Review Reports - Mpox Clade IIb Virus Introduction into Kinshasa, Democratic Republic of the Congo, July 2025

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The report on clade IIb transmission in Kinshasa by the authors is highly relevant for ongoing genomic surveillance efforts and public health interventions.

The authors place the sequence in a tree showing relationship with the current SL outbreak. However, the individual had no travel history to SL.

If the authors could elaborate on their hypothesis where the outbreak might have spread to and the potential for undetected transmission other countries that would help the reader. Otherwise the presentation in the tree and placement of the sequences is not backed by the metadata from the patient.

Author Response

The report on clade IIb transmission in Kinshasa by the authors is highly relevant for ongoing genomic surveillance efforts and public health interventions.

Response: Thank you.

The authors place the sequence in a tree showing relationship with the current SL outbreak. However, the individual had no travel history to SL. If the authors could elaborate on their hypothesis where the outbreak might have spread to and the potential for undetected transmission other countries that would help the reader. Otherwise the presentation in the tree and placement of the sequences is not backed by the metadata from the patient.

Response: We appreciate this comment from the reviewer. A recent analysis by Campbell AKO and colleagues have provided additional insights into the linkages between ongoing Clade IIb MPXV circulation in West Africa and the public health impacts of Clade IIb in Sierra Leone in 2025 (Genomic epidemiology uncovers the origin of the mpox epidemic in Sierra Leone | https://www.medrxiv.org/content/10.1101/2025.10.15.25337823v1). This genomic epidemiology investigation identified linkages between Clade IIb circulation in Sierra Leone with a common ancestor from Nigeria, and linkages to additional regions in West Africa including Togo. This has been included in the revised manuscript in Lines 166-176.

Reviewer 2 Report

Comments and Suggestions for Authors

The authors describe the first two Clade IIb/sh2017 mpox cases identified in the DRC, one imported from West Africa and one resulting from local transmission, both phylogenetically linked to the ongoing outbreak in Sierra Leone.

The major points that will need to be addressed are detailed below:

It is not entirely clear whether the primary aim of the study is to investigate the epidemiological and pathogenic characteristics of Clade IIb or to focus on the genetic characterization of the newly identified lineage G1. If the former, additional demographic and clinical data should be provided, including patient age.
The manuscript mentions that the male patient had a prior smallpox vaccination, but the timing of this vaccination should be specified. In fact, infection and disease in previously vaccinated individuals is not unexpected and has been already discussed in the literature (e.g., doi:10.1128/jcm.01365-22, doi.org/10.1016/j.jve.2022.100078, doi: 10.1093/immadv/ltad020 doi: 10.3201/eid2903.221231). However, an important distinction should be made between recent vaccination as part of current prophylactic campaigns and vaccination received during childhood. These scenarios imply different interpretations, such as potential immune evasion by Clade IIb in the former case or waning immune memory in the latter. What about the women patient? Was she vaccinated? if not, was the severity of the disease diffferent? Further discussion of this point is will be needed.
In relation to the analysis of epidemiological and pathogenetic features of Clade IIb, the authors should clarify which biological matrices were examined in addition to vesicles, blood, and nasopharyngeal swabs. Specifically, was semen analyzed? Moreover, information on the duration of viral positivity across the different matrices would strengthen the manuscript.
Regarding the characterization of lineage G1, the authors should explain why the sequences were not deposited in GISAID (not present at time of the review), as this would facilitate inclusion in phylogenetic analyses by researchers who primarily use this database. Which were the criteria employed for selecting the genomic sequences included in the phylogenetic analysis? This point should be also clarified.
Given that lineage G1 is newly described, an analysis of APOBEC-associated mutations would be particularly informative a tool to facilitate the analysis is provided by from doi:10.1038/s41598-025-11855-5, as would a more detailed discussion of the identified SNPs in comparison with other African Clade IIb sequences. The authors may also consider incorporating all these insights to enrich the discussion.

