SARS-CoV-2 genetic diversity in Venezuela: Predominance of D614G variants and analysis of one outbreak

Carmen L. Loureiro; Rossana C. Jaspe; Pierina D´Angelo; José L. Zambrano; Lieska Rodriguez; Víctor Alarcon; Mariangel Delgado; Marwan Aguilar; Domingo Garzaro; Héctor R. Rangel; Flor H. Pujol

doi:10.1371/journal.pone.0247196

Peer Review History

Original SubmissionOctober 10, 2020
30 Oct 2020 Decision Letter - Marta Giovanetti, Editor PONE-D-20-31900 SARS-CoV-2 genetic diversity in Venezuela: predominance of D614G variants and analysis of one outbreak PLOS ONE Dear Dr. Pujol, 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 Dec 14 2020 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. 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Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information. [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: Yes Reviewer #2: Partly Reviewer #3: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: No Reviewer #3: N/A ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? 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Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: No Reviewer #3: 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 Prevalence and Hearing Outcome of Congenital CMV Infection in an Urban Area of East Asia: Population-based Study" by Yang et al. is well-written and provides a relatively comprehensive view about Congenital cytomegalovirus prevalence in Taipei, Taiwan. Although the study has limitations already recognized by the authors, this manuscript provide advances in our understanding about the relevance the early identification of cCMV in neonates increasing the quality of life of patients and better follow-up of the public service to these patients telling a nice story. I think it would be suitable for publication in PLoS One after careful revision of English language. Reviewer #2: - Highlights sentences do not reflect the content of this article. - Today there are many Sars-CoV-2 genomes in different databases. We still need to insert a larger number of genomes for this type of analysis. - Complementary methodologies are required for phylogenetic and phylogenomics analysis. - Explain the parameters that have been selected for the models used in this article. - Provide the criteria used for the analysis of these variants. - The data presented do not support the conclusion suggested by the authors. - Improve the image resolution in all figures. Reviewer #3: In this study, the authors analyzed full genomes sequences obtained from different regions of Venezuela. They found that the majority of the isolates belongs to lineage B and they bear the mutation D614G in the Spike protein. They also found an in-frame deletion (amino acids 83-85) of the nsp1. - The authors mentioned both in the abstract and in the results section that nsp1 exhibits high frequency of deletions. The authors should add the following references of recent studies that showed deletions in nsp1 of SARS-CoV-2: o F. Benedetti, G.A. Snyder, M. Giovanetti, S. Angeletti, R.C. Gallo, M. Ciccozzi, D. Zella, Emerging of a SARS-CoV-2 viral strain with a deletion in nsp1, Journal of Translational Medicine 18, (11) (2020) 3-29. o T. Phan, Genetic diversity and evolution of SARS-CoV-2, Infect Genet Evol 81, (11) (2020)104- 260. o M.R. Islam, Genome-wide analysis of SARS-CoV-2 virus strains circulating worldwide implicates heterogeneity, Scientific Reports 10, (12) (2020) 14-40. - Does the deletion in nsp1 analyzed by the authors correlate with potential decreased viral pathogenicity? Does it impact the core structure of nsp1? The authors should better clarify how this deletion can impact the functionality and the pathogenesis of SARS-CoV-2. - What are the percentage of mutations? The authors should revise Table 1 and add the number of sequences carrying the mutations/deletions and the related percentages to make the table better informative. - A few typos need to be corrected ****** 6. 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Revision 1
27 Nov 2020 Author Response PONE-D-20-31900 SARS-CoV-2 genetic diversity in Venezuela: predominance of D614G variants and analysis of one outbreak 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_authaut_affiliations.pdf We have edited our manuscript according to the style requirements. 2. We note that Figure 2 in your submission contain map 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 (a) present written permission from the copyright holder to publish these figures specifically under the CC BY 4.0 license, or (b) remove the figures from your submission: The map was edited and modified from https://freevectormaps.com, with no copyright. This information is included in Fig 2. 3. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information. Note: HTML markup is below. Please do not edit.] There is no more supporting information since the cited paper is now published. 