PONE-D-22-17948Genomic epidemiology of SARS-CoV-2 in Russia reveals recurring cross-border transmission throughout 2020PLOS ONE

Dear Dr. Klink,

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 Sep 30 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,

Mohd Adnan, 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 and 

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf.

2. Thank you for stating in your Funding Statement: 

"This study was partially supported by RFBR project 20-04-60556. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript".

Please provide an amended statement that declares *all* the funding or sources of support (whether external or internal to your organization) received during this study, as detailed online in our guide for authors at http://journals.plos.org/plosone/s/submit-now.  Please also include the statement “There was no additional external funding received for this study.” in your updated Funding Statement. 

Please include your amended Funding Statement within your cover letter. We will change the online submission form on your behalf.

3. In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found. PLOS defines a study's minimal data set as the underlying data used to reach the conclusions drawn in the manuscript and any additional data required to replicate the reported study findings in their entirety. All PLOS journals require that the minimal data set be made fully available. For more information about our data policy, please see http://journals.plos.org/plosone/s/data-availability.

"Upon re-submitting your revised manuscript, please upload your study’s minimal underlying data set as either Supporting Information files or to a stable, public repository and include the relevant URLs, DOIs, or accession numbers within your revised cover letter. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories. Any potentially identifying patient information must be fully anonymized.

Important: If there are ethical or legal restrictions to sharing your data publicly, please explain these restrictions in detail. Please see our guidelines for more information on what we consider unacceptable restrictions to publicly sharing data: http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions. Note that it is not acceptable for the authors to be the sole named individuals responsible for ensuring data access.

We will update your Data Availability statement to reflect the information you provide in your cover letter.

4. Your ethics statement should only appear in the Methods section of your manuscript. If your ethics statement is written in any section besides the Methods, please move it to the Methods section and delete it from any other section. Please ensure that your ethics statement is included in your manuscript, as the ethics statement entered into the online submission form will not be published alongside your manuscript. 

5. We note that Figures 1, 4 and S1 in your submission contain map/satellite images which may be copyrighted. All PLOS content is published under the Creative Commons Attribution License (CC BY 4.0), which means that the manuscript, images, and Supporting Information files will be freely available online, and any third party is permitted to access, download, copy, distribute, and use these materials in any way, even commercially, with proper attribution. For these reasons, we cannot publish previously copyrighted maps or satellite images created using proprietary data, such as Google software (Google Maps, Street View, and Earth). For more information, see our copyright guidelines: http://journals.plos.org/plosone/s/licenses-and-copyright.

We require you to either (1) present written permission from the copyright holder to publish these figures specifically under the CC BY 4.0 license, or (2) remove the figures from your submission:

a) You may seek permission from the original copyright holder of Figures 1, 4 and S1 to publish the content specifically under the CC BY 4.0 license.  

We recommend that you contact the original copyright holder with the Content Permission Form (http://journals.plos.org/plosone/s/file?id=7c09/content-permission-form.pdf) and the following text:

“I request permission for the open-access journal PLOS ONE to publish XXX under the Creative Commons Attribution License (CCAL) CC BY 4.0 (http://creativecommons.org/licenses/by/4.0/). Please be aware that this license allows unrestricted use and distribution, even commercially, by third parties. Please reply and provide explicit written permission to publish XXX under a CC BY license and complete the attached form.”

Please upload the completed Content Permission Form or other proof of granted permissions as an "Other" file with your submission.

In the figure caption of the copyrighted figure, please include the following text: “Reprinted from [ref] under a CC BY license, with permission from [name of publisher], original copyright [original copyright year].”

b) If you are unable to obtain permission from the original copyright holder to publish these figures under the CC BY 4.0 license or if the copyright holder’s requirements are incompatible with the CC BY 4.0 license, please either i) remove the figure or ii) supply a replacement figure that complies with the CC BY 4.0 license. Please check copyright information on all replacement figures and update the figure caption with source information. If applicable, please specify in the figure caption text when a figure is similar but not identical to the original image and is therefore for illustrative purposes only.

