Genomic Evolution and Clade-Specific Mutation Signatures of Monkeypox Virus (Mpox) from 2022 to 2024 Reveal Distinct African Lineage Divergence

Genomic Evolution and Clade-Specific Mutation Signatures of Monkeypox Virus (Mpox) from 2022 to 2024 Reveal Distinct African Lineage Divergence | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Genomic Evolution and Clade-Specific Mutation Signatures of Monkeypox Virus (Mpox) from 2022 to 2024 Reveal Distinct African Lineage Divergence Mukantwari Enatha, Nzungize Lambert, Nzitakera Augustin, Ndoricyimpaye Ella Larissa, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8175950/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The genomic diversity of the Monkeypox virus (Mpox), mainly observed in Africa, remains poorly characterized despite the global outbreak from 2022 to 2024. This study aims to determine the genomic evolution and clade-specific variations of Mpox using 70 complete genomes from 2022 to 2024, predominantly from Africa. Phylogenetic analysis revealed 1,038 parsimony-informative sites, with clade Ia dominance at 44.4%. The A-T-rich genomes exhibit a 3.7-fold transition/transversion bias. We also identified 20,219 amino-acid substitutions, with nearly half occurring within clade Ia (48.70%). Clade-specific mutation profiling uncovered distinct signatures: clade Ia possessed unique indel-driven mutations (J1L_ins74DDEVSE, J3R_ins74DDEVSE), clade Ib showed recurrent substitutions (A25R_R273Q, A15L_P39H), and the globally dominant clade IIb harbored deletions absent in clade I (A33R_Y119del). These mutational profiles highlight a clear genomic divergence between African clades (Ia and Ib) and global clade IIb. Our findings demonstrate that African Mpox genomes exhibit strong clade Ia dominance and lineage-specific evolutionary patterns, suggesting distinct selective pressures. This marked difference underscores the critical need for enhanced Africa-focused genomic surveillance and development of region-specific diagnostics, therapeutics, and vaccines. Biological sciences/Computational biology and bioinformatics Biological sciences/Evolution Biological sciences/Genetics Biological sciences/Microbiology Monkeypox Mpox Genomic evolution Amino-acid substitutions Clade Ia Clade Ib Clade IIb Africa Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Introduction Mpox is a zoonotic, double-stranded (ds) Deoxyribonucleic Acid (DNA) virus classified within the Orthopoxvirus genus of the Poxviridae family. Although genetically related, poxviruses exhibit notable differences in epidemiology and host interaction [ 1 ]. In July 2022, the World Health Organization declared Mpox a public health emergency of international concern [ 2 ]. Earlier in 2003, an outbreak in the United States, linked to imported rodents from West Africa, marked the first confirmed human monkeypox virus (MPXV) cases outside Africa [ 3 ]. The first human case was reported in 1970 in a child from the Democratic Republic of the Congo (DRC) (then known as Zaire) [ 4 ]. Between 1970 and 1986, 404 cases of human monkeypox were identified in Africa. Of these, 394 cases belonging to Clade I were reported from the Congo Basin countries, including Cameroon, the Central African Republic (CAR), and the DRC. In contrast, 10 cases belonging to Clade II were reported from Western African countries such as Nigeria, Sierra Leone, Côte d'Ivoire, and Liberia [ 5 , 6 ]. Several European nations have documented a steadily rising number of Mpox infections and related illnesses in 2022 and 2023, including case clusters linked to possible super-spreading occurrences that extend to non-endemic countries such as Belgium, Spain, Portugal, France, Germany, and the United Kingdom [ 7 ]. Traditionally, Mpox transmission was zoonotic, occurring through direct contact with infected animals or their carcasses [ 8 ]. However, recent data suggest enhanced human to human transmission, particularly with clade I and subclade IIa, and subclade IIb, through close contact with infected persons, respiratory droplets, and sexual transmission. This shift may reflect genomic alterations driving viral adaptation [ 9 ]. While the majority of Mpox infections are self-limited, severe disease can occur in children, pregnant women, and immunocompromised individuals [ 10 ]. Viral clades or lineages arise from the accumulation of mutations over time [ 11 ]. Historically, the MPXV exhibited a low evolutionary rate of approximately 6.5 × 10⁻⁶ substitutions/site/year[ 12 ]. Recent studies, however, reported significantly higher rates ranging from 1.0 × 10⁻⁵ to 1.5 × 10⁻⁵ substitutions/site/year [ 13 ]. During the 2022 Mpox outbreak, the mutation rate rose further, with an estimated nine substitutions per genome per year, likely reflecting sustained human-to-human transmission and viral adaptation [ 14 , 15 ]. Notably, O'Toole et al. identified distinct mutation patterns in Clade IIb genomes ( 2017–2022 ), including a high prevalence of C→T mutations occurring within a TC dinucleotide consistent with APOBEC3-mediated editing property [ 16 ]. These accounted for 90.8% of APOBEC3-associated mutations, primarily G→A and C→T substitutions, which may reinforce the virus's rapid adaptation. Similarly, Suspène et al. reported APOBEC3F-driven G→A hypermutations in 42% of 2022 outbreak genomes, demonstrating the virus's ability to adapt and evade genetic drift [ 14 ]. Such findings highlights the importance of genomic surveillance, which has been pivotal in understanding evolutionary dynamics of Mpox [ 17 , 18 ]. This study aims to identify evolutionary clades and lineages, investigate geographic spread, transmission dynamics, and assess genetic variations with potential clinical implications to inform public health interventions in Africa. For this, we analyzed genomic data from 59 Mpox isolates collected across Africa between 2022 and 2024, alongside 11 global representative genomes. 2. Results 2.1. Genome sequence metadata Among the retrieved genome sequences, metadata were available for 37 out of 70 cases. Of these 24 (43.29%) and 15 (21.43%) patients were male and female respectively. For 31 (44.29%) patients’ data about gender and age were not recorded. The mean age of patients with available data was 22.97 years, (SD ± 14.43), with median age of 22 years, and interquartile range (IQR, Q1–Q3) of 11–32 years. Regarding clinical outcomes, 61 out of 70 cases (87.14%), had an unknown status, while 5 cases (7.14%) were documented as live or fully recovered, and 4 cases (5.71%) as hospitalized or severely ill (Table 1 ). Table 1 Demographic characteristics and clinical outcomes of patients with Mpox genome sequences ( n = 70). Variable Category Count Percentage (%) *Age (years old) Mean 22.97 – SD 14.43 – Median age 22.00 – IQR (Q1–Q3) 11.00–32.00 ( width = 21.00) – Gender Male 24 34.29 Female 15 21.43 Unknown 31 44.29 Patient outcomes Live and fully recovered 5 7.14 Hospitalized/severely ill 4 5.71 Unknown 61 87.14 *Cases with available age information ( n = 37) 3.2 Geographical and temporal distribution of Mpox in Africa 2022–2024 Since 1970, DRC has remained as the main hotspot and epicenter of Mpox (Fig. 2 ). Between 2022 and 2024, we explored the distribution of genomes by geographical location, with the majority originating from the DRC 35 (50%), and the Central Africa Republic (CAR) 10 (12.29%). The sequences were purposively selected from nine other African countries (59) and smaller numbers from North America (4), South America (2), Europe (1), and Asia (4) for comparative analysis. Figure 2 illustrates the temporal distribution of genomes by year of collection, and by countries, with most sampled between 2022 and 2024 during the recent outbreak period, while a limited number of older sequences were included to provide historical context. Together, these distributions highlight the dataset’s strength in capturing both the geographical diversity of African outbreaks and the temporal dynamics of the recent epidemic, while also incorporating global comparators. 