Generational mutation patterns in a honey bee Deformed wing virus via infectious clones

Anthony Nearman; Alriana Buller-Jarrett; Dawn Boncristiani; Eugene Ryabov; Yanping Chen; Jay D. Evans

doi:10.1371/journal.pone.0337191

Peer Review History

Original SubmissionJuly 23, 2025
28 Sep 2025 Decision Letter - Olav Rueppell, Editor Generational mutation patterns in a honey bee Deformed Wing Virus via infectious clones PLOS ONE Dear Dr. Nearman, Thank you for submitting your manuscript to PLOS ONE. Both reviewers evaluated the manuscript as a valuable contribution that was well presented, and I agree. There are only some minor points raised by reviewer #1 that are worth considering. Please submit your revised manuscript by Nov 12 2025 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). 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If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.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 (if provided): [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? Reviewer #1: Yes Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? -->?> Reviewer #1: Yes Reviewer #2: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available??> The PLOS Data policy Reviewer #1: Yes Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English??> Reviewer #1: Yes Reviewer #2: Yes ****** Reviewer #1: In “Generational mutation patterns in a honey bee Deformed Wing Virus via infectious Clones”, Nearman and colleagues explore viral quasispecies dynamics of a critical pathogen of honey bees, Deformed wing virus (DWV). Using previously developed infectious clones, the researchers passaged these viral population through three generations of bees and used qPCR and amplicon sequencing to examine viral load and mutation accumulation through the passaging paradigm. Researchers also infected bees sourced from multiple colonies to measure any possible host effects on virus dynamics. While the infectious clone did not reveal a high amount of variation through passaging, the researchers were able to compare these dynamics to the passaging dynamics of a co-passaged wild type DWV. This wild type DWV demonstrated higher viral titers and a greater rate of silent mutation accumulation through the experiment. The introduction is a great justification to the study. I only have minor points to improve the manuscript. Methods : Why was only VP2 the focus? I know for example full viral genome sequencing incurs higher costs and more computational demand, but justification for this region for these initial experiments would be beneficial. More details are needed concerning the homogenate prep need to be included for replicability; for example, are pooled homogenates suspended in PBS or some other solution? L265-272 what specific programs were used for variant filtering and for amino acid translation? Results/Discussion : Is it expected that there’s simply more sub-consensus variation in WT vs clones (or detection lower than threshold), which is why you can see the accumulation of silent mutations? This may be addressed by the point made that further passaging beyond F2 may reveal more mutations in the clone, but just to highlight. In Figure 5, you highlight a higher proportion of reads with mutation by F2, but also F2 viral loads is lower than F1. Is it suspected that accumulation of silent mutations could be interfering with the replication rates of the virus? I think some additional support into the utility of using these clones for examining virus dynamics would be helpful. The work is centered around the infectious clones (e.g. the title) but it was the lack of variation in the clones compared to the WT virus that emphasized the importance of the WT virus in the passaging paradigm. Or, as authors, are you arguing that these sorts of virus dynamics experiments should instead be conducted with WT virus? Reviewer #2: The manuscript entitled: "Generational mutation patterns in a honey bee Deformed Wing Virus via infectious clones" exploits the power and novelty of using clones to investigate mutation rates accumulated by viruses in an in vivo setting. The authors focused on a conserved region for obvious applied reasons but it would have been informative if they also contrasted this data set with that obtained from a variable region. That said, the data produced is convincing and therefore has merit in isolation. Congratulations on a really nicely presented paper. ****** 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? 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Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0337191.r001
Revision 1
4 Nov 2025 Author Response Reviewer #1: In “Generational mutation patterns in a honey bee Deformed Wing Virus via infectious Clones”, Nearman and colleagues explore viral quasispecies dynamics of a critical pathogen of honey bees, Deformed wing virus (DWV). Using previously developed infectious clones, the researchers passaged these viral population through three generations of bees and used qPCR and amplicon sequencing to examine viral load and mutation accumulation through the passaging paradigm. Researchers also infected bees sourced from multiple colonies to measure any possible host effects on virus dynamics. While the infectious clone did not reveal a high amount of variation through passaging, the researchers were able to compare these dynamics to the passaging dynamics of a co-passaged wild type DWV. This wild type DWV demonstrated higher viral titers and a greater rate of silent mutation accumulation through the experiment. The introduction is a great justification to the study. I only have minor points to improve the manuscript. Response: Thank you for your thoughtful review. Your comments certainly helped point out a few shortcomings as well as inspire some deeper thought on the system at hand. We really