Review 2: "The emergence of new lineages of the Monkeypox virus could affect the 2022 outbreak"

RR:C19 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:

By September 20, 2022, the ongoing monkeypox outbreak has led to more than 64,000 confirmed cases and 20 deaths in over 105 countries. Because of its fast spread, WHO declared the monkeypox outbreak a global public health emergency on July 23, 2022. Understanding the origin of the current monkeypox virus and its evolution and relationship with previous outbreak virus strains is critical to control and preventing the further spread of the current outbreak. Furthermore, these studies should provide important insights into developing novel drugs and new strategies for the treatment of the monkeypox virus.  In this report, the authors studied the emergence of new lineages of the Monkeypox virus during the current 2022 outbreak. Thus, the report is important and may provide new insights into our understanding of the current outbreak of monkeypox virus isolates/strains. 

In this report, the authors analyzed the currently available sequenced monkeypox genomes of clades B.1, A1.1., and A.2.  They specifically focused on analyses of 9 viral proteins known to be important in viral pathogenesis and 4 proteins important for interacting with the host’s immune response. Their analyses revealed the surprising results that the viral C3L gene was not found in the sequenced genomes of the monkeypox virus isolated from the current outbreak. Furthermore, the authors have identified several synonymous mutations and amino acid changes among these genes and proteins.  They concluded that the mutations identified in the current report have not been associated with genetic alterations and significant changes in transmission and clinical manifestations.

Overall, the method and analysis procedures are appropriate. The analyses are well conceived. The conclusions of the manuscript are supported by the results. However, one major concern about the manuscript is that the current report is very preliminary and can be further improved substantially with simple and additional analyses.  For example, the authors are encouraged to analyze the more than 180 genes encoded by the virus in addition to the 13 genes/proteins they have analyzed.  Global genomic and functional analyses of the current outbreak viral isolates/strains in comparison to those isolates/strains from the previous outbreaks will provide much need information about the similarities and differences among these viral isolates/strains.  

A minor concern is the presentation of the results in the study. For example, the author stated their surprising findings that the viral C3L gene was not found in the sequenced genomes of the monkeypox virus isolated from the current outbreak. Is the entire C3L sequence absent from the current outbreak of monkeypox virus isolates or is a part of the C3L sequence deleted or are point mutations at C3L that are found to block the translation of the protein?  It may be helpful and convincing to include a figure to show the C3L locus from the virus isolates/strains found in the current outbreak and from previous outbreaks so the readers and audiences can fully appreciate the nature of the mutations at the C3L locus.