Comparative Time–Space Evolution of Bundibugyo Ebolavirus Glycoprotein Across the 2007, 2012, and 2026 Outbreaks

Bundibugyo ebolavirus (BDBV) is one of the recognized human-pathogenic ebolaviruses and has caused three documented outbreaks in Uganda (2007), the Democratic Republic of the Congo (2012), and the Democratic Republic of the Congo/Uganda (2026). Although complete glycoprotein (GP) sequences are available from these outbreaks, comprehensive comparative analyses integrating their temporal, geographical, and functional evolutionary characteristics remain limited. In this study, 54 complete GP amino acid sequences (676 amino acids) representing the three outbreak periods were comparatively analyzed using a unified amino acid coordinate framework. Complete sequence alignment enabled direct comparison of homologous residues across all isolates without coordinate renumbering. Core amino acid substitution sites were identified, classified according to their evolutionary behavior, and mapped onto established GP functional domains. Comparative analysis identified 21 core amino acid substitution sites distributed throughout the GP sequence. These substitutions were classified into five evolutionary patterns: fixation (n = 10), reversion (n = 4), emergence (n = 2), within-outbreak polymorphism (n = 4), and a rare variant (n = 1). Most substitutions were concentrated within the mucin-like domain (MLD) and the GP1 C-terminal region, whereas the furin cleavage site, internal fusion loop, immunosuppressive motif, and transmembrane domain remained completely conserved throughout all three outbreak periods. Temporal comparison demonstrated that GP evolution occurred primarily through amino acid substitutions rather than insertions or deletions, indicating substantial structural conservation despite continued viral evolution. These findings provide an integrated time–space overview of BDBV GP evolution across the three documented outbreaks and establish a standardized comparative framework for monitoring future genetic variation. The analytical approach presented here may facilitate genomic surveillance and support future comparative investigations of ebolavirus evolution, antigenic diversity, and potential therapeutic targets.