appreciate it! ----------------------- Methods : Why was only VP2 the focus? I know for example full viral genome sequencing incurs higher costs and more computational demand, but justification for this region for these initial experiments would be beneficial. Response: Great question. VP2 is adjacent to the clone insert, allowing us to design primers that span across the insert into VP2 and select specifically for the clone. We have adjusted the introduction to clarify. ----------------------- More details are needed concerning the homogenate prep need to be included for replicability; for example, are pooled homogenates suspended in PBS or some other solution? Response: Certainly! Two previous papers have been published detailing the methods for this system. They are referenced in the introduction on lines 64 and 72, and further throughout the paper. ----------------------- L265-272 what specific programs were used for variant filtering and for amino acid translation? Response: Great catch. As the sequence was short, I performed all calculations and sequence translations in R. The variant filtering is detailed in the methods but the translation required custom R code. We will include the code in the final submission and reference it in the methods. ----------------------- Results/Discussion : Is it expected that there’s simply more sub-consensus variation in WT vs clones (or detection lower than threshold), which is why you can see the accumulation of silent mutations? This may be addressed by the point made that further passaging beyond F2 may reveal more mutations in the clone, but just to highlight. Response: This is an interesting thought. It would seem we lack the resolution here to determine exactly when the variant explosion occurred, either at low detection at the end of F1 or the beginning of replication during F2. I suppose we also don’t know what the lag between clone vs wt precisely is, generationally speaking. Certainly, both points can be addressed in future work to better characterize this system. For now, we have provided additional analyses that may help elucidate these points. ----------------------- In Figure 5, you highlight a higher proportion of reads with mutation by F2, but also F2 viral loads is lower than F1. Is it suspected that accumulation of silent mutations could be interfering with the replication rates of the virus? Response: Excellent question. We had to think about this for a bit. We landed on two possibilities. One is that sequence diversity may have exceeded the efficacy of our PCR primers by F2 for some quasi species. Another is that there may be some accumulation of non-viable strains in the inoculum. We have updated the discussion with this thinking. ----------------------- I think some additional support into the utility of using these clones for examining virus dynamics would be helpful. The work is centered around the infectious clones (e.g. the title) but it was the lack of variation in the clones compared to the WT virus that emphasized the importance of the WT virus in the passaging paradigm. Or, as authors, are you arguing that these sorts of virus dynamics experiments should instead be conducted with WT virus? Response: We are arguing for the use of the cloned species. Given the nature of this particular virus, however, there is always the possibility of latent infection, so it may be that experiments such as these should always be prepared for both. The used of the clone strain allows us to more easily track mutation rates and we expect that, given enough generations, the cloned strain would demonstrate equally high levels of variation. Still, much more work is needed to measure the precise dynamics of this system. ----------------------- Reviewer #2: The manuscript entitled: "Generational mutation patterns in a honey bee Deformed Wing Virus via infectious clones" exploits the power and novelty of using clones to investigate mutation rates accumulated by viruses in an in vivo setting. The authors focused on a conserved region for obvious applied reasons but it would have been informative if they also contrasted this data set with that obtained from a variable region. That said, the data produced is convincing and therefore has merit in isolation. Congratulations on a really nicely presented paper. Response: Thank you for the insights and the review! We really appreciate it! Attachments Attachment Submitted filename: Reviewer_Responses.docx https://doi.org/10.1371/journal.pone.0337191.r002
6 Nov 2025 Decision Letter - Olav Rueppell, Editor Generational mutation patterns in a honey bee Deformed Wing Virus via infectious clones PONE-D-25-40188R1 Dear Dr. Nearman, 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. For questions related to billing, please contact billing support . 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, Olav Rueppell Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0337191.r003
Formally Accepted
Acceptance Letter - Olav Rueppell, Editor PONE-D-25-40188R1 PLOS ONE Dear Dr. Nearman, 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 You will receive further instructions from the production team, including instructions on how to review your proof when it is ready. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few days 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. You will receive an invoice from PLOS for your publication fee after your manuscript has reached the completed accept phase. If you receive an email requesting payment before acceptance or for any other service, this may be a phishing scheme. Learn how to identify phishing emails and protect your accounts at https://explore.plos.org/phishing. 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. Olav Rueppell Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0337191.r004

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