David Lu, University of Manitoba

David Lu (1,2), Wenguang Cao (2), Logan Banadyga (1,2). 1 - Department of Medical Microbiology and Infectious Diseases, University of Manitoba, Winnipeg, MB, Canada. 2 - Special Pathogens Program, National Microbiology Laboratory, Pubic Health Agency of Canada, Winnipeg, MB, Canada

Expressing & Multiplying

Sudan virus (SUDV), a filovirus closely related to Ebola virus, causes fatal hemorrhagic disease in humans and continues to pose a public health threat, most recently causing an outbreak in Uganda in 2025. Since SUDV is classified as a Risk Group 4 pathogen, working with the virus requires biosafety level (BSL) 4 containment. To enable safer and more accessible investigation of SUDV, we developed and characterized a plasmid-based SUDV transcription and replication modelling system operable under BSL-2. We replaced all SUDV coding sequences in the genome with a NanoLuc luciferase reporter while retaining the viral leader and trailer regulatory regions required for transcription/replication. This "minigenome" (MG) was cloned under a T7 promoter and co-transfected into mammalian cells alongside plasmids expressing T7 RNA polymerase and the essential SUDV replication complex proteins, NP (nucleoprotein),L (polymerase), VP35 and VP30 (co-factors). Primary transcription of the MG is driven by T7, after which viral proteins support subsequent replication and transcription, producing luciferase as a quantitative readout. Assay conditions, including transfection and harvesting conditions, were optimized to yield luciferase signals spanning four orders of magnitude. Component omission experiments demonstrated strict dependence on the viral replication machinery. Utility of the MG system was demonstrated by dose-dependent inhibition of MG activity following treatment with favipiravir, a viral polymerase inhibitor, or co-expression of VP24, an inhibitor of virus replication. Together, these results establish the SUDV MG system as a robust platform for modeling transcription and replication, thus enabling viral protein characterization and preliminary antiviral drug screening in BSL-2.