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 Reviewer #3: Yes We modified the text to a less affirmative style because of some limitations of this study (see below), in Abstract and Conclusions. 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: No Reviewer #3: N/A More information was included the model used for phylogenetic analysis (see Materials and Methods, page 6 lines 100-103). There is no other statistical analysis in this study. 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 Reviewer #3: Yes In terms of Data Availability, we submitted previously all our sequences to GenBank. 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 Reviewer #3: Yes We have revised the manuscript for English accuracy. 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 Prevalence and Hearing Outcome of Congenital CMV Infection in an Urban Area of East Asia: Population-based Study" by Yang et al. is well-written and provides a relatively comprehensive view about Congenital cytomegalovirus prevalence in Taipei, Taiwan. Although the study has limitations already recognized by the authors, this manuscript provide advances in our understanding about the relevance the early identification of cCMV in neonates increasing the quality of life of patients and better follow-up of the public service to these patients telling a nice story. I think it would be suitable for publication in PLoS One after careful revision of English language. These comments do not refer to our manuscript and then were not addressed. Reviewer #2: - Highlights sentences do not reflect the content of this article. We do not know exactly which highlights sentences the reviewer is referring to. We have modified the abstract in order to better reflect the content of the article, including the new information of modelling. As stated before, we modified the text to a less affirmative one. - Today there are many Sars-CoV-2 genomes in different databases. We still need to insert a larger number of genomes for this type of analysis. We acknowledge the comment of the reviewer and agree that this study involves a relative low number of sequences produced from Venezuelan isolates. However, since the sequences were chosen at the beginning of the epidemic and from different locations, representative of the initial outbreaks occurring in the country, we think that the study is at least a partial reflection of the initial viral diversity found in Venezuela. Indeed, none of the isolates were genetically identical, supporting this hypothesis. The manuscript also includes the analysis of 48.635 SARS-CoV-2 sequences available in GISAID data base and described previously by other authors, but we analyzed in detail the frequency of deletions in these sequences. A new figure is added to better describe this analysis (Fig 6). In addition, the revised version includes a more in-deep analysis of the strain exhibiting a deletion in NSP1, with a new figure (Fig 7). - Complementary methodologies are required for phylogenetic and phylogenomics analysis. We acknowledge the comment. Several phylogenetic inferences were performed (see below in parameters), arising basically the same topology for the phylogenetic tress. Modelling of the deleted NSP1 protein was included in this new version and discussed (Figure 7, Abstract, end of Results in page 15 and Discussion in page 18). - Explain the parameters that have been selected for the models used in this article. The parameters used for the models for phylogenetic inference were described in Materials and Methods (Page 6). A short description was added in the legend of Figure 3 (Page 10). In our work, the phylogenetic tree was constructed based on the GTR+G model according to the Akaike criterion. The phylogenetic inference was also performed with the Bayesian criterion (TN93). The results were similar, but the tree generated with the GTR+G model exhibited better bootstrap values. - Provide the criteria used for the analysis of these variants. The criteria of selection of samples were added in Page 5, lines 79-81: samples from different part of the country and from the beginning of the epidemic, with low Ct value to guarantee efficient amplification. This information was included in the first paragraph of Page 5. - The data presented do not support the conclusion suggested by the authors. As sated before, we have modified the conclusions to a less affirmative style. - Improve the image resolution in all figures. Image resolution of figures was improved. Reviewer #3: In this study, the authors analyzed full genomes sequences obtained from different regions of Venezuela. They found that the majority of the isolates belongs to lineage B and they bear the mutation D614G in the Spike protein. They also found an in-frame deletion (amino acids 83-85) of the nsp1. - The authors mentioned both in the abstract and in the results section that nsp1 exhibits high frequency of