GenEpi-BioTrain Virtual Training 29 - Genomic epidemiology of mpox virus (MPXV)

Topic outline

• Welcome to the virtual training on Genomic epidemiology of mpox virus (MPXV)

  Welcome to the virtual training on Genomic epidemiology of mpox virus (MPXV)

  Course description

  This course introduces the participants to the use of genomic data analysis to gain a deeper understanding of the epidemiology of mpox, an emerging viral pathogen. We will introduce the current state-of-the-art in mpox ecology and epidemiology and explain the application of whole genome sequencing for surveillance and outbreak response. Using a realistic scenario, we will explore which methods and analysis tools can be used, and how to interpret the results. The exercise will allow the participants to get hands-on experience in how to construct and interpret phylogenetic trees and how to perform outbreak investigations in real world settings.

  Target Audience

  This training is primarily designed for bioinformaticians involved in infectious disease surveillance and outbreak response. It is also intended for epidemiologists, microbiologists/virologists, and public health professionals with bioinformatics expertise, who are seeking to incorporate genomic data into outbreak investigations.

  Background & Rationale

  Mpox has emerged as a significant public health concern with different clades currently circulation worldwide. These outbreaks demonstrate the potential for zoonotic viruses to spread rapidly across international borders and generate complex outbreak scenarios. For mpox and other emerging viruses, genomic data can reveal transmission links that are not apparent from epidemiological investigations alone. When combined with clinical, demographic, and epidemiological metadata, WGS provides valuable insights into outbreak dynamics, the geographic spread of infections, and the emergence of viral variants. Consequently, genomic epidemiology is increasingly being integrated into routine public health practice at national and international levels.

  Despite the growing availability of sequencing data, many public health professionals face challenges in interpreting genomic information and translating sequencing results into actionable public health decisions. A practical understanding of phylogenetic analyses, sequence quality assessment, and outbreak investigation approaches is therefore becoming an increasingly important competency for epidemiologists, microbiologists, bioinformaticians, and public health practitioners.

  This training introduces the participants to the principles and practical applications of genomic epidemiology using mpox as a realistic case study. Through a combination of lectures, guided exercises, and discussion sessions, participants will gain hands-on experience in analysing whole genome sequence data, constructing and interpreting phylogenetic trees, identifying APOBEC-3 specific mutations and integrating genomic evidence into outbreak investigations. The course emphasizes real-world applications and provides participants with the knowledge and skills necessary to evaluate genomic data critically and apply it effectively in surveillance and outbreak response activities.

  The training addresses the following questions:

  • How can whole genome sequencing support surveillance and outbreak investigations of mpox and other emerging viruses?
  • What information can genomic data provide about pathogen transmission, introductions, and viral evolution?
  • How are phylogenetic trees constructed, and how should they be interpreted in a public health context?
  • What are the strengths, limitations, and practical considerations of using WGS in routine public health surveillance and response?
  • How can genomic evidence be translated into actionable public health insights during outbreak investigations?

  Format

  This virtual training consists of two half-day sessions held on the mornings of Mondays 5 October and 12 October 2026, from 09:00 to 12:30 (CEST).

  Outline

  Session 1: Introduction on emerging viruses, mpox and outbreak investigations using whole genome sequencing and includes six topics

  1. Introduction into the world of emerging viruses
  2. Whole genome sequencing for outbreak response
  3. The emergence of mpox
  4. Methods for whole genome sequencing and the importance of metadata
  5. Phylogenetic analysis
  6. Introduction into the exercise

  Session 2: Interpretation of the results and guided discussion on the use of WGS for mpox outbreak investigations and includes five topics

  1. Guided discussion
  2. Comparative analysis
  3. Troubleshooting
  4. EU-level surveillance
  5. Reflections

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  Aim and Objectives

  Aim and Objectives

  Upon completion of this workshop, participants will be able to:

