the quality of our work. We also thank the editor for his/her careful reading of our manuscript. Comment 1: Line 83, suggest changing "Therefore, presently,..." to "Presently..." Response: This has been changed (Line 83). Comment 2: Line 171, delete "of" Response: This has been deleted (Line 171). Comment 3: Line 268, suggest changing "Further, the..." to "The..." Response: This has been changed (Line 268). Comment 4: Line 268 - 269, It seems that table S1 and S2 only listed representative samples of pdmH1N1, and H3N2, not all the test samples. Please clarify. Response: The editor is correct regarding this issue. Table S1 and S2 only listed representative samples of pdmH1N1, and H3N2, not all the test samples. In response to this suggestion, we have rephrased the statement as:” The threshold cycle (CT) values of some representative tested samples diagnosed …” (Line 268-269). Additionally, the titles of tables S1 and S2 have been changed to “… detected from representative clinical samples…”(Supplemental Materials). Comment 5: Line 271, suggest deleting "data" Response: This has been deleted (Line 271). Comment 6: Line 309 - 310, need to rephrase this sentence for clarity. Does the second set of primers and probe remedy the situation mentioned in the previous sentences? Response: We thank the reviewer for reminding us this important issue. In response to the suggestion, the statement has been rephrased and reads as:” This situation can be remedied by our second set of primers and probe (InfAF and InfAR primers, and InfA probe), which targeted a more conserved region of the M gene sequence of Influenza A H3N2 virus” (Line 309-311). Comment 7: Line 369, suggest changing "...sometimes causes..." to "...caused by..." Response: This has been changed (Line 369). Comment 8: Line 370, suggest deleting "simultaneously applied" Response: This has been deleted (Line 370). Comment 9: Line 371-372, suggest deleting "...with early CT values..." Response: This has been deleted (Line 372). Comment 10: Line 419, suggest deleting "...previous..." Response: This has been deleted (Line 419). Attachments Attachment Submitted filename: Responses to Comments from Editor_R3 .pdf https://doi.org/10.1371/journal.pone.0283074.r006
2 Mar 2023 Decision Letter - Baochuan Lin, Editor A highly efficient and accurate method of detecting and subtyping Influenza A pdm H1N1 and H3N2 viruses with newly emerging mutations in the matrix gene in Eastern Taiwan PONE-D-22-10495R3 Dear Dr. Chen, 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 for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. 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, Baochuan Lin, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0283074.r007
Formally Accepted
13 Mar 2023 Acceptance Letter - Baochuan Lin, Editor PONE-D-22-10495R3 A highly efficient and accurate method of detecting and subtyping Influenza A pdm H1N1 and H3N2 viruses with newly emerging mutations in the matrix gene in Eastern Taiwan Dear Dr. Chen: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. 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. If we can help with anything else, please email us at plosone@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. Baochuan Lin Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0283074.r008

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 .