2.3. The percentage distribution of clades/lineages The preliminary analysis of the 70 selected genomes revealed that clade Ia was the largest single clade, comprising 35% of sequences, while clade IIb accounts 50%, including subclades IIb, IIb A, IIb A.2, IIb A.2.2, and IIb A.2.3 as shown in Fig. 3 . 2.4. Distribution of clades /lineages of African genomes (2022–2024) by country This python script-based exploratory revealed that the DRC exhibited the highest clade/lineage diversity and dominance, particularly within Clade I (sub-clades Ia and Ib), with Clade Ib accounting for 35 occurrences (Fig. 4 A). Clades Ia and Ib exhibit the greatest accumulation of substitutions which might have contributed for the observed transmission and clinical diseases observed in DRC, CAR and Congo. However, clade IIb and its sub-lineages (A.3, A.2.1, B.1 variants) show comparatively fewer but unique mutations (Fig. 4 B). 2.5. Maximum Likelihood Phylogenetic tree. Phylogenetic reconstruction determined three major clades (Ia, Ib, IIb) with bootstrap values > 95%. Clade Ia comprised 44.4% of the genomes, clade Ib 31.9%, and clade IIb 23.6%. 2.6. Nexclade phylogenetic tree. This figure shows the distribution of Monkeypox sequences across clades and lineages. This phylogenetic tree portrays clades I (subclades Ia and Ib) and clade II (subclades IIa and IIb), with clustering patterns showing the predominance of clade Ia that might indicate secondary transmission efficiency in DRC, CAR and Congo. In contrast, clade IIb exhibits greater mutational diversity with multiple lineages (A.1, A.1.1, and B.1). The x-axis represents the number of mutations relative to the reference genomes, highlighting the genetic divergence and evolutionary dynamics of circulating strains in Africa (Fig. 6 ). 2.7. Amino Acid Substitution Patterns and Clade-Specific Mutation Profiles Across the 70 African Monkeypox virus genomes analyzed, amino acid substitution analysis revealed marked heterogeneity between clades Ia, Ib, and IIb (Fig. 7 ). 2.8 Clade-specific amino-acid mutation signatures To further illustrate the evolutionary structure of Mpox lineages and the mutation patterns that differentiate African clades (Ia and Ib) from the globally dominant clade IIb, we generated a two-panel figure summarizing (A) the conceptual evolutionary relationship between clades and (B) the clade-specific amino-acid substitution signatures identified in our dataset. 3. Discussion Our findings indicate that A↔G and C↔T transitions occur significantly more frequently than other substitution types, approximately 3.7 times higher, consistent with the elevated mutation rates reported during the 2022 Mpox outbreak. Previous studies analyzing APOBEC3 (apolipoprotein B mRNA editing enzyme, catalytic polypeptide) deaminase activity support this pattern, particularly in Clade IIb, where transitions dominate [ 27]. Because these transitions are less disruptive to DNA structure, they may confer a selective advantage, potentially driving viral adaptation during human-to-human transmission. Our analysis estimated a total branch length of 0.0105, corresponding to an average of 1.05 substitutions per 100 nucleotide sites, which lies within the higher range of reported evolutionary rates for MPXV [ 26 ]. Previously reported MPXV evolutionary rates ranged from 6.5 × 10⁻⁶ to 1.5 × 10⁻⁵ substitutions per site per year, with peaks during the period of sharp human-to-human transmission [[ 27 ]. Our findings support the view that MPXV’s evolutionary accelerate under transmission pressure, as seen during the 2022 outbreak, which exhibited an estimated 9 substitutions per genome per year, likely associated to increased human-to-human spread and viral adaptation [ 13 , 28 ]. Consistent with this, our analysis revealed a total phylogenetic tree branch length of 0.0105, corresponding to an average of 1.05 substitutions per 100 nucleotide sites. The findings further highlight that most evolutionary divergences occurred between major clades, with internal branches accounting for 66.84% of the total tree length and divergence leading to clades Ia (44.4%), Ib (31.9%), and IIb (23.6%), underscoring the evolutionary pressures shaping the virus's genomic diversity, which is different from the widely accepted genetic population structure traditionally associated with the Mpox virus [ 29 ]. Published studies further attribute the higher mutation rates in Clade IIb to APOBEC3-associated G→A and C→T mutations [ 28 ]. Our findings highlight an evident transition-transversion bias across African Mpox isolates. Specifically, A↔G and C↔T transitions occurred 3.7 times more frequently than the other substitution types, whereas A↔T and C↔G transversions were less frequent occurring at roughly 64% as the rate of A↔C or G↔T transversions. This likely reflects evolutionary conservation at purine-pyrimidine pairing sites, which is critical for maintaining DNA structural integrity during replication. These results are consistent with previous study by Rodolphe et al ., who reported 42% of human Mpox virus genomes during the 2022 outbreak exhibited APOBEC3F-driven G→A hypermutations, demonstrating the virus's ability to repair mutations, evade genetic drift, and enhance evolutionary potential [ 29 ]. Clade Ia dominates the amino acid mutation landscape (Fig. 7 ) and has more amino acid substitutions. Considering a high clustering of clade Ia (Fig. 6 ), the observed high amino acid mutation might be associated with the recently observed person-to-person transmission of the subclade [ 30 ] and over all high number of cases and fatalities observed in DRC and the other 11 African countries [ 31 ]. The enrichment of A47R and B21R mutations within clade IIb correlates with the lineage’s global expansion post-2022, suggesting adaptive evolution potentially linked to immune escape. Conversely, conserved substitutions in clade I lineages may maintain viral stability within endemic reservoirs. Overall, phylogenetic analysis reveals three main clades, Ia, Ib, and IIb, among circulating African Mpox strains, diverging from the historically accepted genetic population structure. The identification of these suggests genetic and antigenic diversification from historical Mpox strains, on which existing vaccines such as modified vaccinia Ankara-Bavarian Nordic and ACAM2000 were based [ 29 ]. Our