Anomalous Emergence of D614 Reverse Mutations in the Delta and Omicron BA.2 Variants

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The analysis provides a comprehensive survey of D614G reversal across multiple VOCs of SARS -CoV2 using the Genbank data. Considering that the sequence provided by NCBI is accurate, we should also understand that data provided by Gen bank could be having more data from urban areas rather than giving data on viral prevalence across the country. 

Line 75, the author reduced burden by not considering deletion and insertion sequences which may bias mutational frequency as these viruses go through mutations very frequently especially the Omicron variants which has many indels. This removal of sequences could also affect the overall statistical comparison of mutation ratios across VOCs. 

The term 'repeated reverse mutation' is a strong claim as its highly possible for the virus to have the D614 sequence by recombination and the presence of ancestral sequence from the beginning.  

Author Response

Dear Reviewer,

We sincerely thank you for your careful review of our manuscript and for your constructive comments. We have revised the manuscript accordingly, and we believe that the revision has improved the clarity and rigor of the work.

All changes are highlighted in the revised manuscript (revisions in red; unchanged text in black). Our detailed, point-by-point responses to your comments are provided in the attached response document, where your comments are reproduced and our responses are given directly beneath each point. Please refer to the section titled “Reviewer 1”.

Thank you again for your time and consideration.

Sincerely,
Hideki Kakeya

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

No phylogenetic trees are constructed.
No ancestral state reconstruction is performed.
Without phylogenetic context, it is impossible to distinguish: true reversion, recombination, sequencing artefacts, contamination, or misassignment of lineages.
This omission alone is fatal to the manuscript’s central claim.

Author Response

Dear Reviewer,

We sincerely thank you for your thorough review of our manuscript and for raising important methodological concerns.

We agree that phylogenetic reconstruction and ancestral state inference are essential to causally distinguish true reversion from alternative explanations such as recombination, sequencing artefacts, contamination, or lineage misassignment. After careful consideration, however, we did not add new phylogenetic analyses in this revision. The primary aim of the present study is not to infer phylogenetic ancestry or transmission routes, but to identify and characterize non-random population-level patterns—specifically variant-specific enrichment, co-mutation structure, and spatiotemporal clustering—within publicly available genomic surveillance data.

Accordingly, we have revised the manuscript to ensure that our conclusions are strictly descriptive and hypothesis-generating, and we explicitly state the relevant limitations, including that the present study does not establish causal mechanisms underlying the observed D614-containing sequences. We also clarify that phylogenetic analyses represent an important next step for follow-up investigations prompted by the anomalous patterns reported here.

Please find our responses to all reviewers’ comments in the attached response document.

Thank you again for your time and consideration.

Sincerely,
Hideki Kakeya

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Dear Authors,

After Carefully Reviewing your communication paper entitled "Anomalous emergence of D614 reverse mutations in the Delta and Omicron BA.2 variants"

This work is very important since it highlights an important medical and microbiological topic in the research aiming to follow the reverse mutations of the D614 in different libraries of the sequences of different SARS-CoV-2, mainly Delta and Omicron Variants, isolated from COVID-19 patients from different geographical regions. Where Results showed high prevalence of d614 reverse mutations in different variants.

The Paper is very well written in English Language, very well designed and presented. Figures and Tables are very clear and are free from any additional modifications.

Kindly find below some remarks regarding this nice work:

01- The Abstract is very long for a communication paper, so please try your best to make it shorter.

02- The list of keywords is long, you are kindly invited to remove at least two words of it.

03- The Introduction section is very very short, you are invited to make it longer, maybe by talking about the impact of different mutations in S gene (Coding for Spikes) on the transmission rate of SARS-CoV-2. You can refer to the following paper concerning this point:

-- The Emergence of SARS-CoV-2 Variant(s) and Its Impact on the Prevalence of COVID-19 Cases in the Nabatieh Region, Lebanon.

04- In the whole manuscript, it is not clear when you talked about D614G and G614D I think that here you have a TYPO mistake, please correct it.

05- The paper lacks a conclusion section, please add a Conclusion section at the end of your Paper.

Best Regards,

Author Response

Dear Reviewer,

We sincerely thank you for your careful review of our manuscript and for your constructive comments. We have revised the manuscript accordingly, and we believe that the revision has improved the clarity and rigor of the work.

All changes are highlighted in the revised manuscript (revisions in red; unchanged text in black). Our detailed, point-by-point responses to your comments are provided in the attached response document, where your comments are reproduced and our responses are given directly beneath each point. Please refer to the section titled “Reviewer 3”.

Thank you again for your time and consideration.

Sincerely,
Hideki Kakeya

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

For me, a better decision is to reject this manuscript

Reviewer 3 Report

Comments and Suggestions for Authors

Dear Authors,

After Carefully Reviewing the revised version of your communication paper entitled "Anomalous emergence of D614 reverse mutations in the Delta and Omicron BA.2 variants"

I see that this paper is suitable for publication in its present form thanks to the modifications you made.

Best Regards