Background
Sierra Leone confirmed its first Mpox case on January 10 2025, with the Minister of Health declaring a Public Health Emergency shortly thereafter. Cases have surged in recent weeks, with over 2,800 new infections reported as of May 22, 2025, strongly indicating ongoing human-to-human transmission similar to patterns observed in the early stages of the ongoing outbreaks in Nigeria and the DRC (Parker et al. 2025; O’Toole et al. 2023; Vakaniaki et al. 2024). Sierra Leone accounted for up to half of all confirmed cases in Africa in early May, with weekly suspected and confirmed cases increasing by 71% and 61% relative to previous weeks.
The scarcity of full-length MPXV genomes from the region has left several critical questions unresolved, including whether a new lineage capable of sustained human transmission has emerged, and whether it is connected to ongoing epidemics in West or Central Africa. In this study, we analyze 33 newly generated, high-quality sequences collected between January 10 and May 5 2025 from four districts in Sierra Leone.
Results
Of the 33 MPXV genome sequences we generated, 32 fall within the hMPXV-1 (A) lineage of Clade IIb, which emerged in southern Nigeria in August 2014 and has since sustained the Nigerian epidemic through ongoing human-to-human transmission (Figure 1; Parker et al. 2025). According to the nomenclature proposed by Happi et al. (2022), hMPXV-1 is designated as Lineage A, with direct descendants designated as e.g. A.1, and subsequent subdivisions identified as e.g. A.1.1, similar to the Pango nomenclature for SARS-CoV-2. Within hMPXV-1, our sequences form a well-supported monophyletic group (posterior support -1 in Delphy analysis) descended from Lineage A.2.2 (Figure 1). In accordance with the nomenclature, the newly identified lineage from Sierra Leone is likely to be designated as A.2.2.1 or the alias G.1 (Figure 1).
The only non-Clade IIb sequence we identified was a Clade IIa sequence, collected in mid-January 2025 in the earliest phase of the outbreak. Although highly divergent, it clusters with some of the earliest Clade IIa sequences, including those from 1958, 1961, and 1968, originating from the DRC and historical export events (Figure 2). This sequence has 33 non-APOBEC and 2 APOBEC-like mutations along its terminal branch, and is an additional 6 mutations (4 non-APOBEC, 2 APOBEC) away from its closest sequence. The lack of a APOBEC-like mutational signature in the sequence suggests that this case represents a zoonotic spillover rather than being part of the ongoing human outbreak.
The closest sequence to the novel G.1 lineage is PP_0015JY8.1, a sequence sampled from Chicago in the USA in mid-February 2025. G.1 is separated from its shared ancestor with PP_0015JY8.1 by 8 APOBEC-like and 2 non-APOBEC-like mutations along its stem branch, and an additional 9 APOBEC-like and 1 non-APOBEC-like mutation on PP_0015JY8.1’s terminal branch (Figure 1). PP_0015JY8.1 clusters with three sequences that were also sampled in the US from January 2024 - January 2025 (Figure 1). Based on the long branches separating the US sequences, it is more likely they represent independent viral imports from the Nigerian A.2.2 lineage than an established lineage that has been cryptically diversifying in the USA. The four US sequences were sampled from three different states: Illinois (2), California and Massachusetts, with confirmed travel history to Nigeria for at least two of the sequences. This suggests that G.1 is descendant from the ongoing epidemic in Nigeria, although we cannot infer a direct import to Sierra Leone as we cannot exclude the possibility of unsampled intermediates. The closest Nigerian sequence to G.1 is TRM326, sampled in Rivers state in September of 2022, which has a zero-length branch to its common ancestor with the USA-sampled A.2.2 diversity. There is also evidence of G.1 viral export from Sierra Leone, including two sequences sampled in the USA in March and April 2025, and two samples from Germany in March 2025.
A preliminary Bayesian analysis using Delphy estimated the tMRCA (time to the most recent common ancestor) of lineage G.1 as 5 July 2024 [95% HPD: 7 April 2024 to 8 September 2024] (Figure 3). The tMRCA represents the time at which the sampled G.1 lineage started to diversify in humans in Sierra Leone, suggesting that the lineage circulated undetected in Sierra Leone for at least six months before detection in January 2025 based on current sampled diversity. A period of cryptic circulation is consistent with the observed diversification within lineage G.1 in Sierra Leone, largely driven by APOBEC-mediated evolution across sustained human transmission, as well as the four viral exports. Within G.1, sequences differ by a total of 56 APOBEC-like and 6 non-APOBEC substitutions (Figure 1). The tMRCA estimate may be updated as additional early samples are added to the tree, since there is currently only one sequence from the early phase (January) of the epidemic. Additionally, one sequence from Kenema is separated on a terminal branch of 7 APOBEC-like mutations, which suggests the period of circulation may be underestimated based on expectation from previous APOBEC3 accumulation rates of 6.05 substitutions per year (95% credible interval: 5.8 to 6.3) from Parker et al. (2025), or 6.2 (95% credible interval: 5.2 to 7.2) from O’Toole et al. (2023), though the credible intervals may account for the observed range. The tMRCA between G.1 and PP_0015JY8.1 is estimated at 29 August 2023 [95% HPD: 23 April 2023 to 24 December 2023], indicating when G.1 diverged from its most recent common ancestor with the USA A.2.2 diversity. This ancestor was likely circulating in Nigeria, not the USA as PP_0015JY8.1 represents a likely viral export from Nigeria. This date does therefore not represent the date of the viral import to Sierra Leone. G.1 diverged from the closest A.2.2 Nigeria sequence (TRM326) around 29 August 2022 [tMRCA 95% HPD: 16 June 2022 to 24 September 2022].
