A Mouse-Adapted CHIKV Strain Harboring E2-K200R and Non-Structural Mutations Exhibits Enhanced Pathogenicity in Multiple Rodent Models

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Chikungunya virus (CHIKV) has been recognized as a re-emerging pathogen and a public health threat causing long-lasting joint pain. To develop effective antivirals, understanding of their detailed pathogenic mechanism is thought as crucial. For this purpose, establishment of the animal model mimicking human disease is urgent issue for CHIKV research. Authors obtained the mouse adapted virus exhibiting higher and durable viremia with severe joint inflammation comparing to wild type virus. This virus can be used as a useful tool for various CHIKV research. The manuscript is essentially well prepared. Several points were raised.

 

1. Description of methods of viral titration and neutralization test are not appeared, although those results were shown in fig 1 and fig 5. Please describe those in Materials and Methods section.
2. Fig1D; The CHIKV-Adapt possesses the mutation in NSp2 in addition to K200R. Why did authors ignore this mutation?
3. Fig1D; If authors can describe “CHIKV-WT” along with MH670649, it should be helpful for future readers.
4. Fig2; If authors can indicate where the K200R residue locate in this model, it should be helpful for future readers.
5. Fig4; There are the scale bar representing 500um, however in the legend, authors described as 50um. Which is correct?
6. Fig5; Please indicate (A), (B) and (C).
7. Fig5; Relating to the comment#1, which virus did authors use for neutralization test? If authors used homologous virus, why were the titers induced by WT higher than Adapt?
8. Fig5; Have authors conducted re-challenge experiment by using WT virus?
9. Line 331-332; Where can we find the data that inflammation was caused by mononuclear cells and lymphocytes? Please describe those findings in the Result section.
10. Reference #22 and #23 were identical.

Author Response

Dear Reviewer:

Thank you very much for your thoughtful and constructive comments on our manuscript entitled "A Mouse-Adapted CHIKV Strain Harboring E2-K200R and Non-structural Mutations Exhibits Enhanced Pathogenicity in Multiple Rodent Models". We greatly appreciate your positive assessment of our work and your recognition that the adapted virus provides "a useful model for studying CHIKV pathogenesis and evaluating vaccines". Your insightful suggestions have been invaluable in helping us improve the clarity and quality of our paper.

In the revised manuscript, all changes have been marked for your convenience: English language polishing is indicated in red, while revisions made in response to reviewer comments are highlighted in yellow.

We have prepared a detailed point-by-point response to all your comments, which is attached as a PDF file for your review.

We hope that the revised manuscript now addresses your concerns satisfactorily and meets the standards for publication in your esteemed journal. Thank you once again for your time and valuable insights.

Yours sincerely,
Junbing Wang, Shuaiyao Lu

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Please clarify the sequencing results, rewrite your discussion and conclusions regarding the role of K200R, and discuss the potential contributions of other mutations that arose during the adaptation process. See the attached pdf for more comments.

Comments for author File: Comments.pdf

Comments on the Quality of English Language

The English could be improved to more clearly express the research.

Author Response

Dear Reviewer,

We would like to thank you and the reviewers for the thoughtful and constructive comments on our manuscript entitled "A Mouse-Adapted CHIKV Strain Harboring E2-K200R and Non-structural Mutations Exhibits Enhanced Pathogenicity in Multiple Rodent Models" (Manuscript ID: 54292386). We appreciate the reviewer's recognition that our work provides " a useful model for studying CHIKV pathogenesis and evaluating vaccines." We have carefully considered all comments and have revised the manuscript accordingly.

In the revised manuscript, all changes have been marked for your convenience: English language polishing is indicated in red, while revisions made in response to reviewer comments are highlighted in yellow.

We have prepared a detailed point-by-point response to all your comments, which is attached as a PDF file for your review.

We hope that the revised manuscript now addresses your concerns satisfactorily and meets the standards for publication in your esteemed journal. Thank you once again for your time and valuable insights.

Yours,

Sincerely

Junbing Wang, Shuaiyao Lu

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript by Tang et al. describes the generation of a mouse-adapted Chikungunya virus strain (CHIKV-Adapt) through serial passaging in A129 and C57BL/6 mice. The authors identify an E2-K200R mutation in this adapted strain and demonstrate that it exhibits enhanced viremia, broader tissue distribution, and exacerbated joint pathology across multiple immunocompetent rodent models (C57BL/6, BALB/c, and hamsters) compared to the wild-type virus. The development and characterization of robust immunocompetent models for CHIKV pathogenesis are valuable contributions to the field. However, there are significant limitations regarding the attribution of the entire in vivo phenotype solely to the E2-K200R mutation, as well as questions regarding the mechanistic novelty of the findings given prior literature. Addressing these issues will be critical for strengthening the manuscript.

