Preliminary analysis of 98 genomic sequences of MPXV clade Ib cases from Burundi, July to September 2024
Introduction
A large mpox clade Ib virus outbreak started in the province of South Kivu in the Democratic Republic of the Congo (DRC) and is rapidly geographically expanding to other areas within the DRC as well as to other countries surrounding the DRC. Especially Burundi is severely affected with close to 1531 confirmed cases of mpox Clade Ib virus infections since the first detection at the end of July 2024. In addition, travel related cases linked to east African countries have been identified in Germany, Kenya, India, Sweden and Thailand.
Previously, we have described the MPXV Clade Ib introduction into Burundi and the phylogenetic analysis of the first two whole genome sequences (Nzoyikorera et al., 2024). Here, we describe the sequencing and phylogenetic analysis of an additional 96 near-complete genome sequences generated in the first three months to better understand the ongoing mpox Clade Ib virus outbreak in Burundi.
Methods
Swab samples from vesicular lesions were confirmed for MPXV using a generic RT-PCR and Clade Ib specific RT-PCR assay (Li et al., 2010; Schuele et al., 2024). Samples from different health districts and a timeframe between 22-07-2024 and 13-10-2024 and below Ct 30 were selected for amplicon sequencing (GitHub - pha4ge/primer-schemes: Tiling PCR primer scheme definitions; mpxv/rigshospitalet/2500/v1.0.0). Sequencing libraries were prepared using the Native Barcoding kit 24 v14 (Oxford Nanopore Technologies) and run on R10.4.1 flowcells (ONT) with the high accuracy model on Dorado Basecall Server v7.4.13 (ONT). Generation of consensus sequences and phylogenetic analysis was performed as previously described (Murhula et al., 2024 (a)). Briefly, sequencing reads were quality controlled with fastp (GitHub - OpenGene/fastp: An ultra-fast all-in-one FASTQ preprocessor (QC/adapters/trimming/filtering/splitting/merging...)) and primers were trimmed using cutadapt (GitHub - marcelm/cutadapt: Cutadapt removes adapter sequences from sequencing reads) and Ampliclip (GitHub - dnieuw/Ampliclip: Tool to softclip reads in bam files based on amplicon primers). Reads were mapped against NC003310.1 using minimap2, and consensus was generated using Virconsens (GitHub - dnieuw/Virconsens) with a minimal coverage of 30x. Generated consensus sequences and available Clade Ib sequences available on GenBank and GISAID were aligned using Squirrel v1.0.11 making use of the WHO recommended CladeI masking option (GitHub - WHO-Collaboratory/collaboratory-mpox-genomics-phylomasking) (GitHub - aineniamh/squirrel). Phylogenetic analysis was performed with IQ-TREE v2 with model K3Pu+F+I and visualised with a custom R script.
Results
We have generated 96 additional genome sequences from the first three months of the outbreak from different geographical areas in Burundi. RT-PCR and WGS confirmed all MPXV positive samples as Clade Ib. These sequences mainly originated from patients in the most densely populated province of Bujumbura Nord (n=69), Bujumbura Centre (n=15), Bujumbura Sud (n=3) but also from the provinces Gitega (n=1), Kabezi (n=2), Kayanza (n=3), Kiremba (n=1), Mpanda (n=1) and Muramvya (n=2). From one sample the location was unknown.
Phylogenetic analysis showed that there were at least two different introductions of the virus into Burundi with one introduction leading to further spread within the country (top panel Figure 1). Sample number 362 showed alternative clustering suggesting a different introduction into the country or perhaps this case reflects a recent travel history to the DRC.
Figure 1: Phylogenetic analysis of the newly generated sequences and mpox clade Ib sequences available in GISAID. Tip shapes are coloured by country. Linking mutations and unique munitions are plotted next to the phylogenetic tree and APOBEC-3 signature mutations are highlighted with a black box.
Conclusion
Here we show sustained circulation of the clade Ib mpox virus in Burundi, most likely after initial introduction from South Kivu in DRC. The virus acquired several additional APOBEC-3 mediated mutations, in line with ongoing human to human transmission. More genomic sequencing is needed from neighbouring countries and more recent time points but it seems that the outbreak in Burundi is caused by in-country transmission after the initial introduction into Burundi.
Figure 1: Phylogenetic analysis of the newly generated sequences and mpox clade Ib sequences available in GISAID. Tip shapes are coloured by country. Linking mutations and unique munitions are plotted next to the phylogenetic tree and APOBEC-3 signature mutations are highlighted with a black box.
Contacts:
For more information on the ongoing studies, sequences and data analysis, please contact Nehemie Nzoyikorera ([email protected]) or Bas Oude Munnink ([email protected]).
The following individuals are involved in this work:
Burundi: Nehemie Nzoyikorera, Cassien Nduwimana, Théogène Ihorimbere, Denis Niyomwungere, Armstrong Ndihokubwayo, Alexis Niyomwungere, Dionis Nizigiyimana, Marie Noelle Uwineza, Joseph Nyandwi
Morocco : Idrissa Diawara
Denmark: Saria Otani, Frank M. Aarestrup
Netherlands: David F. Nieuwenhuijse, Leonard Schuele, Hayley Cassidy, Marjan Boter, Bas B. Oude Munnink, Marion Koopmans
Funding:
Sequencing and data analysis were made possible by funding provided by the GREAT-LIFE consortium (grant number 101103059).
Ethical statement:
Ethical approval was given by the National Ethics Committee in Burundi (CNE/10/2024).
References:
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