In vitro one-pot construction of influenza viral genomes for virus particle synthesis based on reverse genetics system

Ryota Tanaka; Kenji Tamao; Mana Ono; Seiya Yamayoshi; Yoshihiro Kawaoka; Masayuki Su’etsugu; Hiroyuki Noji; Kazuhito V. Tabata

doi:10.1371/journal.pone.0312776

Peer Review History

Original SubmissionJuly 24, 2024
21 Aug 2024 Decision Letter - Herman Tse, Editor PONE-D-24-30909In vitro one-pot construction of influenza viral genomes for virus particle synthesis based on reverse genetics systemPLOS ONE Dear Dr. Tabata, 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 Oct 05 2024 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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If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. Additional Editor Comments: The reviewer has pointed out a few minor issues, particularly with regards to some technical descriptions. Otherwise, this is a well-written manuscript which I would be ready to accept once the revisions are made. [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 ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: 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 ****** 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 ****** 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 by Tanaka et al entitled “In vitro one-pot construction of influenza viral genomes for virus particle synthesis based on reverse genetics system“ describes a novel approach to generate influenza A viruses from plasmid DNA. Using the in vitro one-pot plasmid construction (IVOC) reverse genetics approach, which exploits the E.coli DNA replication machinery in vitro, the authors show that they are able to generate all influenza A virus rescue plasmids within 8 hours instead of 3 days with conventional cloning techniques. Therefore, the IVOC approach offers the advantage to rapidly generate virus mutants that can be used for high throughput screens. The manuscript and the method described therein describe a novel cloning approach for the rescue of influenza A viruses and thus represent a valuable resource; however, there are some points listed below that should be addressed by the authors. Specific comments: In the abstract: It is unclear to the reviewer why the authors expect that this method “potentially advance further understanding of influenza viruses…”. In the introduction: Please clarify that only influenza A viruses have caused known pandemics. General comments: The reviewer understood the IVOC approach to mean that the authors generated pPolI and pCAGGS plasmids with an E. coli oriC. For clarity, the authors should consider naming these modified plasmids, e.g. as pPolI-oriC, rather than referring to them as "these plasmids". In addition, Figure 1 should be adjusted accordingly to distinguish the plasmids with oriC (upper part of the figure) from the conventional pPolI and pCAGG plasmids without the oriC. In the Materials and Methods section: Authors should provide the manufacturers of all reagents, especially the E. coli enzyme mix required for the IVOC method. Is it commercially available? Because it is unclear to the reviewer why segments of similar length (e.g. PB2 and PB1) would generate a different amplicon size, the authors should provide a list of the primer pairs (and show were they bind in the plasmid backbone) used to generate the PCR products shown in Figure 2B. In this study, the authors generated PR8 viruses using available plasmids as a template. The authors should comment on whether or not they tested isolated vRNA from infectious cell culture supernatant as a template for the IVOC approach. The authors should also mention whether or not this approach can be used to rapidly generate a plasmid rescue system for virus isolates from infectious patient/animal material. ****** 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 ******** [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.0312776.r001
Revision 1
5 Oct 2024 Author Response Reviewer #1 Specific comments: 1. In the abstract: It is unclear to the reviewer why the authors expect that this method “potentially advance further understanding of influenza viruses…”. At the end of the abstract, the following text was included to explain how the IVOC method can be used in influenza virus research. This method enables the pathogenicity analysis and vaccine development using genetically modified viruses, and it is expected to allow for faster analysis of newly emerging variants than ever before. Furthermore, its application to other RNA viruses is also expected. 2. In the introduction: Please clarify that only influenza A viruses have caused known pandemics. We have made the following changes to the introduction section you pointed out. In particularthe 20th century, influenza A viruses caused there were 3 three pandemics in the 20th century (9), and in the 21st century, there was a pandemic was caused by a swine-origin influenza A virus in 2009(10). Considering the potential emergence of new pathogenic viruses(11), it is urgent to advance our biological understanding of the influenza A virus through research and the development of tools to counter its threat to humanity. General comments: 1. The reviewer understood the IVOC approach to mean that the authors generated pPolI and pCAGGS plasmids with an E. coli oriC. For clarity, the authors should consider naming these modified plasmids, e.g. as pPolI-oriC, rather than referring to them as "these plasmids". In addition, Figure 1 should be adjusted accordingly to distinguish the plasmids with oriC (upper part of the figure) from the conventional pPolI and pCAGG plasmids without the oriC. As you suggested, in order to make the names of the plasmids easier to understand, we have changed the plasmids used as templates for PCR to pPolI and pCAGGS, and the plasmids that have oriC to pPolI-oriC and pCAGGS-oriC. Therefore, we have made several corrections to the main text. In addition, we have made corrections to Figures 1 and 5 to distinguish between plasmids that do and do not have oriC. Please confirm the relevant sections. 2. In the Materials and Methods section: Authors should provide the manufacturers of all reagents, especially the E. coli enzyme mix required for the IVOC method. Is it commercially available? All the reagents required for RA-RCR were prepared by Su’etsugu et al. The text has been modified as follows to make this clear. Plasmid construction using the RA-RCR system followed the protocol outlined in a previous report(30). The RA mix and RCR mix required for the RA-RCR system were prepared by Su’etsugu et.al according to that report(30). 3. Because it is unclear to the reviewer why segments of similar length (e.g. PB2 and PB1) would generate a different amplicon size, the authors should provide a list of the primer pairs (and show were they bind in the plasmid backbone) used to generate the PCR products shown in Figure 2B. The primer sets used in the PCR in Fig. 2B are listed in the Supporting Information as Fig. S1 and S2, along with the sequences and annealing positions for each plasmid. This is also shown in the legend for Fig. 2. As a result of this change, the previous Fig. S1 has been moved to Fig. S3. 4. In this study, the authors generated PR8 viruses using available plasmids as a template. The authors should comment on whether or not they tested isolated vRNA from infectious cell culture supernatant as a template for the IVOC approach. The authors should also mention whether or not this approach can be used to rapidly generate a plasmid rescue system for virus isolates from infectious patient/animal material. In this study, we did not perform IVOC using vRNA isolated from clinical specimens. However, because general reverse genetics methods reconstitute viruses from vRNA derived from clinical specimens, we believe that IVOC is also possible. We will perform such experiments in the future. In this regard, we have also added the following text to the main text. In this study, viral RNA isolated and purified from infectious cell culture supernatants or patient/animal samples was not tested as a template in the IVOC approach. However, it is anticipated that this would be feasible within the IVOC system, as viral RNA has successfully served as a template for virus production in the conventional methods. Attachments Attachment Submitted filename: Response to Reviewers.pdf https://doi.org/10.1371/journal.pone.0312776.r002
14 Oct 2024 Decision Letter - Herman Tse, Editor In vitro one-pot construction of influenza viral genomes for virus particle synthesis based on reverse genetics system PONE-D-24-30909R1 Dear Dr. Tabata, 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 will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, 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, Herman Tse Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0312776.r003
Formally Accepted
28 Oct 2024 Acceptance Letter - Herman Tse, Editor PONE-D-24-30909R1 PLOS ONE Dear Dr. Tabata, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks to review your paper and let you know the next and final steps. Lastly, 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 customercare@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. Herman Tse Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0312776.r004

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