  1. Explain the principles of genomic epidemiology and their role in infectious disease surveillance and outbreak response.
  2. Describe the ecology, epidemiology, and transmission dynamics of zoonotic and sustained human-to-human transmissible mpox lineages.
  3. Access and utilize appropriate reference genomic datasets and analytical approaches for investigating mpox outbreaks.
  4. Perform basic quality assessment and interpretation of mpox genome sequence data including the use of masking strategies to exclude potential misguiding parts in the alignment.
  5. Use bioinformatics tools to perform basic genomic and phylogenetic analyses.
  6. Apply phylogenetic methods to investigate the evolution and spread of mpox viruses.
  7. Interpret phylogenetic trees to infer genetic relatedness, transmission patterns, and outbreak clusters.
  8. Identify APOBEC-3 specific mutations and thereby discriminate between zoonotic infections and ongoing human-to-human transmission.
  9. Evaluate the strengths, limitations, and uncertainties of phylogenetic analyses in public health decision-making.
  10. Formulate evidence-based conclusions from genomic epidemiology investigations.

  
  
• Prepare for the training

  Prepare for the training

  Suggested Readings

  • World Health Organisation (WHO). Fact sheets on mpox, 2024. Available at: https://www.who.int/news-room/fact-sheets/detail/mpox

  • European Centre for Disease Control. Mpox overview worldwide, 2026. Available at: https://www.ecdc.europa.eu/en/mpox-worldwide-overview
  • Otieno, J.R., Ruis, C., Onoja, A.B. et al. Global genomic surveillance of monkeypox virus. Nat Med 31, 342–350 (2025). https://doi.org/10.1038/s41591-024-03370-3
  • Ruis, C., Lusamaki, E., O’Toole, A. et al. A systematic nomenclature for mpox viruses causing outbreaks with sustained human-to-human transmission. Nat Med 31, 2854–2858 (2025). https://doi.org/10.1038/s41591-025-03820-6
  • Masirika, L.M., Udahemuka, J.C., Schuele, L. et al. Epidemiological and genomic evolution of the ongoing outbreak of clade Ib mpox virus in the eastern Democratic Republic of the Congo. Nat Med 31, 1459–1463 (2025). https://doi.org/10.1038/s41591-025-03582-1
• Software Requirements

  Software Requirements

  Equipment and Tools

  To enable smooth participation in the practical session of the course make sure the following requirements are met:

  Equipment:

  • Laptop with internet access.
  • macOS, Linux, or Windows with WSL enabled is highly recommended.

  Tool Setup:

  Participants may choose to use web-based tools to some extent. Command line-based tools are essential for parts of the course, but intermediate analysis results are also provided to also enable participation of individuals without command line knowledge.

  • It is recommended to have the tools required for the exercise installed before the start of session 1.

  • Web tools require no installation, and links are provided in the ECDC Learning Portal.

  Description of the exercise:

  After module 1, the participants will be assigned an exercise using real life sequencing data on mpox. You will receive a set of 6 mpox genomes and their corresponding metadata.

  Your task is to investigate this data using the bioinformatic tools recommended below or other freely available bioinformatic tools.

  • Retrieve relevant reference sequences from:
  • NCBI: https://www.ncbi.nlm.nih.gov/
  • GISAID: https://gisaid.org/ (requires a – free – account)
  • Pathoplexus: https://pathoplexus.org/
  • Perform multiple sequence alignment
  • Consider which sequence to include in the alignment
  • Masking
  • Construct the alignment using MAFFT (https://mafft.cbrc.jp/alignment/server/index.html)
  • Construct the alignment using squirrel (https://github.com/aineniamh/squirrel)
  • Build phylogenetic trees
  • squirrel (https://github.com/aineniamh/squirrel)
  • MEGA (https://www.megasoftware.net/)
  • IQTREE-3 (https://github.com/iqtree/iqtree3)
  • Determine the number of APOBEC-3 specific mutations
  • squirrel (https://github.com/aineniamh/squirrel)
  • View the phylogenetic tree
  • iTOL (https://itol.embl.de/)
  • Figtree (https://tree.bio.ed.ac.uk/software/figtree/)
  • PearTree (https://github.com/artic-network/peartree)
  • Interpret the phylogenies
  • What do the tree and the APOBEC-3 specific mutations tell us?
• Session 1: Introduction on emerging viruses, mpox and outbreak investigations using whole genome sequencing

