A panel of biologically contained orthoebolaviruses for the screening of broad-spectrum antivirals

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Abstract
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.

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