Author Response

It is not entirely clear whether the primary aim of the study is to investigate the epidemiological and pathogenic characteristics of Clade IIb or to focus on the genetic characterization of the newly identified lineage G1. If the former, additional demographic and clinical data should be provided, including patient age.

Response: We thank the reviewer for this comment. In this study, we aimed to describe the first and second Clade IIb mpox cases identified in the DR Congo. Accordingly, we built a phylogenetic tree including Clade IIb genomes from these cases. As suggested by the reviewer, we have included the age range of these patients in the revised manuscript.

Line 80: Between 21 and 26 July 2025, a 45-49-year-old man presented himself to …

Lines 102-103: index case, a nasopharyngeal swab was collected from the patient’s wife -aged 35-39 years olds- on day 2…

The manuscript mentions that the male patient had a prior smallpox vaccination, but the timing of this vaccination should be specified. In fact, infection and disease in previously vaccinated individuals is not unexpected and has been already discussed in the literature (e.g., doi:10.1128/jcm.01365-22, doi.org/10.1016/j.jve.2022.100078, doi: 10.1093/immadv/ltad020 doi: 10.3201/eid2903.221231). However, an important distinction should be made between recent vaccination as part of current prophylactic campaigns and vaccination received during childhood. These scenarios imply different interpretations, such as potential immune evasion by Clade IIb in the former case or waning immune memory in the latter. What about the women patient? Was she vaccinated? if not, was the severity of the disease different? Further discussion of this point is will be needed.

Response: Thank you very much for raising this concern.

“A prior history of smallpox vaccination” refers to an individual who had received smallpox vaccine during childhood, most likely before the discontinuation of routine smallpox vaccination in 1980. Indeed, the index patient aged between 45-49 years old had been vaccinated against smallpox and had a smallpox vaccination scar, while his wife aged 35-39 years had not received this vaccine since she was born after the cessation of routine smallpox vaccination. We have added more detail regarding smallpox vaccination in the revised manuscript:

Lines 87-88: He also reported a prior history of smallpox vaccination and had a smallpox vaccination scar.

In relation to the analysis of epidemiological and pathogenetic features of Clade IIb, the authors should clarify which biological matrices were examined in addition to vesicles, blood, and nasopharyngeal swabs.

Response: As mentioned in the manuscript, we analyzed blood and vesicle swab specimens collected from the index patient, as well as vesicle and nasopharyngeal swabs from the index patient’s wife. These samples were sufficient to confirm mpox and describe Clade IIb genomes.

Specifically, was semen analyzed? Moreover, information on the duration of viral positivity across the different matrices would strengthen the manuscript.

Response: Semen samples were not collected as part of the clinical assessment for this patient. As such, assessment of viral positivity across different specimens was not the goal of our study.

Regarding the characterization of lineage G1, the authors should explain why the sequences were not deposited in GISAID (not present at time of the review), as this would facilitate inclusion in phylogenetic analyses by researchers who primarily use this database.

Response: Many other public databases, including NCBI, ENA, Pathoplexus etc … can be used for genome data sharing. Genomes from our study were submitted to Pathoplexus database and accession numbers are provided in the Data availability section.

Which were the criteria employed for selecting the genomic sequences included in the phylogenetic analysis? This point should be also clarified.

Response: We selected Clade IIb sequences from Ivory Coast, Togo, Republic of Congo, Benin, Nigeria, Sierra Leone, USA, UK, and Austria to construct the phylogenetic tree. Sequences were selected to encompass lineage A.2.2 as well as the newly identified Clade IIb lineage G.1, first reported in Sierra Leone.

Given that lineage G1 is newly described, an analysis of APOBEC-associated mutations would be particularly informative a tool to facilitate the analysis is provided by from doi:10.1038/s41598-025-11855-5, as would a more detailed discussion of the identified SNPs in comparison with other African Clade IIb sequences. The authors may also consider incorporating all these insights to enrich the discussion.

