Internal gene segments from a mouse-adapted influenza B virus confer increased pathogenicity to mice

Arne Matthys; Laura Amelinck; Anouk Smet; Tine Ysenbaert; Thorsten U. Vogel; Xavier Saelens; João Paulo Portela Catani

doi:10.1371/journal.pone.0335324

Peer Review History

Original SubmissionOctober 16, 2025
9 Dec 2025 Decision Letter - Victor Huber, Editor Dear Dr. Portela Catani, 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. During the revision process, please address all reviewer comments while paying close attention to those associated with experimental design and the inclusion/justification of appropriate controls. Please submit your revised manuscript by Jan 23 2026 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If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. [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: Matthys and colleagues developed a mouse-adapted IBV strain based on a previously published B/Memphis/12/1997 backbone. They assessed how acquisition of mutations in all viral segments contribute to enhanced pathogenicity in mice, and rescued an reassortant IBV strain which contained the most recent B/Austria/2021 HA and NA. Given the low pathogenicity of IBV strains in mice in general, this is a useful tool which can be used to study IBV pathogenesis in vivo. I have a few minor comments which I hope will improve the quality of the manuscript: - Line 33: add complete lineage names, e.g. B/Victoria/2/1987-like (B/Victoria) and B/Yamagata/16/1988-like - Line 36/37: there is an issue with the bibliography (numbers vs last authors name in some citations) - Line 45: is the 6 IBV strains in the last 10 years in Victoria only? If so, please comment on this. - Line 71 ff (Figure 1 legend): were four or three mice used per group? This is currently unclear. Also more appropriate to speak of 'weight change over time' instead of 'weight loss'. - Line 83 ff (Figure 2 legend): Change pathogenicity to something like 'Kaplan Meier survival curve'. Pathogenicity could also be reflected in weight loss, viral replication across respiratory organs etc, and this was not assessed here. - Line 122 ff (Figure 4). The figure and the used colours are confusing. As said in Line 124, the HA, NP and NS segments are identical between the Mem97 WT and Mem97 m.a., then why are the colours different? I think it would be helpful to split the figure up and first show what segments are generated and how they differ between the Mem97 WT and the MEM97 m.a., and then show in a subsequent figure what RG viruses have been generated and tested. Line135 ff: I find the usage of the word 'heterologous' confusing in this context, because the B/HwNw-Mem97 is already per se an heterologous virus, as it contains the interal segments from B/Mem/1997 and HA/NA from B/Washington/2019. Reviewer #2: The manuscript addresses an important topic in influenza B virus research by generating mouse-adapted viruses and examining how different viral gene segments contribute to pathogenicity. The study is thoughtfully designed, and the combination of reverse genetics, sequencing, and mouse infection models offers useful insights into virulence determinants. The experiments appear carefully performed, and the use of multiple reassortant viruses adds strength to the work. That said, several areas need clarification or additional detail to improve transparency and reproducibility. For the virus that could not be rescued (B/HwNw-Mem97 m.a.), please provide a hypothesis for why rescue failed and discuss possible technical or biological reasons. PFU measurements were used to monitor virus rescue and replication, and the rescued viruses were confirmed by sequencing. However, no independent positive control was included. Please discuss this limitation and explain how sequencing combined with PFU measurements ensured that the rescue system worked reliably. No negative control (such as mock-transfected cells or PBS-treated mice) was included. Please clarify how background effects or potential contamination were ruled out. Please clarify whether PFU or viral titers were measured after each mouse passage during adaptation, or whether cytopathic effect (CPE) was the only readout. Quantitative titers across passages would strengthen conclusions about viral adaptation. Were lung viral titers measured during the adaptation passages or in the BALB/c and DBA/2J infection studies? If not, please consider adding these data, as they would support the morbidity and mortality observations. Were histopathological analyses performed on lung tissue to confirm pathology and correlate with clinical signs? If not, please discuss this as a limitation. Were cytokine profiles or other immune markers