Fig. 3.

BoHV-4-syEBOVgD106ΔTK constructs and characterization. a Diagram (not to scale) illustrating the re-targeting event (i.e., replacement of the Kana/GalK cassette with the CMV-syEBOVgD106 cassette) obtained by heat-inducible homologous recombination in SW102 E. coli cells containing pBAC-BoHV-4-A-TK-KanaGalK-TK. b Representative, 2-deoxy-galactose resistant colonies, tested by HindIII restriction enzyme analysis and southern blotting performed with a specific probe for syEBOVgD106 ORF. The 2650 bp band (indicated by a green arrow) corresponding to the non-retargeted pBAC-BoHV-4-A-TK-KanaGalK-TK control (lane 1) is replaced by 3460 bp band (indicated by a red arrow) in pBAC-BoHV-4-syEBOVgD106ΔTK (lanes 2 and 3). Phase contrast and fluorescent microscopy images of the plaques formed by viable, reconstituted recombinant BoHV-4-syEBOVgD106ΔTK (c) after electroporation of the corresponding BAC DNA clones into BEK or BEKcre cells (magnification, ×10). d Replication rate of BoHV-4-syEBOVgD106ΔTK grown in BEK cells and compared with that of the parental BoHV-4-A isolate. The data are the mean ± standard error of triplicate measurements (P > 0.05 for all time-points; Student’s t test). e Immunoblotting analyses conducted on extracts from cells infected with BoHV-4-syEBOVgD106ΔTK (numbers indicate the micrograms of total protein loaded). BoHV-4-A infected cells served as negative controls