analysis also demonstrated clear separation between clades I (Ia, Ib) and II (IIa, IIb), with the majority of recent isolates clustering within clade Ia. Complementary analysis further categorized genomes into three sub-groups: the Western African clade (Sub-group 1) and two Central African clades (Sub-groups 2 and 3). While the Western African clade encompasses most sequences linked to widespread international transmission, the Central African clades exhibit greater genetic diversity within localized clusters and remain associated with higher virulence and mortality. High-frequency amino acid substitutions are A47R_Y221H, B21R_I1741M, B21R_N209D, C18L_K125E, which occur predominantly in clade IIb B.1 genomes, suggesting ongoing evolution since the 2022 global outbreak, whereas clade I mutations, such as A14L_T17A, A28L_M14T, remain phylogenetically conserved (Fig. 8 ). Together, these findings illustrate a dual evolutionary pattern: clade IIb drives global dissemination, whereas Central African clades maintain localized but clinically significant circulation. This underscores the need for continued genomic surveillance and tailored therapeutics and vaccines for African Mpox lineages to monitor both the expansion of clade IIb and the potential re-emergence of Central African variants. 4. Methods 4.1. Genome sequence retrieval This study was conducted using a structured and comprehensive bioinformatics pipeline, as illustrated in Fig. 1 , ensuring methodological rigor and reproducibility throughout the genomic analysis. As of September 20, 2024, a total of 70 complete or near-complete Mpox genome sequences were retrieved from the publicly accessible GISAID database, a trusted global platform for sharing influenza and emerging pathogen data [ 19 ]. Genomic sequences metadata, including geographical locations, were also retrieved for subsequent analysis. 4.2. Genome sequence alignment Following data retrieval, all selected sequences underwent multiple sequence alignment using the Multiple Alignment Fast Fourier Transform (MAFFT) tool, a robust method known for its accuracy in aligning large genomic datasets in a command-line environment, using default parameters, against reference genomes [ 20 ]. We used two reference genomes retrieved from the National Center for Biotechnology Information (NCBI): the Central African Mpox Zaire strain (NC_003310.1), consisting of ~ 197 kb of dsDNA and encoding over 190 open reading frames (ORFs) which belong to Clade I [ 21 ], and the West African Mpox strain (NC_063383.1), which serves as the reference for Clade II [ 22 ]. A total of 59 Mpox genomes from patients across Africa, 11 representative genomes from global cases, and the two reference genomes were aligned using MAFFT in bash command line mafft --auto --thread 12 –leavegappy region. The resulting alignment contained 184,965 nucleotide sites, of which 183,321 (99.11%) were constant, 1,038 (0.56%) were parsimony-informative, and 5,879 (3.18%) represented distinct site patterns. Upon completion, the alignment was thoroughly checked for quality and duplicate sequences using Aliview [ 23 ] and Raxml-ng [ 24 ]. This step was crucial to ensure the integrity of the dataset by removing potential errors or redundant entries that could skew downstream analyses. In order to pinpoint specific genetic changes, mutations were identified using BioEdit v7.2.5 [ 25 ], R packages, and Python libraries. This analysis allowed for the detailed characterization of unique single-nucleotide polymorphisms, amino acid substitutions, and other genetic variations across the circulating strains, which was visualized by R packages and Python libraries, including R photomap and ggplot2, as well as Python matplotlib and seaborn libraries. 4.3. Phylogenetic analysis From this quality-controlled alignment, the workflow diverged into two parallel but complementary paths. Firstly, to infer evolutionary relationships, a phylogenetic tree was constructed using IQ-TREE, a computationally efficient tool chosen for its ability to handle large sequence datasets and perform robust maximum likelihood analysis. The tree was generated from 72 aligned sequences using the bash script ./run_iqtree.sh, with 1000 bootstrap replicates and the best-fit model (BIC: K3Pu + F + I), resulting in a consensus tree with a log-likelihood of -260572.09. Additionally, Nextclade was used to complement the analysis by visualizing mutations and clade assignments. The final phylogenetic tree was visualized using Figtree v1.4.4, enabling clear interpretation of evolutionary relationships and identification of distinct clades or lineages among African Mpox strains. Declarations 6. Acknowledgment of data contributors. We gratefully acknowledge the Authors from the originating laboratories responsible for obtaining the specimens, as well as the submitting laboratories where the genome data were generated and shared via GISAID, on which this research is based. All data submitters can be contacted directly via www.gisaid.org. Full details of accession IDs, laboratories, and authors are provided in Supplementary Table S1. 7. Author Contributions E.M. (Enatha Mukantwari) conceived the study, designed the analysis, downloaded and curated genomic datasets, performed descriptive statistics, phylogenetic, and mutational profiling analyses, generated all figures and drafted the manuscript. L.N. (Lambert Nzungize ) contributed to data analysis and interpretation, verified analytical workflows and outputs, and reviewed and edited the manuscripts, including figures. A.N. (Nzitakera Augustin) supported in genomic data cleaning, metadata standardization, quality-control checks, interpretation of clade-specific mutations, and critically revised the manuscript. E.L.N. (Ndoricyimpaye Ella Larissa) contributed to interpretation of genomic and epidemiological findings and reviewed the manuscript. T.N. (Nshimiyimana Thaddee) assisted with computational workflows and supplementary analysis verification. E.N. (Nsengiyumva Emmanuel) participated in figure review, and supported in manuscript revisions. J.C.T. (Tuyishimire Jean Claude) supported in manuscript revisions. O.M. (Majyambere Onesphore) assisted in literature review, background framing, and manuscript editing. S.A. (Solomon Ali) provided advanced methodological guidance, validated evolutionary and mutational interpretations, and critically revised the manuscript. N.R. (Rujeni Nadine) supervised the project, provided conceptual and scientific guidance, and approved the final manuscript. A.G.W. (Araya Gebreyesus Wasihun) contributed to study design, reviewed analytical strategies, and critically revised the manuscript. 8. Data Availability Statement All genome sequences analyzed in this study were accessed and downloaded through GISAID under its data-use agreement. Accession identifiers, originating laboratories, and submitting laboratories are provided in Supplementary Table S1. Reference genomes were retrieved from NCBI. All codes used for genomic analysis will be made available upon request. 