The following resources for replacing copyrighted map figures may be helpful:

USGS National Map Viewer (public domain): http://viewer.nationalmap.gov/viewer/

The Gateway to Astronaut Photography of Earth (public domain): http://eol.jsc.nasa.gov/sseop/clickmap/

Maps at the CIA (public domain): https://www.cia.gov/library/publications/the-world-factbook/index.html and https://www.cia.gov/library/publications/cia-maps-publications/index.html

NASA Earth Observatory (public domain): http://earthobservatory.nasa.gov/

Landsat: http://landsat.visibleearth.nasa.gov/

USGS EROS (Earth Resources Observatory and Science (EROS) Center) (public domain): http://eros.usgs.gov/#

Natural Earth (public domain): http://www.naturalearthdata.com/

Additional Editor Comments:

Manuscript does not fulfill the standards established for the journal to be considered for publication in its current form. I agree with the reviewers that manuscript requires substantial revision and additional work to support the conclusion and improve the quality of the publication. Please see the detailed comments made by the reviewers.

[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

Reviewer #3: Partly

Reviewer #4: Yes

Reviewer #5: Yes

**********

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

Reviewer #1: N/A

Reviewer #2: Yes

Reviewer #3: N/A

Reviewer #4: Yes

Reviewer #5: Yes

**********

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

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: 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: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: No

Reviewer #5: 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: I would recommend addressing the following issues before it can be considered for publication on this journal.

Major revisions:

General comments: I’m concerned about the heterologous stringency for sequence selection, since up to 10% Ns were allowed in Russian sequences whilst non-Russian sequences were required to have far fewer (1%). The former are actually quite low, so I fear this may impact phylogenetic reconstruction.

Also, only by accessing figure S2 and the methods did the IBT and OBT become clear. As these were central to the study, they should be clearly defined earlier in the results. The reader should be provided a clear explanation on why the authors assume inbound or outbound transmission and why is it relevant.

Also, figures are almost peripheric to the main manuscript, they are not really used for support. They should be linked to the main study and discussed accordingly.

Ultimately, the manuscript could use a better explanation on the importance of this analysis and why was it limited to the first year? If the answer is “demonstrating that transfrontier transmission was ongoing throughout 2020”, then why stop there and how can the authors be sure of the actual direction since sequenced genomes represent only a very small percentage of total confirmed cases?

Finally, no statistical comparisons were shown at all, which could prove useful to determine differences within regions or countries of origin. Why was nothing compared, was there not enough data?

Introduction section

L47: The authors should provide a figure depicting total daily cases in Russia with a sliding average window (7 or 14-day avg) including 2020 (perhaps as a supplementary figure) for anyone who is not familiar with Russia’s situation during the pandemic. The figure should also include the total sequenced genomes (different y axis, perhaps) from the same time period as the first part tend to have very few samples. Finally, it would be rather useful if the authors could mark up some of the main events that are relevant for the current study (first documented case, border closure and opening).

L69: Please briefly specify how were non-Russian sequences selected (randomized, preselected, etc.) and perhaps reference the methods section here as well.

Results section:

Figure 1: The authors should include international borders in the map as land frontiers are important determinants of the actual dynamics.

Figure 2: Nothing is said in the main text about this figure, even though it is quite interesting. Also, perhaps the authors should include different y axes as to clearly define in which periods were these most prevalent. Also, area plots tend to be misleading when only few samples are available, as may be the case with the first introductions. It may be a good idea to include stacked area plots (not as frequencies but as raw totals, maybe in a different supplementary figure).

Also, consider moving this to supplementary and bringing up S2 to the main figures as it makes the next part easier to understand.

L70-L77: I found this paragraph to be somewhat confusing, perhaps it could be improved by using some more precise terms:

-L70: First, please refer to sequences, rather than samples, to avoid confusion, or make sure the reader understands they are used interchangeably here. Also, please be clear as to what the authors mean by “Russian transmission lineage” (were all of them monophyletic clades containing only sequences from Russian samples? Were there any non-Russian sequences in Russian transmission lineages? To what extent?). Also, other clade limits are not entirely clear in the text. (From Fig S2 it is clear that, apart from transmission lineages, others were actually paraphyletic) but, again, was there a minimum quota in the prevalence of Russian sequences within?

