Fig. 2: The construction of recombinant ASFV virus—GeorgiaΔBDKE-CmutQ96R/K108D.

a Before deleting B125R from the parental virus GeorgiaΔDP148RΔK145RΔEP153R-CD2v_mutQ96R/K108D (here designated GeorgiaΔBDKE-CmutQ96R/K108D), the existing fluorescent reporter gene markers flanked by loxP (TagRFP-T, mNeonGreen were removed (top panel). PBMs were infected with the parental virus and then transfected with a plasmid expressing Cre recombinase. After two days, cells not expressing any fluorescent markers were bulk sorted via FACS into complete RPMI and these cells were subjected to two rounds of limiting dilutions. The final selected virus, GeorgiaΔDP148RΔK145RΔEP153R-CD2v_mutQ96R/K108D-NoFP, which was non-haemadsorbing and non-fluorescent, but still containing the GUS marker was grown and titrated before being used as template to delete the B125R gene (bottom panel). b Using the newly produced, GeorgiaΔDP148RΔK145RΔEP153R-CD2v_mutQ96R/K108D-NoFP as parental virus, the B125R gene was deleted from nucleotides 106,585–106,886 (ASFV Georgia 2007/1: FR682468.2). The transfer plasmid, pΔB125R-tdT, contains left and right flanking regions of the B125R gene, either side of the fluorescent marker tdTomato, under the control of ASFV VP30 promoter. Purified PBMs were infected with GeorgiaΔDP148RΔK145RΔEP153R-CD2v_mutQ96R/K108D-NoFP, then transfected with pΔB125R-tdT. After 36–48 post-infection-transfection, recombinants expressing tdTomato⁺ were isolated by single-cell sorting into PBMs using FACS. Following subsequent rounds of single-cell isolation and limiting dilutions, GeorgiaΔBDKE-CmutQ96R/K108D was obtained.