Recovery of mutant rHAZV targeting multiple caspase motifs. (A) Schematic showing locations of caspase cleavage motifs on the apex of the arm domains of HAZV and CCHFV, with amino acid positions indicated numerically. (B) Western blot detection of HAZV-N protein for rHAZV-DQVE, ENKE, and AQVA mutants posttransfection (p.Tr) of BSR-T7 cells and 48 h postinfection (p.Inf) of SW13 cells, using supernatants harvested 72, 96, and 120 h posttransfection. Recovery of all mutants was carried out alongside independent complete control recovery of WT rHAZV. Detection of GAPDH abundance was included as a loading control. (C) Representative plaque assays of supernatants taken 120 h posttransfection, displaying plaque morphology for recovered viruses. (D) Titers of infectious WT rHAZV versus DQVE and ENKE at 24-h intervals following infection of SW13 cells at an MOI of 0.001. (E) Detection of HAZV-N and associated cleavage products at 30 and 32 kDa (*) and 20 kDa (**) following a 48-h infection at an MOI of 0.01.