-L71: The term “Phylogenetic positions” on its own may be mistaken with actual mutations per se. Maybe use “On the basis of their position in the phylogeny (ML tree)”?

-L72: How were “characteristic changes” defined and according to what? “At its base” refers to the actual last common ancestor (LCA) of a monophyletic clade. Please use standard phylogenetic terms to avoid confusion (e.g. monophyletic clades having x mutations in common).

-L73: Variants, rather than strains (as of 2022, no serotypes nor strains have been defined officially).

-L74: Rather, these were phylogenetically closer to non-Russian clades than to other Russian sequences. It would be better if the authors used standard evolutionary terms instead.

L81: How can the authors be sure that these were actually imported and not the other way around? Can they confirm community transmission from these data? What if some of these remained undetected even though they were in Russia first?

Figure 3: “gray circles” also refer to lineages A* and B* according to the legend, so please be careful in the description. Also do the gray circles with dashed lines refer to actual sequences within the same clades that were also found in Russia earlier as basal/stem nodes in the tree (this is what I get from the figure description) or could these also be from non-Russian basal sequences as I think they do? This wasn’t clear to me but would explain them closely resembling other international items. Also, what are those four gray lines with no color terminal nodes on top of the figure (first 4 lines)?

L84: Rarefactions with no plateau do not necessarily mean that they were constantly imported (even though that might be a potential explanation) but only that some were missing due to sampling, so be careful when addressing this in the manuscript.

L89: Were bordering regions more prone to exchange variants? Also, were there variants exclusively seen in Russia or heavily present compared to the world during this period? It would be useful if the authors could provide some table containing those in a much higher prevalence in Russia than in other parts of the world.

L95: What about clades having sequences from both Russian and non-Russian origin in outgroups, were these discarded for this estimation?

L100: Could the authors very briefly describe how Treetime (also ML) differs from their method from a technical perspective?

L107: Were there any differences during border closure?

L110: Each of these particular cases should include some information on the actual variants they belonged to and why they are actually relevant for the study.

L121: Adding upon the last comment, which other variants are exemplified here and why was this particular mutation (S:P681H) relevant?

L124: What were the variants in this clade? Why was their relevance?

L131: What about these two clusters, why are they mentioned?

Discussion

L140: This substantial increase is with respect to existing sequences (how many were there?)? I’m assuming these were the ~1600 sequences mentioned above? The authors could mention them explicitly here as it is an important result.

L142: The authors should further discuss the rationale behind inbound and outbound transmissions in this section (how should outgroups be defined in order to determine a clade may be an IBT/OBT). Also, IBTs and OBT topologies could arise from undersampled regions and other contextual artifacts and this should be included here. How can the authors be sure of the actual direction of the transmission (or else mention these as limitations)?

L149: What are some examples of other countries? Since the percentage of crossing passengers that are sampled is always low, probability of picking out the exact event is almost nonexistent. Instead, this might get sequenced by chance but only after they are already rather common. Ultimately, I don’t think these events in particular may be important, but their dynamics are.

L153: Please elaborate on which epidemiological simulations the authors refer to as plenty have been shown before (just what kind of simultation and what variables are considered).

L161: Will the authors continue this study or expand it beyond 2020? What are the current perspectives?

References:

Ref 5 was not working. Please check all others. When were they last accessed?

L63: When the authors mention the Diamond Princess patient, they should include a reference for context.

Materials and Methods:

L204: As a recommendation for further studies, cutadapt may be used to trim the adapters more finely so there is no need to trim 25 nt from each end, which may improve mapping.

L214: Just another recommendation: Consensus by bcftools may be upgraded with ivar,to determine which variants are most common. This can be used to reduce Ns where polymorphic positions are found but not prevalent.

L220-221: These heterologous filters (~10% Ns for Russian items and 1% for non-Russian ones) may lead to issues in the downstream analyses and artifacts in phylogenetic trees due to some positions being missing from the alignment. I understand if this was selected as to avoid losing data but the consensus may be improved to prevent this by a major vote rule over SNP positions instead (ivar can be used to work this out).

