Review 4: "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:

• Reliable. The main study claims are generally justified by its methods and data. The results and conclusions are likely to be similar to the hypothetical ideal study. There are some minor caveats or limitations, but they would/do not change the major claims of the study. The study provides sufficient strength of evidence on its own that its main claims should be considered actionable, with some room for future revision.

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Review: The manuscript entitled “Clade I Mpox virus genomic diversity in the Democratic Republic of the Congo, 2018 - 2024: Predominance of Zoonotic Transmission” by Kinganda-Lusamaki presents a timely and relevant study, providing insights into the genomic diversity and zoonotic transmission of Mpox virus in the Democratic Republic of the Congo (DRC). While the research is valuable, several key concerns need to be addressed:

1. Methodology - Reference-based Genome Assembly: The authors used a reference-based genome assembly, but why weren’t de novo assembly included? Also considering that, all these strains have been sequenced recently, the amount of sequencing data per sample should also be provided and the raw data should be submitted in the public domain.

2. APOBEC3 Mutations & Phylogeny: The use of multiple outgroup references for APOBEC3 mutation analysis was appropriate. However, the same approach should be adopted in constructing the phylogeny to ensure robust results. Additionally, the citation of Vakaniaki et al., 2024, references mutations, yet a table summarizing these mutations is missing and should be included for clarity. A phylogenetic analysis based on single nucleotide polymorphisms (SNPs) using a globally representative genome dataset would provide a more accurate understanding of lineage clustering (PMID: 37043267).

3. For the subset of 22 patients with multiple isolated strains, the manuscript lacks clarity on the completeness and coverage of these genomes. Further, the criteria used to determine that these strains represent distinct variants based on mutational sites should be incorporated.

4. Cluster Analysis in Figure 2a: The authors claim that "three main clusters were observed within group II," but this is not clearly visualized in Figure 2a. This reviewer recommends a reevaluation of the figure or clearer explanation within the text.

5. Discussion - The statement "provide insight into the potential drivers underlying the increasing mpox cases" is misleading. The paper does not seem to identify any new genetic drivers but rather validates previous findings. This should be clarified to reflect that no novel genetic determinants were discovered.

6. While the manuscript indicates that sequences are publicly available in nextclade, it would be helpful to provide more details on data accessibility, such as links to sequence repositories or accession number.

7. The authors use "APOBEC3-related mutations" but without sufficient explanation for readers who may not be familiar to these specific mutations.

8. Incorporating clinical outcomes data would provide valuable perspective and significantly enhance the study's impact.