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. 2019 Jul 17;93(15):e00616-19. doi: 10.1128/JVI.00616-19

FIG 2.

FIG 2

Recovery of rHAZV. (A) Schematic showing workflow for recovery of rHAZV. Plasmids were transfected into BSR-T7 cells for the indicated period prior to harvest of supernatant and subsequent 48-h infection of SW13 monolayers. (B) Detection of HAZV-N protein posttransfection (p.Tr) of BSR-T7 cells and 48 h postinfection (p.Inf). Supernatant samples collected from transfected BSR-T7 cells at 72, 96, and 120 h posttransfection (hpt) were used to infect monolayers of SW13 cells. Following a 48-h infection, lysates were collected (p.Inf, lanes 2 to 4) and analyzed for N expression by Western blotting, alongside lysates collected from the initial transfected BSR-T7 cells (p.Tr, lane 1). Recovery of WT rHAZV (Complete) was carried out alongside a control recovery (Control) omitting transfection of the essential pMK-RQ-L plasmid (p.Tr and p.Inf, lanes 5 to 8). Detection of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) abundance was included as a loading control. (C) Titers of infectious rHAZV released into the supernatant at 72, 96, and 120 h posttransfection, as assessed via plaque assay. Error bars represent averages of two repeats.