L222: Why were minks left in the study?

Minor revisions

L47: Why January 19, 2021? It would be better if the authors reported a more recent figure, even if the study is limited to 2020.

L49: “from four-five million” Are these averages? Could the authors provide a reference for this?

L60: March 2.

L76: Unique mutations instead of characteristic changes or else describe what are those.

L80: All were sequenced from samples, maybe specify them as coming from the original Russian set.

L104: Please specify that inferred IBTs and OBTs refer to the the former 82/43.

L148: case*.

Reviewer #2: This is a well-written manuscript about cross-border transmissions of SARS-CoV2 variants. The authors attempted to establish a link between travel restrictions and the transmission of variants in Russia. Although, it is important to note that travel restrictions were mainly imposed broadly to contain the spread of the SARS-CoV-2 virus rather than variants. It is challenging for our increasingly interdependent world to impose complete border closure. For example, the presence of the SARS-CoV-2 virus in water bodies shared by countries and the movement of sub-clinically infected animals (White-tailed deer) can also contribute to the transmission of new variants. That said, the authors have provided enough evidence to confirm their findings. Please address these minor comments,

1. Please include the limitations to this study.

2. Line 149: “ ..corresponding countries,” What are these countries?

3. Line 153: “ radical reduction of the air passenger flow.” please refer to results.

4. I believe it may be Federal Air transport Agency webpage is not accessible outside Russia, at least, it did not work for me. Therefore, please provide the data used for analysis as supplementary information

5. Line 183: “ The presence of SARS-CoV-2 RNA…” please provide manufacturer information and test protocol/ reaction conditions for RT-qPCR. Why were Ct below 25 considered positive, explain? How many cycles were the samples run?

6. What was the quality and quantity of RNA used for WGS? Please include the information in the manuscript.

7. There were several references to websites and news outlets, some of which are local websites. It is important to include the date and time they were accessed.

Reviewer #3: The manuscript appears to have been sent for publication very late. Actually, there are much information about the epidemiology of SARS-CoV2 . Thus the results of this work -in my opinion -are obsolete and lack of novelty.

Reviewer #4: Some of the grammar can be improved. An email with comments is to be sent.

The paper is well written and understandable and the methodology has given all tools used so should be replicated if one desires to.

The paper is a bit technical in analysis but that is expected because of the analysis tools available.

A revision of the grammar should allow the paper to be published

In the discussion, the author should compare IBTs and OBTs introduced during the travel ban versus when the ban was lifted to be able to clearly conclude that there was no major impact.

.

Reviewer #5: The article: "Genomic epidemiology of SARS-CoV-2 in Russia reveals

recurring cross-border transmission throughout 2020." explains in an organized way

the effect of border closures on the transmission of SARS-COV2 infection during the first year of the pandemic.

This information is relevant because it can serve as a guiding element in decision-making by the authorities.

in health in the face of the eminent threat of a rapidly transmitted respiratory disease, as it turned out to be human coronaviruses.

**********

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

Reviewer #3: No

Reviewer #4: Yes: Rachel Achilla

Reviewer #5: 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.

Genomic epidemiology of SARS-CoV-2 in Russia reveals recurring cross-border transmission throughout 2020

PONE-D-22-17948R1

Dear Dr. Klink,

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,

Mohd Adnan, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Manuscript is significantly improved by the authors and now can be accepted in its current form.

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

**********

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: N/A

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: 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

Reviewer #2: Yes

**********

6. Review Comments to the Author

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

Reviewer #1: I am glad to see all my comments (and there were quite a few of them) have been addressed by the authors or were at least clarified in the manuscript. Images now flow better with the text as well, and authors have shown good disposition to make their manuscript more accessible in general. I think the current version is well-rounded for most readers to follow now and will prove valuable as a retrospective study for anyone looking for information on the pandemic in Russia during that period. With no further ado, I will now proceed to recommend your manuscript for publication. I wish you success.

Reviewer #2: For consistency, please double check typos. Follow MIQE guidelines for PCR data and their acronyms.

**********

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: Yes: Rodrigo García-López

Reviewer #2: No

**********