Background
The Democratic Republic of the Congo (DRC) is currently facing concurrent outbreaks including mpox, cholera and malaria (1-4). The Ministry of Public Health, DRC has declared the 16th Ebola Virus Disease (EVD) outbreak on 04 September 2025 after PCR confirmation of Ebola virus (EBOV), formely Zaïre ebolavirus, in patient’s specimens from Bulape Heath Zone, in Kasai Province.
The re-emergence of EBOV in remote areas with very limited access is not unprecedent since 15 Ebola outbreaks have previously occurred in the country (5). In late August 2025, the local health authorities in Bulape Heath Zone, Kasai Province, DRC raised an alert about cases presenting with haemorrhagic fever symptoms and associated fatalities among both infected individuals and healthcare workers. Suspecting EVD, a buccal swab (from a fatal case) and five blood specimens were collected from six suspected cases then shipped to the national reference laboratory, Institut National de Recherche Biomédicale (INRB) in Kinshasa, for diagnosis confirmation and whole genome sequencing (WGS).
Methods
On 03 September 2025, six samples were tested at INRB using the point-of-care GeneXpert Ebola assay, the automated BioFire FilmArray System (Global Fever Panel), and real-time PCR with Altona RealStar Filovirus RT-PCR kit. EBOV PCR positive samples were subsequently sequenced on a nanopore sequencing device (Oxford Nanopore Technology).
Briefly, viral RNA was extracted from 140 µL of inactivated specimens using viral RNA extraction kit (QIAGEN) according to the manufacturer’s instructions. Reverse transcription was performed with LunaScript RT SuperMix kit (New England Biolabs). Multiplex PCR was performed on cDNA using Q5 Hot Start High-Fidelity 2X master mix (New England Biolabs), and EBOV-specific primer pools, designed for EBOV Mangina variants, were used to amplify the EBOV genome. Library was prepared in triplicate using the rapid sequencing DNA V14 barcoding kit (SQK-RBK114.96; Oxford Nanopore Technologies (ONT), Oxford, UK), following the manufacturer’s instructions. Sequencing library was loaded on a R10.4.1 flow cell and run on a GridION sequencer. Reads were base called with the High accuracy model from Guppy. Subsequently, iVar tool ( GitHub – andersen-lab/ivar: iVar is a computational package that contains functions broadly useful for viral amplicon-based sequencing. ) was used for trimming sequencing adapters, primers, and low-quality bases, and for generating the consensus genome.
Results and Discussion
We were able to generate an accurate consensus genome in less than one hour starting from the library preparation. The newly EBOV genome is 99.97% complete and consists of 18,819 nucleotides. Multiple sequence alignment of 71 complete EBOV genomes, including the newly generated genome, was performed using MAFFT (6). A phylogenetic tree (Figure 1) was constructed using IQ-TREE v2.1.4 (7). The new EBOV genome has a nucleotide similarity of 99.52% to the most closely related genome, Ebola virus/H.sapiens-tc/COD/1976/Yambuku-Mayinga (GenBank accession number NC_002549.1), suggesting that this case represents a new zoonotic spillover event and is not directly linked to the 2007 Luebo or 2008/2009 Mweka EVD outbreaks.
Authors (key contributors to data collection/molecular testing/data interpretation/whole genome sequencing/bioinformatics analysis/phylogenetic analysis, and manuscript writing):
Adrienne Amuri-Aziza (INRB, Kinshasa, DRC)
Gradi Luakanda-Ndelemo (INRB, Kinshasa, DRC)
Jean-Claude Makangara-Cigolo (INRB, University of Kinshasa, Kinshasa, DRC; Institute of Social and Preventive Medicine, University of Bern, Bern, Switzerland)
Prince Akil-Bandali (INRB, Kinshasa, DRC)
Servet Kimbonza (INRB, Kinshasa, DRC)
Fiston Cikaya-Kankolongo (INRB, Kinshasa, DRC)
Princesse Paku-Tshambu (INRB, Kinshasa, DRC)
Elzedek Mabika-Bope (INRB, Kinshasa, DRC)
Emmanuel Lokilo (INRB, Kinshasa, DRC)
André Citenga (INRB, Kinshasa, DRC)
Raphael Lumembe (INRB, University of Kinshasa, Kinshasa, DRC)
Gabriel Kabamba (INRB, University of Kinshasa, Kinshasa, DRC)
Christian Ngandu (Institut National de Santé Publique (INSP), Kinshasa, DRC)
Louis Tshulo (Division Provinciale de la Santé, Kasaï, DRC)
Mathias Mossoko (Institut National de Santé Publique (INSP), Kinshasa, DRC)
Dieudonné Mwamba (Institut National de Santé Publique (INSP), Kinshasa, DRC)
Elisabeth Pukuta (INRB, Kinshasa, DRC)
Eddy Kinganda-Lusamaki (INRB, University of Kinshasa, Kinshasa, DRC; TransVIHMI, Université de Montpellier, INSERM, IRD, Montpellier, France)