deletions. The authors should add the following references of recent studies that showed deletions in nsp1 of SARS-CoV-2: o F. Benedetti, G.A. Snyder, M. Giovanetti, S. Angeletti, R.C. Gallo, M. Ciccozzi, D. Zella, Emerging of a SARS-CoV-2 viral strain with a deletion in nsp1, Journal of Translational Medicine 18, (11) (2020) 3-29. o T. Phan, Genetic diversity and evolution of SARS-CoV-2, Infect Genet Evol 81, (11) (2020)104- 260. o M.R. Islam, Genome-wide analysis of SARS-CoV-2 virus strains circulating worldwide implicates heterogeneity, Scientific Reports 10, (12) (2020) 14-40. The references were included and discussed (Page 18, line 271 and Page 19, line 291-292). Thank you very much. - Does the deletion in nsp1 analyzed by the authors correlate with potential decreased viral pathogenicity? Does it impact the core structure of nsp1? The authors should better clarify how this deletion can impact the functionality and the pathogenesis of SARS-CoV-2. We wish to thank the reviewer for this comment. We include a modelling analysis of the deleted NSP1 proteins and found indeed some differences compared to the wt protein. Information was included in Abstract, Materials and Methods, Results, Discussion and a Figure 6 was added, as stated before. A new co-author, responsible for this analysis, is also included. - What are the percentage of mutations? The authors should revise Table 1 and add the number of sequences carrying the mutations/deletions and the related percentages to make the table better informative. The Table was substituted by a figure which is more informative (Fig 4). - A few typos need to be corrected. The manuscript was corrected for English accuracy. Attachments Attachment Submitted filename: Response to reviewers.docx https://doi.org/10.1371/journal.pone.0247196.r002
19 Jan 2021 Decision Letter - Ahmed S. Abdel-Moneim, Editor PONE-D-20-31900R1 SARS-CoV-2 genetic diversity in Venezuela: predominance of D614G variants and analysis of one outbreak PLOS ONE Dear Prof. Pujol, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, 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 Mar 05 2021 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: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols We look forward to receiving your revised manuscript. Kind regards, Ahmed S. Abdel-Moneim, Ph.D. Academic Editor PLOS ONE 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 #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 #2: Partly ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #2: N/A ****** 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 #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 #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 #2: We noticed that the authors responded to the reviewers' requests. Note that there are still two points that need to be revised: increase the number to 1000 bootstrap iterations for better analysis and all Figures need improvement in resolution and colors, in particular Figure 7. The Figure 6 needs to be done with two scales in order to identify the lowest values. ****** 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 #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. https://doi.org/10.1371/journal.pone.0247196.r003
Revision 2
29 Jan 2021 Author Response PONE-D-20-31900R1 SARS-CoV-2 genetic diversity in Venezuela: predominance of D614G variants and analysis of one outbreak There are still two points that need to be revised: increase the number to 1000 bootstrap iterations for better analysis Bootstrap iterations were increased to 1000. New Figure is included and the bootstrap replicas was corrected to1000 in Materials and Methods, page 6. and all Figures need improvement in resolution and colors, in particular Figure 7. Figures are now included with better resolution and colors were slightly modified in Figure 7, for better appreciation. The Figure 6 needs to be done with two scales in order to identify the lowest values. The scale of Figure 6 was changed to a logarithmic one for better evaluation of lowest values. Attachments Attachment Submitted filename: Response to reviewers.docx https://doi.org/10.1371/journal.pone.0247196.r004
3 Feb 2021 Decision Letter - Ahmed S. Abdel-Moneim, Editor SARS-CoV-2 genetic diversity in Venezuela: predominance of D614G variants and analysis of one outbreak PONE-D-20-31900R2 Dear Dr. Pujol, 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, Ahmed S. Abdel-Moneim, Ph.D. Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0247196.r005
Formally Accepted
8 Feb 2021 Acceptance Letter - Ahmed S. Abdel-Moneim, Editor PONE-D-20-31900R2 SARS-CoV-2 genetic diversity in Venezuela: predominance of D614G variants and analysis of one outbreak Dear Dr. Pujol: 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. Ahmed S. Abdel-Moneim Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0247196.r006

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