9. Additional Information Competing Interests Statement The authors declare no competing interests. Funding The author received No Funding for this work. References Lefkowitz, E., Wang, C. & Upton, C. Poxviruses: past, present and future. Virus Res. 117 (1), 105–118 (2006). Peng, Q. et al. Structure of monkeypox virus DNA polymerase holoenzyme. Science 379 (6627), 100–105 (2023). Likos, A. M. et al. A tale of two clades: monkeypox viruses. J. Gen. Virol. 86 (10), 2661–2672 (2005). Ladnyj, I., Ziegler, P. & Kima, E. A human infection caused by monkeypox virus in Basankusu Territory, Democratic Republic of the Congo. Bull. World Health Organ. 46 (5), 593 (1972). Ravichandran, N. Monkeypox in Europe: Epidemiology, Risk Factors and Implications for Public Health Actions–A Scoping Review Study. medRxiv, : p. 2024.08. 28.24312706. (2024). Khodakevich, L., Ježek, Z. & Messinger, D. 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18:23:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8175950/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8175950/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":102534254,"identity":"43e45886-5180-401b-8f98-b42973c491bb","added_by":"auto","created_at":"2026-02-12 17:04:52","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":114625,"visible":true,"origin":"","legend":"\u003cp\u003eWorkflow of the Mpox genome sequence analysis\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8175950/v1/bbd24f3f88e73b682b204888.png"},{"id":102534249,"identity":"3916414d-a556-4355-a35d-9f356e17cdf4","added_by":"auto","created_at":"2026-02-12 17:04:52","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":74064,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGeographical and temporal distribution of Mpox in Africa, 2022-2024.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8175950/v1/f05728d2bce8e93bb73cf15a.png"},{"id":102534250,"identity":"f910ab6a-2a67-4991-9468-e18addab534f","added_by":"auto","created_at":"2026-02-12 17:04:52","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":54184,"visible":true,"origin":"","legend":"\u003cp\u003eThe percentage of clade and lineage is categorized. The pie chart indicates the genetic diversity across a pathogen population and the proportion of each distinct genetic clade or lineage.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8175950/v1/119e20f474798f37ff182c9a.png"},{"id":103056437,"identity":"fd797faa-0fa8-4753-b0f7-79796b8beacb","added_by":"auto","created_at":"2026-02-20 09:10:25","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":98775,"visible":true,"origin":"","legend":"\u003cp\u003eDistribution of clades /lineages of African genomes (2022-2024) \u0026nbsp;by country. A) Bar plot showing the distribution of Mpox clades/lineages across various African countries. B) Total amino acid mutation counts per Mpox clade/lineage.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8175950/v1/362fcb0d03ceb39c18f83773.png"},{"id":102534251,"identity":"990e5a30-bd93-4733-89ac-fe4c0e670013","added_by":"auto","created_at":"2026-02-12 17:04:52","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":318726,"visible":true,"origin":"","legend":"\u003cp\u003eMaximum likelihood phylogenetic tree. Sub-group 1 represents the Western African clade, while Sub-groups 2 and 3 correspond to Central African clades 1 and 2, respectively. Strong bootstrap support (100) distinguishes the Western African lineage from Central African lineages, with further branching reflecting intra-clade diversity. The scale bar (6.0E-4 or 6 x 10\u003csup\u003e-4\u003c/sup\u003e) indicates the number of nucleotide substitutions per site per year.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8175950/v1/9f8fcdc1a587e716a1939421.png"},{"id":102962453,"identity":"aa85509d-23af-4ef7-a2af-09546f859b9c","added_by":"auto","created_at":"2026-02-19 04:08:53","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":250753,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNexclade phylogenetic tree\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis phylogenetic tree portrays clades I (subclades Ia and Ib) and clade II (subclades IIa and IIb), with clustering patterns showing the predominance of clade Ia that might indicate secondary transmission efficiency in DRC, CAR and Congo. In contrast, clade IIb exhibits greater mutational diversity with multiple lineages (A.1, A.1.1, and B.1). The x-axis represents the number of mutations relative to the reference genomes, highlighting the genetic divergence and evolutionary dynamics of circulating strains in Africa (Figure 6).\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8175950/v1/debbae53bb3d8bade45aeda8.png"},{"id":102534256,"identity":"e7d2c10c-5bc8-4815-ac54-69d0b2cf5c57","added_by":"auto","created_at":"2026-02-12 17:04:52","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":170494,"visible":true,"origin":"","legend":"\u003cp\u003eTop 20 amino acid substitutions across African \u003cem\u003eMonkeypox virus\u003c/em\u003e genomes. \u0026nbsp;The heatmap color gradient, from light yellow to dark red, represents an increasing mutation frequency, with darker shades indicating a higher frequency of mutation.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-8175950/v1/b83c6b485cc41f4fa4044005.png"},{"id":102534257,"identity":"4ba1413a-5359-4bfb-a0cf-52846ce553b7","added_by":"auto","created_at":"2026-02-12 17:04:52","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":122364,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCombined evolutionary and mutation-signature comparison of Mpox clades.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Schematic evolutionary relationship of Mpox virus, showing divergence of African clade I into Ia and Ib, and the emergence of the globally dominant clade IIb associated with the outbreak 2022–2024.\u003cbr\u003e\n \u003cstrong\u003e(B)\u003c/strong\u003e The top 5 Clade-specific amino-acid mutation signatures. Horizontal bar charts display the top lineage-restricted mutations for each clade. Clade Ia is characterized by indel-driven mutations such as J1L_ins74DDEVSE, and J3R_ins74DDEVSE, while clade Ib shows recurrent substitutions such as A25R_R273Q, and A15L_P39H. Clade IIb genomes harbor a distinct set of low-frequency deletions/insertions absent from clade I.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-8175950/v1/2ae9fcabfccb00062690fc74.png"},{"id":103589726,"identity":"c81df870-8251-4c46-b0cf-c5df8414b79b","added_by":"auto","created_at":"2026-02-27 11:57:17","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2113948,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8175950/v1/aa0657fc-13b8-4c7a-99b6-35fcecb8c18b.pdf"},{"id":102534248,"identity":"523bfd45-3566-4c03-9bdf-6cd7f38ecc94","added_by":"auto","created_at":"2026-02-12 17:04:52","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":40459,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryS1IDsofGenomicdataandmetadata.