The assay was described previously [21 ,22 (link)]. In brief, HEK293T cells (producer cells) were seeded in 6-well plate and transfected with 125 ng pCAGGS-NP, 125 ng pCAGGS-VP35, 75 ng pCAGGS-VP30, 1μg pCAGGS-L, 250 ng EBOV minigenome (encoding Renilla Luciferase, VP40, GP and VP24) and 250 ng pCAGGS-T7 by Mirus TransIT-LT1 (Invitrogen, MIR2300). After 12 h, the cell culture medium was discarded and replaced with a fresh medium. After 72 h of transfection, supernatants containing EBOV-trVLPs were collected, filtered through a 0.22 μm filter (Millipore, SLGPR33RB) and stored at −80°C. To support the infection of the EBOV-trVLPs, HEK293T cells (target cells) were seeded in a 12-well plate and transfected with helper plasmids (50 ng pCAGGS-NP, 50 ng pCAGGS-VP35, 30 ng pCAGGSVP30, 400 ng pCAGGS-L, and 100 ng pCAGGS-Tim-1). Simultaneously, 5 ng pCAGGS-firefly luciferase was used as transfection control. After 24 h of transfection, the medium was discarded, and cells were infected with supernatants containing EBOV-trVLPs. After 12 h of infection, the medium was discarded and replaced with fresh medium. After 48 h of infection, target cells were lysed and analysed with a dual-luciferase assay kit (Promega, E2920) according to the manufacturer’s instructions.