  Session 1: Introduction on emerging viruses, mpox and outbreak investigations using whole genome sequencing

  AGENDA FOR SESSION 1 – Monday, October 5, 2026

  
  

  Hours	Content	Session
  9:00-9:10	ECDC introduction and rules of conduct Dr Priyanka Nannapaneni, ECDC	ECDC introduction and rules of conduct
  9:10-9:35	Emerging viruses in a changing world Prof. Marion Koopmans, EMC	Background on emerging disease outbreaks
  9:35-10:00	Whole genome sequencing for outbreak response Dr. Bas Oude Munnink, EMC	Explain use cases including SARS-CoV-2 on mink farms and the hantavirus outbreak on a cruise ship
  10:00-10:10	Break
  10:10-10:40	The emergence of mpox Dr. Bas Oude Munnink, EMC	Background on the global context from zoonotic spillovers to ongoing human-to-human transmission mainly in sexual networks
  10:40-11:10	Methods for whole genome sequencing and the importance of metadata Dr. David Nieuwenhuijse, EMC	Application of amplicon-based sequencing, bioinformatic tools, importance of masking alignments and interpretation
  11:10-11:20	Break
  11:20-12:00	Phylogenetic analysis Dr. David Nieuwenhuijse, EMC	Overview of phylogenetic analysis and background on how APOBEC-3 mutations can be identified and used
  12:00-12:20	Introduction to the exercise Dr. David Nieuwenhuijse, EMC
  12:20-12:30	Q&A and conclusion Dr. Bas Oude Munnink, EMC, Dr. David Nieuwenhuijse, EMC

  • SeminarOnline session 1 — Virtual training 29

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  • Opening of session 1
  • Emerging viruses in a changing world

     

     
    
  • FileEmerging viruses in a changing world
  • Whole genome sequencing for outbreak response

     
    

  • FileWholeGenomeSequencingForOutbreakResponse
  •  The emergence of mpox

     
    

  • FileECDC TheEmergenceOfMpox
  • Methods for whole genome sequencing and the importance of metadata

  • FileMethods for WGS and importance of metadata
  • Phylogenetic analysis

     
    

  • FilePhylogenetic analysis
  • Introduction to the exercise

     
    

  • Q&A and conclusion
• Homework - between sessions

  Homework - between sessions

  Between January and May 2026, surveillance in South Kivu Province, Democratic Republic of the Congo (DRC), detected circulation of both mpox Clade Ia and Clade Ib viruses. Historically, South Kivu had been heavily affected by Clade Ib, while Clade Ia had not previously been detected in the province.

  A total of 595 laboratory-confirmed mpox cases were identified of which 545 (91.6%) were positive for Clade Ib and 50 (8.4%) were positive for Clade Ia, with an increasing prevelance of Clade Ia during time.

  Cases were reported from 13 health zones. Most infections occurred in children under 15 years of age, particularly those younger than 5 years. Pupils represented the most affected occupational group. 3 Clade Ia positive samples have been sequenced and have been released in GISAID:

  hMpxV/DRC/DPS-KAN0041/2026	EPI_ISL_20453361
  hMpxV/DRC/DPS-KAN0047/2026	EPI_ISL_20453362
  hMpxV/DRC/DPS-0042/2026	EPI_ISL_20453363

   

  The central research question is:

  Are the newly detected mpox Clade Ia cases in South Kivu the result of multiple independent zoonotic spillover events, or do they indicate ongoing human-to-human transmission and establishment of Clade Ia within the community?