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8175950/v1/6a6d1dc6489e505a80ec4651.xlsx"},{"id":102534253,"identity":"c724942b-84e2-4e6a-bdda-2214d605c1b9","added_by":"auto","created_at":"2026-02-12 17:04:52","extension":"xls","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":134144,"visible":true,"origin":"","legend":"","description":"","filename":"supplementaryS2AASubstitutions.xls","url":"https://assets-eu.researchsquare.com/files/rs-8175950/v1/7508b3c4a4411320008ab3bc.xls"}],"financialInterests":"No competing interests reported.","formattedTitle":"Genomic Evolution and Clade-Specific Mutation Signatures of Monkeypox Virus (Mpox) from 2022 to 2024 Reveal Distinct African Lineage Divergence","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eMpox is a zoonotic, double-stranded (ds) Deoxyribonucleic Acid (DNA) virus classified within the Orthopoxvirus genus of the \u003cem\u003ePoxviridae\u003c/em\u003e family. Although genetically related, poxviruses exhibit notable differences in epidemiology and host interaction [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. In July 2022, the World Health Organization declared Mpox a public health emergency of international concern [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Earlier in 2003, an outbreak in the United States, linked to imported rodents from West Africa, marked the first confirmed human monkeypox virus (MPXV) cases outside Africa [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The first human case was reported in 1970 in a child from the Democratic Republic of the Congo (DRC) (then known as Zaire) [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Between 1970 and 1986, 404 cases of human monkeypox were identified in Africa. Of these, 394 cases belonging to Clade I were reported from the Congo Basin countries, including Cameroon, the Central African Republic (CAR), and the DRC. In contrast, 10 cases belonging to Clade II were reported from Western African countries such as Nigeria, Sierra Leone, C\u0026ocirc;te d'Ivoire, and Liberia [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Several European nations have documented a steadily rising number of Mpox infections and related illnesses in 2022 and 2023, including case clusters linked to possible super-spreading occurrences that extend to non-endemic countries such as Belgium, Spain, Portugal, France, Germany, and the United Kingdom [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTraditionally, Mpox transmission was zoonotic, occurring through direct contact with infected animals or their carcasses [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. However, recent data suggest enhanced human to human transmission, particularly with clade I and subclade IIa, and subclade IIb, through close contact with infected persons, respiratory droplets, and sexual transmission. This shift may reflect genomic alterations driving viral adaptation [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. While the majority of Mpox infections are self-limited, severe disease can occur in children, pregnant women, and immunocompromised individuals [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eViral clades or lineages arise from the accumulation of mutations over time [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Historically, the MPXV exhibited a low evolutionary rate of approximately 6.5 \u0026times; 10⁻⁶ substitutions/site/year[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Recent studies, however, reported significantly higher rates ranging from 1.0 \u0026times; 10⁻⁵ to 1.5 \u0026times; 10⁻⁵ substitutions/site/year [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. During the 2022 Mpox outbreak, the mutation rate rose further, with an estimated nine substitutions per genome per year, likely reflecting sustained human-to-human transmission and viral adaptation [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNotably, O'Toole et al. identified distinct mutation patterns in Clade IIb genomes ( 2017\u0026ndash;2022 ), including a high prevalence of C\u0026rarr;T mutations occurring within a TC dinucleotide consistent with APOBEC3-mediated editing property [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. These accounted for 90.8% of APOBEC3-associated mutations, primarily G\u0026rarr;A and C\u0026rarr;T substitutions, which may reinforce the virus's rapid adaptation. Similarly, Susp\u0026egrave;ne et al. reported APOBEC3F-driven G\u0026rarr;A hypermutations in 42% of 2022 outbreak genomes, demonstrating the virus's ability to adapt and evade genetic drift [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Such findings highlights the importance of genomic surveillance, which has been pivotal in understanding evolutionary dynamics of Mpox [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThis study aims to identify evolutionary clades and lineages, investigate geographic spread, transmission dynamics, and assess genetic variations with potential clinical implications to inform public health interventions in Africa. For this, we analyzed genomic data from 59 Mpox isolates collected across Africa between 2022 and 2024, alongside 11 global representative genomes.\u003c/p\u003e"},{"header":"2. Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Genome sequence metadata\u003c/h2\u003e \u003cp\u003eAmong the retrieved genome sequences, metadata were available for 37 out of 70 cases. Of these 24 (43.29%) and 15 (21.43%) patients were male and female respectively. For 31 (44.29%) patients\u0026rsquo; data about gender and age were not recorded. The mean age of patients with available data was 22.97 years, (SD\u0026thinsp;\u0026plusmn;\u0026thinsp;14.43), with median age of 22 years, and interquartile range (IQR, Q1\u0026ndash;Q3) of 11\u0026ndash;32 years. Regarding clinical outcomes, 61 out of 70 cases (87.14%), had an unknown status, while 5 cases (7.14%) were documented as live or fully recovered, and 4 cases (5.71%) as hospitalized or severely ill (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDemographic characteristics and clinical outcomes of patients with Mpox genome sequences (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;70).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVariable\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCategory\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCount\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePercentage (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e*Age (years old)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMean\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e22.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e14.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMedian age\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e22.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIQR (Q1\u0026ndash;Q3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11.00\u0026ndash;32.00 ( width\u0026thinsp;=\u0026thinsp;21.00)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eGender\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e34.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e21.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUnknown\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e44.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePatient outcomes\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLive and fully recovered\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHospitalized/severely ill\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUnknown\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e87.