Response: We thank the reviewer for this suggestion. Using squirrel (https://github.com/aineniamh/squirrel) to estimate the number of APOBEC3-like mutations, we identified two APOBEC3-like mutations (C92702T and C182135T) and one non-APOBEC3 mutation (C161570A); reference genomes PP_00341CY.3 and PP_00341DW.3 in Pathoplexus database. We have now included a table and the following paragraph in the revised manuscript:

Lines 155-163: Although the MPXV Clade IIb/sh2017 sequences obtained from two cases identified in July 2025 in Kinshasa, DRC are genetically identical and cluster with sequences from the 2025 Sierra Leone mpox outbreak, they differ by two APOBEC3-like mutations (C92702T and C182135T) and one non-APOBEC3 mutation (C161570A), when compared with reference genomes PP_00341CY.3 and PP_00341DW.3 (Pathoplexus accession numbers). In addition, Clade IIb/sh2017 sequences from this study exhibit APOBEC3 mutation enrichment, with approximately 69% (67/97) of reconstructed SNPs consistent with APOBEC3 editing (Table 2). Together, this study indicates that transmission of MPXV Clade IIb/sh2017 in Kinshasa resulted from human-to-human transmission.

Reviewer 3 Report

Comments and Suggestions for Authors

Authors representing a wide range of organizations, academic institutions, and research centers warned about the emergence of a new epidemiological threat in Kenya, where a complicated situation already existed with monkeypox: two circulating clades, Clade Ib/sh2023 and Clade Ia/sh2024, and the first detection of Clade IIb/sh2017. Local spread was confirmed, prompting calls for strengthened epidemiological and genomic surveillance, swift case detection, contact tracing, and vaccination to prevent further infection dissemination.

Clinical cases and key phylogenetic findings are described. Tables and the figures are informative, making the article interesting for a broad audience of medicals, virologists, and other specialists.

Unfortunately, the abstract is excessively brief and merely states the factual conclusion, omitting key information about the methodology, results, and significance typically expected in a scientific article.

The manuscript can be published after minor revision.

The following additions could be incorporated into the Abstract section: a) indicate that DRC is an endemic zone for Clade I, and the appearance of Clade IIb in this region represents a new and alarming epidemiological dynamic; b) specify the objective of the study; c) mention the methods used to confirm the case, such as point-of-care PCR and confirmation in a national laboratory; d) add that whole-genome sequencing was conducted for phylogenetic analysis.
Please provide some references properly. For example, in ref. 2: vol. 30(10), p. 2791-2795; in ref. 4: vol. 31(9), p. 2854-2858; etc.

Author Response

Response: We thank the reviewer for this positive feedback.

The manuscript can be published after minor revision.

The following additions could be incorporated into the Abstract section: a) indicate that DRC is an endemic zone for Clade I, and the appearance of Clade IIb in this region represents a new and alarming epidemiological dynamic; b) specify the objective of the study; c) mention the methods used to confirm the case, such as point-of-care PCR and confirmation in a national laboratory; d) add that whole-genome sequencing was conducted for phylogenetic analysis.

Response: Thank you. The abstract has been revised as suggested by the reviewer.

Lines 40-50: Clade I mpox virus (MPXV) is endemic in the Democratic Republic of the Congo (DRC). Recent studies have described the changing epidemiology of mpox in the country, which has been mainly characterized by the emergence of new MPXV lineages, Clade Ib/sh2023 and Ia/sh2024, associated with sustained human-to-human transmission. Both Clade Ib/sh2023 and Ia/sh2024 are co-circulating in Kinshasa, the capital city of the DRC. Here, we report the first two cases of Clade IIb/sh2017 identified in Kinshasa, DRC, imported from West Africa and locally transmitted. Clinical specimens were collected and tested by PCR. We performed whole genome sequencing using a tiled-amplicon sequencing approach with Clade IIb MPXV specific primers. Phylogenetic tree shows that Kinshasa Clade IIb MPXV assigned to Clade IIb/sh2017 within the newly designated lineage G.1, identified in January 2025 in Sierra-Leone.

Please provide some references properly. For example, in ref. 2: vol. 30(10), p. 2791-2795; in ref. 4: vol. 31(9), p. 2854-2858; etc.

Response: Thank you. Corrected