Daniel Mukadi-Bamuleka (INRB, University of Kinshasa, Laboratoires P2/P3 Rodolphe -Merieux INRB-Goma, DRC)
Patrick Mukadi (INRB, University of Kinshasa)
Dieudonné Mumba Ngoyi (INRB, University of Kinshasa, Kinshasa, DRC)
Steve Ahuka-Mundeke (INRB, University of Kinshasa, Kinshasa, DRC)
Jean-Jacques Muyembe-Tamfum (INRB, University of Kinshasa, Kinshasa, DRC)
Tony Wawina-Bokalanga (INRB, University of Kinshasa, Kinshasa, DRC; Department of Clinical Sciences, Institute of Tropical Medicine, Antwerp, Belgium)
Placide Mbala-Kingebeni (INRB, University of Kinshasa, Kinshasa, DRC; South African National Bioinformatics Institute, University of the Western Cape, South Africa)
Acknowledgements section
We are grateful to the ARTIC Network ( ARTIC network | ARTIC network – pathogen genomics from sample to response ) and the University of Nebraska for the provision of the primers. The biofire panels and primers were provided by Culmen International under their cooperative Agreement funded by the US CDC. We also acknowledge the Africa Pathogen Genomic Initiative, Agence Française de Dévelopement through the AFROSCREEN project (grant agreement CZZ3209, coordinated by ANRS-MIE Maladies infectieuses émergentes in partnership with Institut de Recherche pour le Développement (IRD) and Pasteur Institute) ; the Global Funds; the Belgian Directorate-General for Development Cooperation and Humanitarian Aid; the Research Foundation Flanders (“Fonds voor Wetenschappelijk Onderzoek–Vlaanderen”); Institute of Tropical Medicine Structurele Onderzoeksfinanciering (Flemish Government; Science, Technology, and Innovation), and the World Health Organization for their support to genomic surveillance efforts in DRC through equipment acquisition, reagents procurement and logistic support.
Statement on continuing work and analyses prior to publication
This genome is being shared pre-publication. Please note that this data is based on work in progress and should be considered preliminary. Our analyses are ongoing and a publication communicating our findings is in preparation. If you intend to use our data prior to our publication, please contact Prof. Placide Mbala-Kingebeni.
Collaborating institutions and agencies
- Africa Centres for Disease Control and Prevention, Addis Ababa, Ethiopia
- Culmen International
- Institute of Ecology and Evolution, University of Edinburgh, Edinburgh EH9 3FL, UK
- Institute of Tropical Medicine, Antwerp, Belgium
- TransVIHMI, Université de Montpellier, INSERM, IRD, Montpellier, France
- University of Birmingham, Birmingham, UK
- University of California Los-Angeles (UCLA), Los-Angeles, USA
- University of Manitoba, Winnipeg, Manitoba, Canada
- University of Bern, Switzerland
- US Department of Agriculture, Manhattan, KS, USA
- Viral Special Pathogens, US Centers for Disease Control and Prevention, Atlanta, GA, USA
- World Health Organization Country Office, Kinshasa, Democratic Republic of the Congo
- World Health Organization, Geneva, Switzerland
References
- Vakaniaki EH, Kacita C, Kinganda-Lusamaki E, O’Toole A, Wawina-Bokalanga T, Mukadi-Bamuleka D, et al. Sustained human outbreak of a new MPXV clade I lineage in eastern Democratic Republic of the Congo. Nat Med. 2024 Jun 13.
- Wawina-Bokalanga T, Merritt S, Kinganda-Lusamaki E, Jansen D, Halbrook M, O’Toole A, et al. Epidemiology and phylogenomic characterisation of two distinct mpox outbreaks in Kinshasa, DR Congo, involving a new subclade Ia lineage: a retrospective, observational study. Lancet. 2025 Jul 5;406(10498):63-75.
- World Health Organization (WHO). WHO response to challenging cholera outbreak in the Democratic Republic of the Congo.2025; Available from: WHO response to challenging cholera outbreak in the DRC
- World Health Organization. Acute respiratory infections complicated by malaria (previously undiagnosed disease) – Democratic Republic of the Congo 2024. Available from: Acute respiratory infections complicated by malaria (previously undiagnosed disease) – DRC.
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- Katoh K, Rozewicki J, Yamada KD. MAFFT online service: multiple sequence alignment, interactive sequence choice and visualization. Briefings in Bioinformatics July 2019
- Minh BQ, Schmidt HA, Chernomor O, Schrempf D, Woodhams MD, von Haeseler A, et al. IQ-TREE 2: New Models and Efficient Methods for Phylogenetic Inference in the Genomic Era. Mol Biol Evol. 2020 May 1;37(5):1530-4.
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