Major critiques:

1) The authors attribute the enhanced pathogenicity and in vivo fitness of the CHIKV-Adapt strain entirely to the E2-K200R mutation. However, Figure 1D clearly indicates the presence of two additional mutations in the non-structural proteins: NSP1 G230R and NSP2 M466E. Because the authors used a serially passaged virus rather than a reverse-genetics-derived infectious clone, they cannot definitively conclude that E2-K200R is the sole driver of the observed phenotypes. Alphavirus non-structural proteins (such as nsP2) are known to actively antagonize host innate immune responses, such as inhibiting interferon-induced JAK-STAT signaling and causing host transcriptional shutoff. Therefore, the NSP1 and NSP2 mutations could significantly contribute to the enhanced replication observed in vivo. To claim that the E2-K200R mutation drives this phenotype, the authors must generate a recombinant virus containing only the E2-K200R mutation. Alternatively, the manuscript’s title and text must be revised to acknowledge that the phenotype is driven by the combined mutations of the CHIKV-Adapt strain.

2) As the authors acknowledge in their Discussion, previous studies (e.g., Hawman et al. 2017, Carpentier et al. 2019) have already identified E2-K200R as a key mutation that enhances virulence by evading clearance mediated by the MARCO scavenger receptor on liver Kupffer cells. Given this established mechanism, the lack of an in vitro replication difference in Vero and A549 cells (Figure 1E) is entirely expected, as these epithelial cell lines do not express MARCO. To provide novel mechanistic insight rather than just describing a confirmatory in vivo phenotype, the authors should test viral replication or clearance using primary macrophages or Kupffer cells in vitro.

3) The authors use AlphaFold3 to model what they call the "complete E protein (E3-E2-6K-E1)" of the WT and adopted strains. The authors need to clarify if this indeed the un-cleaved polyprotein which includes 6K or if this is the cleaved E2-E1 heterodimer. The subsequent docking experiments with the MXRA8 receptor should be done with the E2-E1 heterodimer and not the un-cleaved polyprotein as this is irrelevant for receptor binding. Moreover, Alphafold may not accurately model the impact of point mutations as it is heavily influenced by existing structures. The authors should discuss this limitation. Furthermore, since the primary known mechanism for the K200R mutation is evasion of the MARCO receptor, structural modeling of E2 interactions with MARCO would be far more relevant and informative than modeling MXRA8.

Minor Comments

1) In Figure 1A, the text "Ankale joint Injection" should be corrected to "Ankle joint Injection".

2) Figure 1, panel E is mislabeled as B in the figure legend.

3) Figure 4, panel A: The authors should indicate on the H&E stains what they consider to be  microscopic lesions, such as synovial hyperplasia and inflammatory cell infiltration.

4) In Section 2.6, the methodology states that a semi-quantitative scoring system of 0-3 was used for each item. However, Figure 4B shows total pathology scores reaching up to ~14. The text should explicitly clarify that the "Total pathology score" presented in the figure is the sum of the individual parameter scores.

5) The authors should clarify how much virus was used to infect the mice in the passaging experiment.

 

 

Author Response

Dear Reviewer,

 

We would like to thank you for the thoughtful and constructive comments on our revised manuscript entitled "A Mouse-Adapted CHIKV Strain Harboring E2-K200R and Non-structural Mutations Exhibits Enhanced Pathogenicity in Multiple Rodent Models" (Manuscript ID: 54292386). We appreciate your recognition that "the development and characterization of robust immunocompetent models for CHIKV pathogenesis are valuable contributions to the field." We have carefully considered the valuable suggestions you raised, which address core concerns of our study, and have further revised the manuscript accordingly.

In the revised manuscript, all changes have been marked for your convenience: English language polishing is indicated in red, while revisions made in response to reviewer comments are highlighted in yellow.

We have prepared a detailed point-by-point response to all your comments, which is attached as a PDF file for your review.

We hope that the revised manuscript now addresses your concerns satisfactorily and meets the standards for publication in your esteemed journal. Thank you once again for your time and valuable insights.

Yours,

Sincerely

Junbing Wang, Shuaiyao Lu

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript is accordingly revised. Thus this manuscript is now considered as acceptable for publication in "Viruses".

Reviewer 2 Report

Comments and Suggestions for Authors

Thank you for your careful and thorough revision of the manuscript. I appreciate the effort taken to address the concerns raised in the initial review.

Overall, the revised version is significantly improved in clarity, presentation, and interpretation. The study presents a valuable contribution by developing a mouse-adapted CHIKV strain and demonstrating its utility across multiple immunocompetent rodent models. The in vivo data are comprehensive and provide a useful framework for future pathogenesis and vaccine studies.

I particularly appreciate the your revisions to the discussion and the more cautious interpretation of the E2-K200R mutation. The clarification that this mutation is associated with, rather than definitively responsible for, the observed phenotype appropriately aligns the conclusions with the available data.

The concerns regarding: additional mutations in NSP1 and NSP2, overinterpretation of computational docking, reliance on RT-qPCR without infectious titration assays, and speculative mechanistic explanations, have been sufficiently acknowledged and appropriately moderated in the revised manuscript.

While experimental dissection of individual mutations (e.g., via reverse genetics) would further strengthen the mechanistic conclusions, I agree that this may be beyond the current scope and can reasonably be addressed in future studies.

At this stage, I have no further major concerns.