PONE-D-19-24652

Epidemic of influenza A(H1N1)pdm09 analyzed by full genome sequences and a first case of oseltamivir-resistant strain in Myanmar 2017

PLOS ONE

Dear Dr. Kyaw Win,

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.

I have received the reviews of your manuscript. While your paper addresses an interesting question, there are several issues raised by the reviewers that needs to be addressed.  Please see reviewers' insightful comments for detail.  On a personal level, I felt the paper is very descriptive and I agreed with the reviewers about the information for in-patients did not appear until "drug resistance testing", please provide more information regarding in-patients.  Also, some details on the phylogenetic analysis on the whole genome will help strengthen the merit of the paper.

Specific comments:

1. Line 58, replace "HI" with "Hemagglutination inhibition (HI)"

2. Line 121, please provide more information regarding in-patients, how many samples? Are they similar to out-patients in characteristics, etc.

3. Line 184:  replace "MUNANA" with "2′-(4-Methylumbelliferyl)-α-D-N-acetylneuraminic acid (MUNANA)".

4. Line 192, please add (Niigata, Japan) after Niigata University.

5. Line 194 - 195, suggest removing "using Sanger Method" from the title

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Kind regards,

Baochuan Lin, Ph.D.

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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

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

Reviewer #1: Yes

Reviewer #2: Partly

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

Reviewer #1: Yes

Reviewer #2: N/A

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3. Have the authors made all data underlying the findings in their manuscript fully available?

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

Reviewer #1: Yes

Reviewer #2: No

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

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

Reviewer #1: Yes

Reviewer #2: No

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5. Review Comments to the Author

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

Reviewer #1: This is a descriptive study on H1N1 influenza viruses isolated from in-patients and out-patients (mostly less than 14 years old) in Myanmar in 2017. It was motivated following a community outbreak during this year and declaration a high level of alert.

The authors investigated the circulation patterns, distribution of influenza subtypes, antigenic and genetic characterization of influenza A(H1N1)pdm09 and susceptibility to neuraminidase inhibitors.

The virus circulating in Myanmar was found to have several HA mutations including S84N, S162N and I216T; and NA mutations including V13I, I34V and I314M substitutions which placed the circulating H1N1 virus in the 6.1B clade.

Minor concerns

1. The introduction is well written, encompasses some aspects of influenza virology relevant to surveillance and focused properly on the work done here.

2. In M& M, Neuraminidase assay section can be written better. It is somehow confusing. For instance in three sequential sentences “FOUR KINDS OF NAI” was repeated.

3. In line 196 (pdf version), “The HA and NA proteins of selected A(H1N1)pdm09 viruses” should be “The HA and NA genes of selected A(H1N1)pdm09 viruses”.

4. In results, some data presented in table 1 are repeated in the text unnecessarily, also, demographic and baseline clinical characteristics of A(H1N1)pdm09 patients are clearly explained in table 2 and no need for more explanation. Please present the data just one time; table or text.

5. In table 6, please refer to appropriate article when mention to play a role in viral oligomerization interfaces and binding of small ligands/ to be related to virulence when combined with V3 and N328K for an avian virus A/quail/Vietnam/36/04 (H5N1) and to be related to drug resistance to amantadine.(line 452 -455 pdf version)

6. Please explain why did you Sanger sequencing while you could obtain the same data from NGS.

Major concern:

How you can explain HI results? The isolated viruses reacted to antisera raised against vaccine strain even more than the vaccine strain virus!! It should be Vice Versa. Do you think the data presented in this part is reasonable? Please clearly argue about this issue in discussion.

Reviewer #2: MAJOR POINTS

1. Title. I would suggest changing the title to something like – influenza full-genome sequencing for surveillance of pathogenic markers and antiviral resistance during the 2017 season in Myanmar.

2. Abstract. There is no mention on the in-patients here

3. L121. Please specify location of the YKCH Hospital (Myanmar, I guess)

4. Besides, one of the problems with this paper is that in-patients were selected from a different center from that of out-patients. Why only 12? This limitation needs to be acknowledged in the discussion. Why did you not include as in-patients those patients who were finally admitted in the two surveillance hospitals? The comparison of mild/severe cases from different sites needs an explanation.