measured to provide mechanistic insight into disease severity? If feasible, please consider including these data or discuss this limitation. Please clarify the rationale for choosing DBA/2J mice for adaptation and BALB/c mice for subsequent infection studies, and discuss how strain-specific susceptibility might influence interpretation. The manuscript states that virulence is independent of HA and NA. Please expand on the mechanism and explain how the data support this conclusion. Finally, please include a more explicit discussion of the study’s limitations. ****** 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 ******** [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.] To ensure your figures meet our technical requirements, please review our figure guidelines: https://journals.plos.org/plosone/s/figures You may also use PLOS’s free figure tool, NAAS, to help you prepare publication quality figures: https://journals.plos.org/plosone/s/figures#loc-tools-for-figure-preparation. NAAS will assess whether your figures meet our technical requirements by comparing each figure against our figure specifications. https://doi.org/10.1371/journal.pone.0335324.r001
Revision 1
28 Jan 2026 Author Response Ref. [PONE-D-25-54336] - [EMID:03d00e8efc403f93] - Multi-segment mouse-adaptation of a recent B/Victoria-lineage virus independent from substitutions in hemagglutinin and neuraminidase We are grateful to the reviewers for their time and valuable comments on our manuscript. Below, please find our point-by-point responses (in blue font) to the comments that were raised. Reviewer #1: Matthys and colleagues developed a mouse-adapted IBV strain based on a previously published B/Memphis/12/1997 backbone. They assessed how acquisition of mutations in all viral segments contribute to enhanced pathogenicity in mice, and rescued an reassortant IBV strain which contained the most recent B/Austria/2021 HA and NA. Given the low pathogenicity of IBV strains in mice in general, this is a useful tool which can be used to study IBV pathogenesis in vivo. I have a few minor comments which I hope will improve the quality of the manuscript: - Line 33: add complete lineage names, e.g. B/Victoria/2/1987-like (B/Victoria) and B/Yamagata/16/1988-like Thank you for the valuable remark. Complete lineage names were added - Line 36/37: there is an issue with the bibliography (numbers vs last authors name in some citations) Thank you for the meticulous review, the bibliography was corrected. - Line 45: is the 6 IBV strains in the last 10 years in Victoria only? If so, please comment on this. We refer to both strains, which we clarified in the revised manuscript as follows: “... (e.g. 6 distinct Victoria or Yamagata strains were recommended in the last 10 years)...” - Line 71 ff (Figure 1 legend): were four or three mice used per group? This is currently unclear. Also more appropriate to speak of 'weight change over time' instead of 'weight loss'. Thank you for highlighting this. We now specify in the figure legend that four mice were used per group. In addition, the caption of the Y-axis in the graphs now reads “% original body weight”. - Line 83 ff (Figure 2 legend): Change pathogenicity to something like 'Kaplan Meier survival curve'. Pathogenicity could also be reflected in weight loss, viral replication across respiratory organs etc, and this was not assessed here. “Pathogenicity” was replaced by: “Survival of mice inoculated with...” - Line 122 ff (Figure 4). The figure and the used colours are confusing. As said in Line 124, the HA, NP and NS segments are identical between the Mem97 WT and Mem97 m.a., then why are the colours different? I think it would be helpful to split the figure up and first show what segments are generated and how they differ between the Mem97 WT and the MEM97 m.a., and then show in a subsequent figure what RG viruses have been generated and tested. Thank you for this comment. All the segments were amplified from B/HwNw-Mem97 m.a. virus, and then used to rescue the reassortants as depicted in the figure. That is the reason why they were colored differently. However, we agree that color distinction is not necessary for the three non mutated segments. The figure was adapted accordingly. Line135 ff: I find the usage of the word 'heterologous' confusing in this context, because the B/HwNw-Mem97 is already per se an heterologous virus, as it contains the interal segments from B/Mem/1997 and HA/NA from B/Washington/2019. We agree with this comment. To improve clarity our statement was rephrased as follows: “To assess whether the 6 internal gene segments of B/HwNw-Mem97 m.a. could confer pathogenicity in mice when combined with HA and NA from another IBV, we