PONE-D-21-23774Molecular characterization of haemagglutinin genes of influenza B viruses circulating in Ghana during 2016 and 2017PLOS ONE

Dear Dr. Yakubu,

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============================== In particular, you need to address reviewer 1's comments about the low number of sequences obtained. Furthermore, addressing all of the other major/minor points will improve the readability of your paper.

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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: Partly

Reviewer #2: Yes

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

Reviewer #1: I Don't Know

Reviewer #2: Yes

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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: Yes

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

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

Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

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

Reviewer #1: The authors characterized the lineages of influenza B viruses that circulated in Ghana between 2016 and 2017. Eight sequences of the complete HA genes (4:Yamagata lineage, 4: Victoria lineage) were included for the phylogenetic analysis. The authors reported eleven amino acids substitutions detected in the B/Victoria lineage and six in the B/Yamagata lineage. However, some issues should be revised to strengthen the manuscript.

Major points:

- The main concern of this manuscript is whether these samples represent the characteristic of influenza B viruses circulating in Ghana. The eight sequences seem a relatively low number for the results obtained from the targeted sequencing (Sanger sequencing), not the whole genome sequencing.

- Are these the only eight sequences positive for influenza B RT-PCR between 2016-2017 from the archive of the study sites? The sample numbers sent to the study sites during 2016-2017 and the samples positive for influenza B viruses during 2016-2017 should be included to calculate the rate of detection. There are no precise inclusion criteria for selecting these eight samples as the representative samples. This information should be included in the materials and methods section.

- The order of figure legends is confusing, starting from Fig. 1, Fig. 4, Fig. 2, Fig. 3.

- Materials and Methods: the method/program used for potential glycosylation analysis should be included.

- There is inconsistent information regarding the analysis of antigenic drift. The materials and methods section stated that the analysis was performed for both lineages using the WHO vaccine candidate virus sequences for the 2018-2019 Influenza season. However, the results section mentioned that the sequences were compared with that of the 2016-2017 WHO trivalent and quadrivalent vaccine candidate.

Minor points:

- Materials and Methods: Line 116-117: These had been previously determined to be Influenza B virus positive by real-time RT-PCR using the standard U.S. CDC protocol [15]. -> If the name of the real-time RT-PCR test kit is available, it should be included.

- Results: The title that states “Nucleotide changes distinguishing influenza B Victoria from Yamagata lineages” provided no new information. It should be revised to present other important points of the study.

- Results: Line 174: Whole genome amplicon sequencing of the HA genes… should be revised to Targeted sequencing of the full HA genes...

- Results: S1 Figure should include the size label of the 100 bp marker.

Reviewer #2: The paper is well written and addresses an important issue such as the choice of the best vaccine component for influenza vaccines. In particular, the problem of choosing component B was addressed.

The samples that have been analyzed are few and this could represent a limit. However, the aim of the work was to identify a path to improve the choice of influenza B vaccine components and not the description of the circulation of influenza viruses in a specific area and in a specific period.

Please make the following changes:

1. In the paper “influenza” sometimes it is written with a capital letter, other times with a lowercase. It is necessary to standardize the way of writing.

2. In the paper sometimes it is reported B/Victoria and B/Yamagata, other times B Victoria and B Yamagata (with or without /). Also, in this case it is necessary to standardize the way of writing

3. It is unclear whether the samples are collected from 2016-17 or during 2016 and 2017 influenza seasons (line 110)

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Reviewer #1: No

Reviewer #2: No

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Molecular characterization of haemagglutinin genes of influenza B viruses circulating in Ghana during 2016 and 2017

PONE-D-21-23774R1

Dear Dr. Yakubu,

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

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

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

Kind regards,

Brian M. Ward, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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

Reviewer #1: (No Response)

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

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

Reviewer #1: I Don't Know

Reviewer #2: Yes

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

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

Reviewer #1: Yes

Reviewer #2: Yes

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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: Yes

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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: On these comments:

1- Are these the only eight sequences positive for influenza B RT-PCR between 2016-

2017 from the archive of the study sites? The sample numbers sent to the study sites

during 2016-2017 and the samples positive for influenza B viruses during 2016-2017

should be included to calculate the rate of detection. There are no precise inclusion

criteria for selecting these eight samples as the representative samples. This

information should be included in the materials and methods section.

Response:

No, the eight samples assessed were just a few and they were selected in line with the

aim of the study“to identify a path to improve the choice of influenza B vaccine

components” and not the description of the circulation of influenza viruses in Ghanain

the specific period.

These samples were selected as representatives from the 3 different zones of the

country, Southern, Northern and Central Ghana. Specimen positive for Influenza B by

real-time RT-PCR during subtyping and with a CT <29 were selected for sequencing

and further analysis. Mostly considered because of their strong Ct values upon

subtyping and have been mentioned in the materials and methods section [Lines 119-

122].

>>Reviewer's comment on the author's response:

Thank you for the explanation. The current title/abstract gives the impression of the aim to characterize the circulating strains during 2016-2017.

To clarify that the eight samples assessed were the representative samples with the aim to identify a path to improve the choice of influenza B vaccine components, the abstract should be revised to state this point.

For example,

" To assess the potential impact in Ghana, we characterized the lineages of influenza B viruses that circulated in Ghana between 2016 and 2017 from different regions of the country: Southern, Northern, and Central Ghana.

Eight representative specimens from the three regions that were positive for influenza B virus by real-time RT-PCR were sequenced and compared to reference genomes from each lineage."

2- The order of figure legends is confusing, starting from Fig. 1, Fig. 4, Fig. 2, Fig. 3.

Response:

The figure legends have been rearranged from Fig. 1, Fig. 2, Fig. 3 and Fig. 4 [Lines

168-179].

>>Reviewer's comment on the author's response:

- The current figures 2 and 3 should still be in the result section but be renumbered to be figures 3 and 4, while the current figure 4 should be renumbered to be figure 2. Please adjust the order of the attached figures accordingly and remove the citing (Fig.2 and 3) from the Method section-Phylogenetic analysis.

Reviewer #2: (No Response)

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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.

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

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