Dear Volker,

Thank you very much for submitting your manuscript "A Hepatitis C virus genotype 1b post transplant isolate with high replication efficiency in cell culture and its adaptation to infectious virus production in vitro and in vivo" for consideration at PLOS Pathogens. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. The reviewers appreciated the attention to an important topic. the quality of the work, and the impact of the study for the HCV field. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the reviewer's recommendations.

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Thank you again for your submission to our journal. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Charles M Rice

Associate Editor

PLOS Pathogens

Guangxiang Luo

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

​orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

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Your manuscript has been reviewed by two experts in the field. Both were highly positive of the quality of the study and its impact. However, the reviewers did raise several points that they felt were important for finalizing the submission.

Reviewer Comments (if any, and for reference):

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: See the attached file.

Reviewer #2: The study by Heuss et al. (PPATHOGENS-D-22-0053), describes a novel cloned hepatitis C genotype 1b strain with intrinsic high levels of replication in cell culture. The authors provide evidence for the mechanism behind this unique phenotype. Through a remarkable process of culture adaptation, the authors then succeed in selecting mutations that overcome a restriction on infectious particle production and perform exhaustive mechanistic analysis. Finally, experiments in human liver chimeric mice show that the cell culture adapted clone retains viability in vivo, and importantly identify a critical mutation in NS5A. Thus, this is the first paper to demonstrate in vivo viability of a highly adapted HCVcc; most existing full-length culture systems depend on a large number of adaptive mutations.

This work is original, highly relevant and represents a significant advance in the field, since most HCV isolates are non-replicative in vitro and genotype 1b, the most prevalent genotype worldwide, have been notoriously difficult to culture. Further, it has important in vivo data. However, there are a few issues that needs to be addressed.

Specific comments.

1. The authors should acknowledge/cite the previous work by Jinqian Li et al., who developed genotype 1b cell culture systems (https://doi.org/10.1016/j.antiviral.2021.105136), and therefore tone down or delete statements referencing GLT1cc as the first efficient infectious cell culture system for genotype 1b throughout the text, including in the abstract.

2. The following statement from lines 75-77: “It also provides novel perspectives towards our understanding how liver transplantation drives the evolution of viral isolates with high replication capacity, which might contribute to direct pathogenesis of HCV infection.”

Although certainly a very relevant topic, the authors should tone down the contribution of the study to the understanding of evolution of viruses and liver transplantation, as none of the experiments and data presented address this aspect.

3. Were the experimental conditions applied in this study identical to those from references #17 and #30? What is the variability of methods assessing the size of DMVs? In other words, can the authors reliably compare the size of GLT1 DMVs with data on Con1 and JFH1 obtained from previous studies? If not, DMVs of transfected cells with Con1, JFH1 and GLT1 replicons should be measured head-to-head.

4. Lines 313-315, the origin of the GLT1-mut4B sample is missing.

5. In Fig 4C, can the residual level of Jc1 spread in CD81 deficient cells be explained?

6. Lines 323-325: “comparing GLT1-20M with JC1 324 and the cell culture adapted gt3a isolate DBN3acc (36), generating similar titers as GLT1-20M (Fig. S6A).”

Could the authors clarify in which conditions were the titers of GLT1-20M and DBN3acc comparable? In Fig.S6A the titers of GLT1-20M present high variability in case that the depicted bar is indeed an error bar, which should be stated in the figure legend.

7. In the legend of figure 4D, please specify time point measured.

8. In figure 4E, correct the typo, both cell lines have the same name.

9. Abstract: “but cell-to-cell spreading efficiency was not higher as in other isolates like JFH-1”

Suboptimal language – rewrite.

10. Introduction: “Meanwhile, additional genomes capable of infectious virus production based on gt1a, gt2, gt3a and gt6a have been generated (reviewed in (26)).”

Update to include gt4a, which were recently published also. Subtypes not specified for gt2 (2a, 2b and 2c have been cultured)

11. Introduction: “GLT1cc therefore closes an important gap in the availability of full-replication cycle models of all major HCV genotypes”

Since there is already published full-length culture systems for at least 3 gt1a and 2 gt1b isolates, this statement should be changed to reflect this fact.

12. Results: “We therefore PCR-amplified the viral genomic sequences from infected Huh7 cells and generated a consensus sequence based on direct sequencing of the PCR products”

The authors should specify here that the 5’ and 3’ terminal sequences were derived from Con1 (and not determined from the patient sequence).

13. Discussion: “So far they have been established for gt1a, gt2a, gt2b, gt3a and gt6a, with varying efficiency, requiring up to 20 mutations for efficient virus production (reviewed in (26)), but no efficient gt1b isolate is available.”

Update to reflect comments above about HCVcc for genotype 4a and other genotype 1b isolates.

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Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: See the attached file.

Reviewer #2: (No Response)

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Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: See the attached file.

Reviewer #2: (No Response)

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Reviewer #1: No

Reviewer #2: No

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References:

Please review your reference list to ensure that it is complete and correct. 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.

Attachments

Attachment

Submitted filename: Comments.docx

Dear Volker,

Many thanks for your thoughtful responses to the reviewers' critiques and the revised manuscript incorporating these changes. We are pleased to inform you that your manuscript 'A Hepatitis C virus genotype 1b post transplant isolate with high replication efficiency in cell culture and its adaptation to infectious virus production in vitro and in vivo' has been provisionally accepted for publication in PLOS Pathogens.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.

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Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Pathogens.

Best regards,

Charles M Rice

Associate Editor

PLOS Pathogens

Guangxiang Luo

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

​orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

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