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e*Cases with available age information (\u003c/b\u003e \u003cb\u003en\u003c/b\u003e\u0026thinsp;\u003cb\u003e=\u0026thinsp;37)\u003c/b\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Geographical and temporal distribution of Mpox in Africa 2022\u0026ndash;2024\u003c/h2\u003e \u003cp\u003eSince 1970, DRC has remained as the main hotspot and epicenter of Mpox (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Between 2022 and 2024, we explored the distribution of genomes by geographical location, with the majority originating from the DRC \u003cem\u003e35\u003c/em\u003e (50%), and the Central Africa Republic (CAR) 10 (12.29%). The sequences were purposively selected from nine other African countries (59) and smaller numbers from North America (4), South America (2), Europe (1), and Asia (4) for comparative analysis. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e illustrates the temporal distribution of genomes by year of collection, and by countries, with most sampled between 2022 and 2024 during the recent outbreak period, while a limited number of older sequences were included to provide historical context. Together, these distributions highlight the dataset\u0026rsquo;s strength in capturing both the geographical diversity of African outbreaks and the temporal dynamics of the recent epidemic, while also incorporating global comparators.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. The percentage distribution of clades/lineages\u003c/h2\u003e \u003cp\u003eThe preliminary analysis of the 70 selected genomes revealed that clade Ia was the largest single clade, comprising 35% of sequences, while clade IIb accounts 50%, including subclades IIb, IIb A, IIb A.2, IIb A.2.2, and IIb A.2.3 as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Distribution of clades /lineages of African genomes (2022\u0026ndash;2024) by country\u003c/h2\u003e \u003cp\u003eThis python script-based exploratory revealed that the DRC exhibited the highest clade/lineage diversity and dominance, particularly within Clade I (sub-clades Ia and Ib), with Clade Ib accounting for 35 occurrences (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Clades Ia and Ib exhibit the greatest accumulation of substitutions which might have contributed for the observed transmission and clinical diseases observed in DRC, CAR and Congo. However, clade IIb and its sub-lineages (A.3, A.2.1, B.1 variants) show comparatively fewer but unique mutations (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Maximum Likelihood Phylogenetic tree.\u003c/h2\u003e \u003cp\u003ePhylogenetic reconstruction determined three major clades (Ia, Ib, IIb) with bootstrap values\u0026thinsp;\u0026gt;\u0026thinsp;95%. Clade Ia comprised 44.4% of the genomes, clade Ib 31.9%, and clade IIb 23.6%.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Nexclade phylogenetic tree.\u003c/h2\u003e \u003cp\u003eThis figure shows the distribution of Monkeypox sequences across clades and lineages.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThis phylogenetic tree portrays clades I (subclades Ia and Ib) and clade II (subclades IIa and IIb), with clustering patterns showing the predominance of clade Ia that might indicate secondary transmission efficiency in DRC, CAR and Congo. In contrast, clade IIb exhibits greater mutational diversity with multiple lineages (A.1, A.1.1, and B.1). The x-axis represents the number of mutations relative to the reference genomes, highlighting the genetic divergence and evolutionary dynamics of circulating strains in Africa (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Amino Acid Substitution Patterns and Clade-Specific Mutation Profiles\u003c/h2\u003e \u003cp\u003eAcross the 70 African \u003cem\u003eMonkeypox virus\u003c/em\u003e genomes analyzed, amino acid substitution analysis revealed marked heterogeneity between clades Ia, Ib, and IIb (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Clade-specific amino-acid mutation signatures\u003c/h2\u003e \u003cp\u003eTo further illustrate the evolutionary structure of Mpox lineages and the mutation patterns that differentiate African clades (Ia and Ib) from the globally dominant clade IIb, we generated a two-panel figure summarizing \u003cb\u003e(A)\u003c/b\u003e the conceptual evolutionary relationship between clades and \u003cb\u003e(B)\u003c/b\u003e the clade-specific amino-acid substitution signatures identified in our dataset.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e "},{"header":"3. Discussion","content":"\u003cp\u003eOur findings indicate that A\u0026harr;G and C\u0026harr;T transitions occur significantly more frequently than other substitution types, approximately 3.7 times higher, consistent with the elevated mutation rates reported during the 2022 Mpox outbreak. Previous studies analyzing APOBEC3 (apolipoprotein B mRNA editing enzyme, catalytic polypeptide) deaminase activity support this pattern, particularly in Clade IIb, where transitions dominate [ 27]. Because these transitions are less disruptive to DNA structure, they may confer a selective advantage, potentially driving viral adaptation during human-to-human transmission. Our analysis estimated a total branch length of 0.0105, corresponding to an average of 1.05 substitutions per 100 nucleotide sites, which lies within the higher range of reported evolutionary rates for MPXV [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Previously reported MPXV evolutionary rates ranged from 6.5 \u0026times; 10⁻⁶ to 1.5 \u0026times; 10⁻⁵ substitutions per site per year, with peaks during the period of sharp human-to-human transmission [[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Our findings support the view that MPXV\u0026rsquo;s evolutionary accelerate under transmission pressure, as seen during the 2022 outbreak, which exhibited an estimated 9 substitutions per genome per year, likely associated to increased human-to-human spread and viral adaptation [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Consistent with this, our analysis revealed a total phylogenetic tree branch length of 0.0105, corresponding to an average of 1.05 substitutions per 100 nucleotide sites. The findings further highlight that most evolutionary divergences occurred between major clades, with internal branches accounting for 66.84% of the total tree length and divergence leading to clades Ia (44.4%), Ib (31.9%), and IIb (23.6%), underscoring the evolutionary pressures shaping the virus's genomic diversity, which is different from the widely accepted genetic population structure traditionally associated with the Mpox virus [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Published studies further attribute the higher mutation rates in Clade IIb to APOBEC3-associated G\u0026rarr;A and C\u0026rarr;T mutations [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOur findings highlight an evident transition-transversion bias across African Mpox isolates. Specifically, A\u0026harr;G and C\u0026harr;T transitions occurred 3.7 times more frequently than the other substitution types, whereas