The development of biologically contained Sudan virus as an alternative and safe tool for fundamental filovirus research.

Sudan virus (SUDV; species Orthoebolavirus sudanense) remains a major public health threat, yet research is limited by restricted access to biosafety level 4 (BSL-4) facilities. To address this, we developed a biologically contained SUDV lacking the essential VP30 gene, restricting replication to VP30-expressing cells. We demonstrate efficient virus rescue, strict functional and genetic containment, and replication kinetics comparable to wild-type SUDV in permissive cells. Using heterologous VP30-expressing cell lines, we observe strong cross-functionality among orthoebolaviruses, whereas Marburg virus VP30 shows minimal activity, highlighting genus-specific constraints. The system supports high-throughput antiviral screening and confirms robust activity of remdesivir. In addition, resistance profiling identified substitutions at residue F549 of viral polymerase L as key determinants for remdesivir resistance, with additional mutations at M675. Together, this biologically contained SUDV system enables safe study of viral replication, antiviral discovery, and resistance evolution under lower biosafety conditions.