Review 5: "Clade I Mpox Virus Genomic Diversity in the Democratic Republic of the Congo, 2018 - 2024: Predominance of Zoonotic Transmission"

RR\ID Evidence Scale rating by reviewer:

• Potentially informative. The main claims made are not strongly justified by the methods and data, but may yield some insight. The results and conclusions of the study may resemble those from the hypothetical ideal study, but there is substantial room for doubt. Decision-makers should consider this evidence only with a thorough understanding of its weaknesses, alongside other evidence and theory. Decision-makers should not consider this actionable, unless the weaknesses are clearly understood and there is other theory and evidence to further support it.

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Review: The authors of this manuscript have sequenced 603 genomes of Mpox virus collected over the period of 6 years from DRC region. They have used 429 of these sequences, which had more than 70% coverage to compare APOBEC enzyme markers across these genomes. The study claims that Clade 1A is the predominant clade during this time period in DRC (2018 to early 2024).

Based on the genome sequences analyzed by the authors, they claim that most of the sequences of MPXV (2018 to2024) belong to Clade Ia, which is associated with zoonotic spread. 

Main comments:

Unfortunately the data appears to be a bit outdated as the virus has moved-on and we already know that Clade1b is the new variant of Mpox that has emerged since the above report. However the authors may be advised to include some information about currently circulating Clad1b to make the manuscript more relevant for publications. 

Major Comments:

1. The authors mention high genetic diversity in Clade Ia, but no tool has been employed to quantitatively assess this diversity. Tools such as DnaSP could be utilized to provide precise measurements of genetic diversity across clades (as shown by Kumar et al. 2024, bioRxiv), strengthening the findings.

2. It is difficult to evaluate whether the APOBEC markers confer any evolutionary advantage to the virus without understanding the genomic context of these mutations. I suggest the authors highlight the regions where these mutations occur (e.g., specific genes or extragenic regions) to gain further insights into their potential impact.

Minor Comments:

1. The authors should deposit the metadata (if available) associated with the new MPXV strain (clade 1b) to help understand how symptoms have evolved.

2. The authors have collected multiple samples from individual patients, particularly from different tissues, which could provide valuable insights into within-host viral evolution. These sequences should be analyzed carefully to better understand how the virus adapts during infection.

3. As previous studies (and the authors themselves) have indicated, disease presentation can vary with different variants. Providing metadata linked to the samples would enable further inference about the MPXV clades, aiding in understanding clinical manifestations. This data will enhance the understanding of mpox disease.

4. In The recent study by Kumar et al. (2024), bioRxiv has shown nucleotide changes across the clades and found enhanced APOBEC markers in Clades Ib and IIb; in addition to this, genetic diversity and unique mutations across the MPXV clades are also presented. Citing this work would enhance the discussion of the current study.