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Peer-reviewedCC BY 4.0International Journal of Molecular Sciences

Development of a Novel Amplicon-Based Whole-Genome Sequencing Approach for Improved Surveillance of Orthoebolavirus bundibugyoense 2026

  • Martina Rueca1
  • Eliana Specchiarello1,2
  • Flavia Smoquina1
  • Francesca Colavita1,2
  • Cesare E. M. Gruber1,2,3
  • Giovanni Chillemi3,4
  • Fabrizio Maggi1
  • Fabrizio Carletti5
5 affiliations
  1. 1Laboratory of Virology and Biosafety Laboratories, National Institute for Infectious Diseases Lazzaro Spallanzani-IRCCS, 00149 Rome, Italy
  2. 2European Union Public Health Reference Laboratories for Emerging, Rodent-Borne and Zoonotic Viral Pathogens (EURL-PH-ERZV)
  3. 3Bioinformatics Research Unit in Infectious Diseases, National Institute for Infectious Diseases Lazzaro Spallanzani-IRCCS, 00149 Rome, Italy
  4. 4Department of Experimental Medicine, University of Rome “Tor Vergata”, 00133 Rome, Italy
  5. 5Head, Sequencing Unit, National Institute for Infectious Diseases Lazzaro Spallanzani-IRCCS, 00149 Rome, Italy
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Abstract

Bundibugyo virus (BDBV) remains one of the least genomically characterized filoviruses, partly due to the absence of primer schemes specifically optimized for its full genome amplification. Existing amplicon-based protocols developed for the Zaire Ebola virus or other orthoebolaviruses provide incomplete coverage when applied to BDBV, limiting genomic surveillance during outbreaks. In this study, we present a newly designed multiplex PCR primer scheme tailored to the genomic features of BDBV and optimized for sequencing on the Ion Torrent platform. The panel comprises 37 overlapping primer pairs spanning the entire ~19 kb BDBV genome, designed to minimize amplicon dropouts and ensure uniform coverage across regions with variable GC content and sequence divergence. Validation using BDBV RNA, derived from a diluted aliquot of a clinical specimen collected from a patient who tested positive for BDBV during the 2026 outbreak, demonstrated >99% genome recovery, high coverage uniformity, and robust performance even in low-titer samples. This represents, to the best of our knowledge, the first experimentally validated primer scheme specifically developed for BDBV. The protocol provides a reliable and accessible tool for genomic surveillance, outbreak investigation, and evolutionary studies, supporting improved diagnostic and public health responses to BDBV disease.

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Cite

BibTeXRIS
BibTeX
@article{rueca2026development,
  title = {{Development of a Novel Amplicon-Based Whole-Genome Sequencing Approach for Improved Surveillance of Orthoebolavirus bundibugyoense 2026}},
  author = {Martina Rueca and Eliana Specchiarello and Flavia Smoquina and Francesca Colavita and Cesare E. M. Gruber and Giovanni Chillemi and Fabrizio Maggi and Fabrizio Carletti},
  journal = {International Journal of Molecular Sciences},
  year = {2026},
  month = oct,
  doi = {10.3390/ijms27219501},
  url = {https://www.mdpi.com/1422-0067/27/21/9501},
  note = {CC BY 4.0},
}
RIS
TY  - JOUR
TI  - Development of a Novel Amplicon-Based Whole-Genome Sequencing Approach for Improved Surveillance of Orthoebolavirus bundibugyoense 2026
AU  - Martina Rueca
AU  - Eliana Specchiarello
AU  - Flavia Smoquina
AU  - Francesca Colavita
AU  - Cesare E. M. Gruber
AU  - Giovanni Chillemi
AU  - Fabrizio Maggi
AU  - Fabrizio Carletti
PY  - 2026
DA  - 2026/10/24
JO  - International Journal of Molecular Sciences
DO  - 10.3390/ijms27219501
UR  - https://www.mdpi.com/1422-0067/27/21/9501
AB  - Bundibugyo virus (BDBV) remains one of the least genomically characterized filoviruses, partly due to the absence of primer schemes specifically optimized for its full genome amplification. Existing amplicon-based protocols developed for the Zaire Ebola virus or other orthoebolaviruses provide incomplete coverage when applied to BDBV, limiting genomic surveillance during outbreaks. In this study, we present a newly designed multiplex PCR primer scheme tailored to the genomic features of BDBV and optimized for sequencing on the Ion Torrent platform. The panel comprises 37 overlapping primer pairs spanning the entire ~19 kb BDBV genome, designed to minimize amplicon dropouts and ensure uniform coverage across regions with variable GC content and sequence divergence. Validation using BDBV RNA, derived from a diluted aliquot of a clinical specimen collected from a patient who tested positive for BDBV during the 2026 outbreak, demonstrated >99% genome recovery, high coverage uniformity, and robust performance even in low-titer samples. This represents, to the best of our knowledge, the first experimentally validated primer scheme specifically developed for BDBV. The protocol provides a reliable and accessible tool for genomic surveillance, outbreak investigation, and evolutionary studies, supporting improved diagnostic and public health responses to BDBV disease.
SN  - 1422-0067
ER  -