rescued recombinant viruses carrying the HA and NA gene segments from B/Austria/1359417/2021 and either the six internal segments from B/HwNw-Mem97 m.a. or from B/Memphis/12/1997 m.a.” Reviewer #2: The manuscript addresses an important topic in influenza B virus research by generating mouse-adapted viruses and examining how different viral gene segments contribute to pathogenicity. The study is thoughtfully designed, and the combination of reverse genetics, sequencing, and mouse infection models offers useful insights into virulence determinants. The experiments appear carefully performed, and the use of multiple reassortant viruses adds strength to the work. That said, several areas need clarification or additional detail to improve transparency and reproducibility. For the virus that could not be rescued (B/HwNw-Mem97 m.a.), please provide a hypothesis for why rescue failed and discuss possible technical or biological reasons. We believe that the failure to rescue the B/HwNw-Mem97 m.a. is technical. The major argument for that is the successful rescue of an IBV carrying the HA and NA gene segments from B/Austria/1359417/2021 and the six internal segments from B/HwNw-Mem97 m.a.. PFU measurements were used to monitor virus rescue and replication, and the rescued viruses were confirmed by sequencing. However, no independent positive control was included. Please discuss this limitation and explain how sequencing combined with PFU measurements ensured that the rescue system worked reliably. Please note that a positive control was included in the rescue: B/Memphis/12/97. The positive control was used to confirm cytopathic effect in HEK/MDCK co-cultures but was not stored or sequenced. This information was not included in Methods section: “The rescue using all 8 parental B/Memphis/12/97 segments was used as positive control, and the negative control consisted of the same plasmids, except for the one encoding NP” No negative control (such as mock-transfected cells or PBS-treated mice) was included. Please clarify how background effects or potential contamination were ruled out. A negative control was included in rescue and comprised a transfection of all segments except NP and no cytopathic effect was observed in this setup. Please clarify whether PFU or viral titers were measured after each mouse passage during adaptation, or whether cytopathic effect (CPE) was the only readout. Quantitative titers across passages would strengthen conclusions about viral adaptation. The titration of mouse passages was not included at the time of the mouse adaptation experiments. Our set up allowed to perform one passage per week, so 13 weeks in total. If done sequentially, the titration would extend this period to at least 26 weeks. We have, however, now titrated individually and pooled lung homogenates of passage 3, 6, 9, and 12. The figure below suggests that there is no consistent increase in lung viral titers with increased passage number. Were lung viral titers measured during the adaptation passages or in the BALB/c and DBA/2J infection studies? If not, please consider adding these data, as they would support the morbidity and mortality observations. Thank you for the suggestion. However, lung virus titers were not measured during the adaptation passage or in the BALB/c and DBA/2J mice. Please see also our response to the previous comment. Were histopathological analyses performed on lung tissue to confirm pathology and correlate with clinical signs? If not, please discuss this as a limitation. Thank you for this remark. We have amended our discussion by including a statement on limitations of our work: “Our study has limitations. First, although we identified new key substitution in the PA and M segments, the precise molecular mechanism underlying the increased pathogenicity remains to be investigated. Second, we did not assess the cytokine profile or lung histopathology, important parameters to understand the mechanism linked to disease severity.” Were cytokine profiles or other immune markers measured to provide mechanistic insight into disease severity? If feasible, please consider including these data or discuss this limitation. Please see response to the previous answer. Please clarify the rationale for choosing DBA/2J mice for adaptation and BALB/c mice for subsequent infection studies and discuss how strain-specific susceptibility might influence interpretation. DBA/2J mice were described to be more susceptible to influenza (10.1371/journal.pone.0004857), a phenotype that is attributed to a dysfunction of alveolar macrophages and increased susceptibility to infection of the airways (10.1089/jir.2014.0237). DBA/2J mice also have other dysfunctionality in immune