5. L164-165. Please provide a table with the sequences for the primers and probes, or an adequate reference for the work describing those.

6. L218. Please, specify details on the software: what is type of treatment to the NGS raw data? What is the output? Only consensus files? Coverage, and variant tables?

7. L231. The authors used MEGA for the phylogenetic analysis but, what was the evolutionary model best fitting the data (for every segment and for the whole genome)? Dis you consider that in the phylogenetic analysis?

8. L282. For the patients included, did you collect the previous vaccination status?

9. L415 segments, no proteins

10. L420. For the NGS whole-genome sequence there are no details on the results obtained (i.e. mean coverage per segment, depth per nucleotide, variants per segment (synonymous, non-synonymous, major and minor) etc.

11. Besides, the analyses performed on NGS data seem limited to the consensus sequences. The authors do not provide a (major and minor) variant analysis for the 8 genome segments, table S3 seems just a summary of alignments using consensus sequences. It would be interesting to see if minor variants were more/less abundant in patients with severe outcome.

12. Further, there is no phylogenetic analysis on the other segments different from HA or NA…nor using the complete genome, data that may be also useful to look for reassortment/recombination.

13. L434-436. I believe this last sentence may be not relevant for the reader.

14. L533. There were in fact some differences between strains from Myanmar and those from India, see table 6!

15. L559-577 these two paragraphs show information that can be parly included in the introduction and that can be significantly streamlined, avoiding some details that may not be interesting to the reader.

16. L581. Here the authors should acknowledge the limitations on patient inclusion raised on the points above.

17. L585. It would be interesting to see if you found any minor variants in NA at positions associated with NAI resistence, discuss here.

18. L591-593. This can be streamlined to something like -2017 season but failed to identify features in the viral genome responsible for the differential severity of influenza between in- and out-patients…

MINOR POINTS

19. L236. A/California/07/2009 (H1N1pdm09). Please specify genebank accession number.

20. L242. Change “alterations” for “implications”

21. L346. …resistance

22. L352 …region did not show the…

23. L388 T314I in HA?

24. L408. …was found in one ….

25. L411 …the D451G…

26. L423 …substitutions (as compared to A/Michigan…)

27. L426…which MAY play a role…

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Reviewer #1: Yes: first name: Fatemeh, last name: Fotouhi

Reviewer #2: No

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PONE-D-19-24652R1

Epidemic of influenza A(H1N1)pdm09 analyzed by full genome sequences and a first case of oseltamivir-resistant strain in Myanmar 2017

PLOS ONE

Dear Dr. Kyaw Win,

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.

Both reviewers agreed that the revised manuscript showed significant improvement, however, there are some issues that need to be addressed.  Also, please have a fluent, preferably native, English-language speaker thoroughly copyedit your manuscript for language usage, spelling, and grammar since PLoS ONE does not perform copyediting of manuscripts at any later stage in the publication process.

We would appreciate receiving your revised manuscript by Feb 21 2020 11:59PM. When you are 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.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

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). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Baochuan Lin, Ph.D.

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

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

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

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

Reviewer #1: Yes

Reviewer #2: (No Response)

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

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

Reviewer #1: Yes

Reviewer #2: No

**********

6. Review Comments to the Author

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

Reviewer #1: (No Response)

Reviewer #2: In point 10 of the answer to reviewer comments, you argue that further analysis and details of the NGS genome sequencing results will be reoprted "in the next paper"...and added that comment in the text (P29, L566-570). Please change that for a more adequate statement, such as "analysis of NGS data is still ongoing and the results will be published elsewhere"

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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.

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Reviewer #1: Yes: Fatemeh Fotouhi

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 be viewed.]

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Epidemic of influenza A(H1N1)pdm09 analyzed by full genome sequences and the first case of oseltamivir-resistant strain in Myanmar 2017

PONE-D-19-24652R2

Dear Dr. Kyaw Win,

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

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

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With kind regards,

Baochuan Lin, Ph.D.

Academic Editor

PLOS ONE

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