A\u0026harr;T and C\u0026harr;G transversions were less frequent occurring at roughly 64% as the rate of A\u0026harr;C or G\u0026harr;T transversions. This likely reflects evolutionary conservation at purine-pyrimidine pairing sites, which is critical for maintaining DNA structural integrity during replication. These results are consistent with previous study by Rodolphe \u003cem\u003eet al\u003c/em\u003e., who reported 42% of human Mpox virus genomes during the 2022 outbreak exhibited APOBEC3F-driven G\u0026rarr;A hypermutations, demonstrating the virus's ability to repair mutations, evade genetic drift, and enhance evolutionary potential [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eClade Ia dominates the amino acid mutation landscape (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003e) and has more amino acid substitutions. Considering a high clustering of clade Ia (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003e), the observed high amino acid mutation might be associated with the recently observed person-to-person transmission of the subclade [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] and over all high number of cases and fatalities observed in DRC and the other 11 African countries [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. The enrichment of A47R and B21R mutations within clade IIb correlates with the lineage\u0026rsquo;s global expansion post-2022, suggesting adaptive evolution potentially linked to immune escape. Conversely, conserved substitutions in clade I lineages may maintain viral stability within endemic reservoirs.\u003c/p\u003e \u003cp\u003eOverall, phylogenetic analysis reveals three main clades, Ia, Ib, and IIb, among circulating African Mpox strains, diverging from the historically accepted genetic population structure. The identification of these suggests genetic and antigenic diversification from historical Mpox strains, on which existing vaccines such as modified vaccinia Ankara-Bavarian Nordic and ACAM2000 were based [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Our analysis also demonstrated clear separation between clades I (Ia, Ib) and II (IIa, IIb), with the majority of recent isolates clustering within clade Ia. Complementary analysis further categorized genomes into three sub-groups: the Western African clade (Sub-group 1) and two Central African clades (Sub-groups 2 and 3). While the Western African clade encompasses most sequences linked to widespread international transmission, the Central African clades exhibit greater genetic diversity within localized clusters and remain associated with higher virulence and mortality. High-frequency amino acid substitutions are A47R_Y221H, B21R_I1741M, B21R_N209D, C18L_K125E, which occur predominantly in clade IIb B.1 genomes, suggesting ongoing evolution since the 2022 global outbreak, whereas clade I mutations, such as A14L_T17A, A28L_M14T, remain phylogenetically conserved (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e8\u003c/span\u003e). Together, these findings illustrate a dual evolutionary pattern: clade IIb drives global dissemination, whereas Central African clades maintain localized but clinically significant circulation. This underscores the need for continued genomic surveillance and tailored therapeutics and vaccines for African Mpox lineages to monitor both the expansion of clade IIb and the potential re-emergence of Central African variants.\u003c/p\u003e"},{"header":"4. Methods","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e4.1. Genome sequence retrieval\u003c/h2\u003e \u003cp\u003eThis study was conducted using a structured and comprehensive bioinformatics pipeline, as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e1\u003c/span\u003e, ensuring methodological rigor and reproducibility throughout the genomic analysis. As of September 20, 2024, a total of 70 complete or near-complete Mpox genome sequences were retrieved from the publicly accessible GISAID database, a trusted global platform for sharing influenza and emerging pathogen data [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Genomic sequences metadata, including geographical locations, were also retrieved for subsequent analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e4.2. Genome sequence alignment\u003c/h2\u003e \u003cp\u003eFollowing data retrieval, all selected sequences underwent multiple sequence alignment using the Multiple Alignment Fast Fourier Transform (MAFFT) tool, a robust method known for its accuracy in aligning large genomic datasets in a command-line environment, using default parameters, against reference genomes [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. We used two reference genomes retrieved from the National Center for Biotechnology Information (NCBI): the Central African Mpox Zaire strain (NC_003310.1), consisting of ~\u0026thinsp;197 kb of dsDNA and encoding over 190 open reading frames (ORFs) which belong to Clade I [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], and the West African Mpox strain (NC_063383.1), which serves as the reference for Clade II [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. A total of 59 Mpox genomes from patients across Africa, 11 representative genomes from global cases, and the two reference genomes were aligned using MAFFT in bash command line mafft --auto --thread 12 \u0026ndash;leavegappy region. The resulting alignment contained 184,965 nucleotide sites, of which 183,321 (99.11%) were constant, 1,038 (0.56%) were parsimony-informative, and 5,879 (3.18%) represented distinct site patterns.\u003c/p\u003e \u003cp\u003eUpon completion, the alignment was thoroughly checked for quality and duplicate sequences using Aliview [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] and Raxml-ng [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. This step was crucial to ensure the integrity of the dataset by removing potential errors or redundant entries that could skew downstream analyses. In order to pinpoint specific genetic changes, mutations were identified using BioEdit v7.2.5 [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], R packages, and Python libraries. This analysis allowed for the detailed characterization of unique single-nucleotide polymorphisms, amino acid substitutions, and other genetic variations across the circulating strains, which was visualized by R packages and Python libraries, including R photomap and ggplot2, as well as Python matplotlib and seaborn libraries.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e4.3. Phylogenetic analysis\u003c/h2\u003e \u003cp\u003eFrom this quality-controlled alignment, the workflow diverged into two parallel but complementary paths. Firstly, to infer evolutionary relationships, a phylogenetic tree was constructed using IQ-TREE, a computationally efficient tool chosen for its ability to handle large sequence datasets and perform robust maximum likelihood analysis. The tree was generated from 72 aligned sequences using the bash script ./run_iqtree.sh, with 1000 bootstrap replicates and the best-fit model (BIC: K3Pu\u0026thinsp;+\u0026thinsp;F\u0026thinsp;+\u0026thinsp;I), resulting in a consensus tree with a log-likelihood of -260572.09. Additionally, Nextclade was used to complement the analysis by visualizing mutations and clade assignments. The final phylogenetic tree was visualized using Figtree v1.4.4, enabling clear interpretation of evolutionary relationships and identification of distinct clades or lineages among African Mpox strains.