system such as a lack of NKG2A receptors (10.1007/s00251-018-01100-x) and C5 complement (10.1016/S0021-9258(19)39817-5). BALB/c mice are very often used to study influenza virus infection, which is why we turned to BALB/c mice to further evaluate the contribution to pathogenicity of reassortant IBVs with the 6-segment mouse-adapted backbone. In the discussion, we added the following to address this comment: “Mouse adaptation was performed by serial passages in DBA/2J mice, which are more susceptible to influenza A virus infection than BALB/cByJ mice (10.1371/journal.pone.0004857 and 10.1371/journal.pone.0004857). This increased susceptibility may explain the difference in LD50 of the B/HwNw-Mem97 m.a. virus in DBA/2J mice (25 PFU) compared to BALB/c mice (810 PFU).” The manuscript states that virulence is independent of HA and NA. Please expand on the mechanism and explain how the data support this conclusion. Thank you for this remark. We realize that the original title of our manuscript was somewhat misleading. We meant to convey that we were able to generate increased pathogenic virus using internal gene segment from a mouse-adapted IBV combined with HA and NA derived from recent IBV strains. To address this remark, we adapted the title of our manuscript as follows: “Internal gene segments from a mouse-adapted influenza B virus confer increased pathogenicity to mice” Finally, please include a more explicit discussion of the study’s limitations. We added the following statement on limitations of our study to the discussion: “Our study has limitations. First, although we identified new key substitution in the PA and M segments, the precise molecular mechanism underlying the increased pathogenicity remains to be investigated. Second, we did not assess the cytokine profile or lung histopathology, important parameters to understand the mechanism linked to disease severity.” ________________________________________ Attachments Attachment Submitted filename: Rebuttal - Matthys et al.docx https://doi.org/10.1371/journal.pone.0335324.r002
2 Mar 2026 Decision Letter - Victor Huber, Editor Internal gene segments from a mouse-adapted influenza B virus confer increased pathogenicity to mice PONE-D-25-54336R1 Dear Dr. Portela Catani, 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, Victor C Huber Academic Editor PLOS One Additional Editor Comments (optional): Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 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??> Reviewer #1: (No Response) Reviewer #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? -->?> Reviewer #1: (No Response) Reviewer #2: Yes ****** 4. Have the authors made all data underlying the findings in their manuscript fully available??> The PLOS Data policy Reviewer #1: (No Response) Reviewer #2: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English??> Reviewer #1: (No Response) Reviewer #2: Yes ****** Reviewer #1: (No Response) Reviewer #2: Thank you for the careful revision and for providing detailed responses to the reviewer’s comments. The manuscript has improved in clarity and transparency following revision. The clarification of positive and negative controls in the reverse genetics rescue system strengthens confidence in the experimental approach. The explanation regarding the unsuccessful rescue of B/HwNw-Mem97 m.a. is reasonable, particularly in light of the successful rescue of reassortant viruses sharing the same internal backbone. The addition of viral titration data across selected mouse passages provides helpful context for the adaptation process. The rationale for the use of DBA/2J mice for adaptation and BALB/c mice for infection studies is now clearly justified and appropriately supported by literature. Importantly, the revision of the manuscript title and clarification of the role of internal gene segments address concerns regarding potential overinterpretation of HA and NA independence. Although measurements of lung viral titers during infection, histopathological analysis, and immune profiling would further strengthen mechanistic insight, the authors have appropriately acknowledged these as limitations in the discussion. Overall, the revisions satisfactorily address the reviewer’s concerns and improve the manuscript. ****** 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: Yes: Mahmuda Yeasmin ******** https://doi.org/10.1371/journal.pone.0335324.r003
Formally Accepted
Acceptance Letter - Victor Huber, Editor PONE-D-25-54336R1 PLOS One Dear Dr. Portela Catani, 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. Victor C Huber Academic Editor PLOS One https://doi.org/10.1371/journal.pone.0335324.r004

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