  Specific steps:

  1. Download the course material​

  2. Acquire mpox reference data (from GISAID, Pathoplexus and/or NCBI)​

  3. Align sequences using squirrel​

  4. Use provided clade Ia masking and compare the difference​

  5. Manually curate the alignment (if necessary)​

  6. Run IQ-TREE on the curated alignment using squirrel​

  7. Determine if there is one or multiple clusters​

  8. Use squirrel to determine the number of APOBEC3 mutations for the new strains​

  
  

  Suggested command line usage:

  To just align your sequences (without masking):​

  squirrel --clade cladei --no-mask <your-sequences.fasta>​​

  To add the additional masking strategy:​

  squirrel --clade cladei --additional-mask cladeIa_mask.tsv <your-sequences.fasta> ​​

  To run IQ-TREE on the generated multiple sequence alignment:​

  squirrel --clade cladei --additional-mask cladeIa_mask.tsv --run-phylo --outgroups KJ642617,KJ642615 <your-sequences.fasta>​

  To add APOBEC3 style mutation analysis to the workflow:​

  squirrel --clade cladei --additional-mask cladeIa_mask.tsv --run-apobec3-phylo --outgroups KJ642617,KJ642615 --interactive-tree <your-sequences.fasta>

  
  

  Data availability:

  ​The specific masking files and the three newly generated sequences can be found here:

  Masking: https://github.com/dnieuw/mpox-genomics-cladeIa-phylomasking

  Sequences are attached below

  • Filesequences
• Session 2: Interpretation of the results and guided discussion on the use of WGS for mpox outbreak investigations.

  Session 2: Interpretation of the results and guided discussion on the use of WGS for mpox outbreak investigations.

  AGENDA FOR SESSION 2 – Monday, 12 October 2026

  
  

  Hours	Session	Content
  9:00-9:10	ECDC introduction and rules of conduct Dr Priyanka Nannapaneni, ECDC	ECDC introduction and rules of conduct
  9:10-10:00	Guided discussion Dr. Bas Oude Munnink, EMC and Dr. David Nieuwenhuijse, EMC	• Recap of the exercise and the use of live polls and questions to start a discussion • Topics include: phylogenetic placement of the sequences, interpretation of the results, identification of potential clusters, evidence of zoonotic transmission or ongoing human-to-human transmission • Explain the relevance of masking
  10:00-10:10	Break
  10:10-11:20	Comparative Analysis and Troubleshooting Dr. Bas Oude Munnink, EMC, Dr. David Nieuwenhuijse, EMC and Dr. Hayley Cassidy	Comparative Analysis: • Live discussion on the advantages and limitations of NGS for outbreak investigations • Discuss the difficulties in doing this in LMIC settings Troubleshooting: • Interactive discussions on challenges encountered
  11:20-11:30	Break
  11:30-11:50	EU/EEA Surveillance of mpox Dr. Katja Siling and Dr. Priyanka Nannapaneni, ECDC	EU/EEA Surveillance of mpox
  11:50-12:20	Reflections Dr. Bas Oude Munnink, EMC and Dr. David Nieuwenhuijse, EMC	• What impact does this have on public health response? • Open Q&A to raise questions on how to use WGS in future outbreak investigations
  12:20-12:30	Q&A and Conclusion Dr. Bas Oude Munnink, EMC and Dr. David Nieuwenhuijse, EMC

  • SeminarOnline session 2 — Virtual training 29

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  • Introduction to Session 2

  • Part 1: Guided Discussion 

     

  • FileGuided Discussion
  • Part 2: Comparative analysis and troubleshooting
  • FileComparative analysis and Troubleshooting
  • Part 3: Exploring the challenges of performing Mpox WGS in LMIC settings
  • FileExploring the challenges of performing Mpox WGS in LMIC settings
  • Part 4: EU/EEA surveillance of Mpox

  • FileEU-EEA Surveillance mpox
  • Part 5: Reflections - Impact on Public Health response 

     
    
  • FileReflections - Impact on public health response
  • Closing remarks 

     
    

• Contacts

  Contacts

  For any questions regarding the this webinar series, please use the discussion forum.

  If you have questions regarding other training initiatives please contact: courses@ecdc.europa.eu.

  If you have technical issues please contact the ECDC Service Desk at servicedesk@ecdc.europa.eu.