Jens Verlinden

Filoviruses, particularly those of the Orthoebolavirus genus, pose ongoing public health threats due to their increasing frequency and geographic spread. However, research has been impeded by the biosafety level 4 classification of filoviruses. We previously reported the generation of biologically contained Ebola, Marburg and Sudan virus as lower biosafety level-compatible filovirus systems. In the present study, we expanded this repertoire to include biologically contained Taï Forest, Bundibugyo and Reston virus, thereby creating a near-complete toolkit for currently recognized human-relevant orthoebolaviruses. VP30-deficient viruses were generated with matching VP30 expressing cell lines. More specifically, we developed and optimized a dual-reporter system in VeroE6, Huh-7 and A549 cells, combining a virus-encoded enhanced green fluorescent protein reporter as readout for viral replication with a stably cellular-expressed nuclear mCherry marker for cytotoxicity assessment. Next, we screened two repurposing-oriented compound libraries comprising 640 small molecules against Taï Forest, Bundibugyo and Sudan virus, and subsequently cross-validated against Reston and Ebola virus. This approach identified multiple candidates with broad-spectrum activity across orthoebolaviruses, while also revealing virus-specific antivirals, with robust activity observed in both primate- and human-derived cell lines. Together, this work establishes a versatile and experimentally tractable lower biosafety level-compatible platform for the study of human-relevant orthoebolaviruses and the systematic discovery of broad-spectrum antiviral countermeasures against filoviruses.

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.