There is no evident geographic structuring in the phylogeny, i.e. sequences do not cluster in lineages according to the district of collection but this may change with more sequencing and phylogenetic resolution. Identical sequences were found in geographically distinct districts, including Bo, Kenema, and Bonthe, which is not unexpected given the short timeline of collection and the estimated evolutionary rate.(O’Toole et al. 2023; Parker et al. 2025)
Our findings indicate that there was cryptic circulation and geographic spread prior to detection in these areas, underscoring the urgent need to strengthen surveillance systems and improve diagnostic and monitoring infrastructure. Enhanced case surveillance is essential to uncover the underlying transmission network and identify associated risk factors including possible sexual networks, enabling the implementation of targeted interventions before the outbreak becomes even more widespread regionally and globally.
Figure 1: Clade IIb phylogeny with reconstructed SNPs mapped onto branches. We performed ancestral state reconstruction across our Clade IIb phylogeny to map SNPs to their relevant branches. We annotated APOBEC3 characteristic substitutions i.e. CT or GA in the correct dimer context along branches and calculated their relative proportion across internal branches. APOBEC3 substitutions along the branches are annotated in yellow and red, with the remainder in gray and black. Our new sequences are annotated in red as enlarged tips and as Lineage A.2.2.1. The tree was rooted to the new zoonotic outgroup identified in (Parker et al. 2025).
Figure 2: Clade IIa phylogeny with reconstructed SNPs mapped onto branches. We performed ancestral state reconstruction across our Clade IIa phylogeny to map SNPs to their relevant branches. We annotated APOBEC3 characteristic substitutions i.e. CT or GA in the correct dimer context along branches and calculated their relative proportion across internal branches. APOBEC3 substitutions along the branches are annotated in yellow and red, with the remainder in gray and black. Our new sequence is annotated in red as an enlarged tip. The tree was rooted to the new Clade IIb zoonotic outgroup identified in (Parker et al. 2025).
Figure 3: Time-resolved global phylogeny of Clade IIb. The new A.2.2.1 lineage is annotated in red and in text.
Methods
We generated 33 high-quality sequences collected between 10 January and 5 May 2025 from the districts of Bo (7), Bonthe (5), Kailahun (4), Freetown(2), and Kenema (15). Samples were sequenced using the Twist Viral Surveillance Panel hybrid capture enrichment kit followed by Illumina sequencing. Sequencing reads were assembled using the viral-ngs assemble_denovo_metagenomic pipeline (Park et al. 2025) with automated reference genome selection from a set of 16 MPXV reference genomes (Park and Mendes 2025). Initial analyses indicated a mixture of Clade IIb and Clade IIa. The mean coverage across all sequences ranged from 25x to 8100x and recovered at least 81.5% of the MPXV genome (32 of the 33 genomes exceeded 97% completeness).
We combined our 33 genomes with all high-quality, publicly available Clade IIb MPXV genomes from Pathoplexus.(Dalla Vecchia 2024) As the multi-country outbreak lineage B.1 was not our research focus, we included only a single representative. Additionally, we included the closest zoonotic outgroup to Clade IIb as an outgroup to root the tree (PP852949.1). In total, the dataset comprises 233 sequences. We aligned our dataset to the Clade IIb reference genome (NC_063383) using the ‘squirrel’ package (GitHub - aineniamh/squirrel) developed by O’Toole et al. (O’Toole et al. 2023). The alignment was trimmed, and the 3′ terminal repeat region, along with regions of repetition or low complexity and clustered mutations, were masked using the squirrel package.
We investigated the initial phylogenetic placement of our sequences within the global mpox genome phylogeny constructed from all available GenBank sequences across clades. The full MPXV phylogeny was reconstructed using IQ-TREE v2.0 under the Jukes-Cantor substitution model (Minh et al. 2020). We also generated a separate phylogeny for Clade IIb using the same parameters as the global tree. The tree was rooted with PP852949.1, the closest zoonotic outgroup, which was subsequently removed. Branches with zero length were collapsed. Ancestral state reconstruction was performed on the Clade IIb phylogeny using IQ-TREE2 (Minh et al. 2020) and all nucleotide mutations were mapped to their related internal branches of the phylogeny(O’Toole et al. 2023). Lineage assignments for our sequences were made using the Nextclade tool, following the nomenclature established by Happi et al.(Happi et al. 2022).
