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

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.