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e6. Acknowledgment of data contributors.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe gratefully acknowledge the Authors from the originating laboratories responsible for obtaining the specimens, as well as the submitting laboratories where the genome data were generated and shared via GISAID, on which this research is based. All data submitters can be contacted directly via www.gisaid.org. Full details of accession IDs, laboratories, and authors are provided in Supplementary Table S1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e7. Author Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eE.M. (Enatha Mukantwari)\u0026nbsp;\u003c/strong\u003econceived the study, designed the analysis, downloaded and curated genomic datasets, performed descriptive statistics, phylogenetic, and mutational profiling analyses, generated all figures and drafted the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eL.N. (Lambert Nzungize\u003c/strong\u003e) contributed to data analysis and interpretation, verified analytical workflows and outputs, and reviewed and edited the manuscripts, including figures.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.N. (Nzitakera Augustin)\u0026nbsp;\u003c/strong\u003esupported in genomic data cleaning, metadata standardization, quality-control checks, interpretation of clade-specific mutations, and critically revised the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eE.L.N. (Ndoricyimpaye Ella Larissa)\u003c/strong\u003e contributed to interpretation of genomic and epidemiological findings and reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eT.N. (Nshimiyimana Thaddee)\u003c/strong\u003e assisted with computational workflows and supplementary analysis verification.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eE.N. (Nsengiyumva Emmanuel)\u003c/strong\u003e participated in figure review, and supported in manuscript revisions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eJ.C.T. (Tuyishimire Jean Claude)\u003c/strong\u003e supported in manuscript revisions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eO.M. (Majyambere Onesphore)\u003c/strong\u003e assisted in literature review, background framing, and manuscript editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eS.A. (Solomon Ali)\u003c/strong\u003e provided advanced methodological guidance, validated evolutionary and mutational interpretations, and critically revised the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eN.R. (Rujeni Nadine)\u003c/strong\u003e supervised the project, provided conceptual and scientific guidance, and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.G.W. (Araya Gebreyesus Wasihun)\u003c/strong\u003e contributed to study design, reviewed analytical strategies, and critically revised the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e8. Data Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll genome sequences analyzed in this study were accessed and downloaded through \u003cstrong\u003eGISAID\u003c/strong\u003e under its data-use agreement. Accession identifiers, originating laboratories, and submitting laboratories are provided in Supplementary Table S1. Reference genomes were retrieved from NCBI. All codes used for genomic analysis will be made available upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e9. Additional Information\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe author received No Funding for this work.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLefkowitz, E., Wang, C. \u0026amp; Upton, C. Poxviruses: past, present and future. \u003cem\u003eVirus Res.\u003c/em\u003e \u003cb\u003e117\u003c/b\u003e (1), 105\u0026ndash;118 (2006).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePeng, Q. et al. Structure of monkeypox virus DNA polymerase holoenzyme. \u003cem\u003eScience\u003c/em\u003e \u003cb\u003e379\u003c/b\u003e (6627), 100\u0026ndash;105 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLikos, A. M. et al. 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Med.\u003c/em\u003e \u003cb\u003e392\u003c/b\u003e (7), 666\u0026ndash;676 (2025).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Monkeypox, Mpox, Genomic evolution, Amino-acid substitutions, Clade Ia, Clade Ib, Clade IIb, Africa","lastPublishedDoi":"10.21203/rs.3.rs-8175950/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8175950/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe genomic diversity of the Monkeypox virus (Mpox), mainly observed in Africa, remains poorly characterized despite the global outbreak from 2022 to 2024. This study aims to determine the genomic evolution and clade-specific variations of Mpox using 70 complete genomes from 2022 to 2024, predominantly from Africa. Phylogenetic analysis revealed 1,038 parsimony-informative sites, with clade Ia dominance at 44.4%. The A-T-rich genomes exhibit a 3.7-fold transition/transversion bias. We also identified 20,219 amino-acid substitutions, with nearly half occurring within clade Ia (48.70%). Clade-specific mutation profiling uncovered distinct signatures: clade Ia possessed unique indel-driven mutations (J1L_ins74DDEVSE, J3R_ins74DDEVSE), clade Ib showed recurrent substitutions (A25R_R273Q, A15L_P39H), and the globally dominant clade IIb harbored deletions absent in clade I (A33R_Y119del). These mutational profiles highlight a clear genomic divergence between African clades (Ia and Ib) and global clade IIb. Our findings demonstrate that African Mpox genomes exhibit strong clade Ia dominance and lineage-specific evolutionary patterns, suggesting distinct selective pressures. This marked difference underscores the critical need for enhanced Africa-focused genomic surveillance and development of region-specific diagnostics, therapeutics, and vaccines.\u003c/p\u003e","manuscriptTitle":"Genomic Evolution and Clade-Specific Mutation Signatures of Monkeypox Virus (Mpox) from 2022 to 2024 Reveal Distinct African Lineage Divergence","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-12 17:04:47","doi":"10.21203/rs.3.rs-8175950/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e11cc4f9-d42b-45d6-a902-acae7808a97f","owner":[],"postedDate":"February 12th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":62771400,"name":"Biological sciences/Computational biology and bioinformatics"},{"id":62771401,"name":"Biological sciences/Evolution"},{"id":62771402,"name":"Biological sciences/Genetics"},{"id":62771403,"name":"Biological sciences/Microbiology"}],"tags":[],"updatedAt":"2026-02-27T11:56:56+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-12 17:04:47","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8175950","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8175950","identity":"rs-8175950","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}