Recombinant filovirus rescue transfections

Recombinant LLOV rescue transfections were performed similar to rescue transfections for other filoviruses Briefly, 1:1 mixtures of Huh7:Vero E6 cells (1x10⁵ cells plated per well of a 12-well plate) were transfected with LLOV support plasmids (500 ng pCAGGS-NP_LLOV, 125 ng pCAGGS-VP35_LLOV, 50 ng pCAGGS-VP30_LLOV, and 200 ng pCAGGS-L_LLOV), pCAGGS-T7 (50 ng; codon-optimized), and a full-length plasmid containing the chimeric LLOV genome (1 μg) using TransIT-LT1 per manufacturer’s recommendations (Mirus Bio LLC). Media was changed approximately 18 hours post-transfection and cells were monitored for cytopathic effect (CPE) and fluorescence for ZsGreen-containing clones. Supernatants of cells showing CPE and/or fluorescence were transferred to T75 flasks of Vero E6 cells approximately 7–11 days post-transfection. rEBOV-ZsGreen and rRESTV-ZsGreen clones were rescued similarly, using the corresponding full-length plasmids (1 μg each), pCAGGS-T7 (50 ng; codon-optimized), plus the EBOV (750 ng pCAGGS-NP_EBOV, 125 ng pCAGGS-VP35_EBOV, 50 ng pCAGGS-VP30_EBOV, and 100 ng pCAGGS-L_EBOV) or RESTV (350 ng pCAGGS-NP_RESTV, 125 ng pCAGGS-VP35_RESTV, 50 ng pCAGGS-VP30_RESTV, and 350 ng pCAGGS-L_RESTV) support plasmids, respectively. Rescue of recombinant filoviruses, including LLOV, was performed in BSL-4 facility of the NEIDL following BSL-4 biosafety procedures.