We reconstructed a time-resolved tree in the Bayesian phylogenetic framework Delphy(Varilly et al. 2025), under the APOBEC-substitution model adapted from O’Toole et al.(O’Toole et al. 2023). Chains were run for 300,000,000 steps, sampling every 100,000 trees. Convergence was assessed using the built-in trace diagnostics, with an estimated effective size of 267.
Data Availability
The new 33 MPXV genomes described here are available on Pathoplexus as SeqSet PP_SS_170.1 (SeqSets | Pathoplexus), and will be replicated soon to INSDC. Short-read Illumina data will be submitted soon to INSDC.
Citations
- Happi, Christian, Ifedayo Adetifa, Placide Mbala, Richard Njouom, Emmanuel Nakoune, Anise Happi, Nnaemeka Ndodo, et al. 2022. “Urgent Need for a Non-Discriminatory and Non-Stigmatizing Nomenclature for Monkeypox Virus.” PLoS Biology 20 (8): e3001769.
- O’Toole, Áine, Richard A. Neher, Nnaemeka Ndodo, Vitor Borges, Ben Gannon, João Paulo Gomes, Natalie Groves, et al. 2023. “APOBEC3 Deaminase Editing in Mpox Virus as Evidence for Sustained Human Transmission since at Least 2016.” Science 382 (6670): 595–600.
- Park, Daniel, and Inês Mendes. 2025. Broadinstitute/viral-References: 1.0.0. Zenodo. broadinstitute/viral-references: 1.0.0.
- Park, Daniel, Chris Tomkins-Tinch, Simon Ye, Irwin Jungreis, Flavia, Ilya Shlyakhter, Hayden Metsky, et al. 2025. Broadinstitute/viral-Pipelines: v2.4.1.1. Zenodo. broadinstitute/viral-pipelines: v2.4.1.1.
- Parker, Edyth, Ifeanyi F. Omah, Delia Doreen Djuicy, Andrew Magee, Christopher H. Tomkins-Tinch, James Richard Otieno, Patrick Varilly, et al. 2025. “Genomics Reveals Zoonotic and Sustained Human Mpox Spread in West Africa.” Nature, May. Genomics reveals zoonotic and sustained human Mpox spread in West Africa | Nature.
- Vakaniaki, Emmanuel Hasivirwe, Cris Kacita, Eddy Kinganda-Lusamaki, Áine O’Toole, Tony Wawina-Bokalanga, Daniel Mukadi-Bamuleka, Adrienne Amuri-Aziza, et al. 2024. “Sustained - Human Outbreak of a New MPXV Clade I Lineage in Eastern Democratic Republic of the Congo.” Nature Medicine, June. Sustained human outbreak of a new MPXV clade I lineage in eastern Democratic Republic of the Congo | Nature Medicine.
- Varilly, Patrick, Mark Schifferli, Katherine Yang, Tim Burcham, Paul Cronan, Olivia Glennon, Olivia Jacks, et al. 2025. “Delphy: Scalable, near-Real-Time Bayesian Phylogenetics for Outbreaks.” bioRxiv. https://doi.org/10.1101/2025.03.25.645253.
Partners and Collaborators
National Public Health Agency (NPHA), Sierra Leone
Sierra Leone Ministry of Health
Kenema Government Hospital, Sierra Leone
Institute of Genomics and Global Health, Redeemer’s University, Ede, Osun State, Nigeria
The Broad Institute of MIT and Harvard, Cambridge, MA, USA
The Scripps Research Institute, La Jolla, CA, 92037, USA
School of Community Health Sciences, Njala University, Sierra Leone
Funding
This work is made possible by support from Flu Lab and a cohort of generous donors through TED’s Audacious Project, including the ELMA Foundation, MacKenzie Scott, the Skoll Foundation, and Open Philanthropy, The Rockefeller Foundation: [Grant Number #2021 HTH 017]; and The World Bank grants projects ACE-019, ACE-IMPACT and HEPR TF0B8412.
Disclaimer and contact information
Please note that this report is based on ongoing work and should be regarded as preliminary findings. If you wish to use this data, please contact:
Prof. Foday Sahr
Executive Director, National Public Health Agency (NPHA), Sierra Leone
Email. [email protected]
Dr. Donald S. Grant (MD, MPH)
District Medical Officer, Kenema and Principal Investigator,
Lassa fever research program, Kenema Government Hospital,
Sierra Leone.
Email. [email protected]
Doris Harding
National Public Health Laboratory Manager, NPHA,
Sierra Leone.
Email. [email protected]
John Demby Sandi
Co-PI and Head of Genomics and Molecular Laboratory
Kenema Government Hospital Lassa fever (VHF) research Lab,
Kenema, Sierra Leone.
Email. [email protected]
Twitter: @john_demby