Figure 1.

Diagram (not to scale); (A) summarizing the heat-inducible homologous recombination in SW102 containing pBAC-BoHV-4-A-TK-KanaGalK-TK, where the Kana/GalK cassette was replaced with the CMV-PPRV-HgD₁₀₆ expression cassette flanked by bovine herpesvirus 4 (BoHV-4) TK sequences, located in pINT2 shuttle plasmid vector. (B) Representative 2-deoxy-galactose resistant colonies tested by HindIII restriction enzyme analysis, agar gel electrophoresis, and Southern blotting performed with specific probes for the peste des petits ruminants virus hemagglutinin (PPRV-H) ORF ORFs. The 2,650 bp band, corresponding to the un-retargeted pBAC-BoHV-4-A-TK-KanaGalK-TK control, has been replaced by a 3240 bp band in pBAC-BoHV-4-A-CMV-PPRV-H-ΔTK. (C) Representative phase contrast microscopic images of plaque formed by viable reconstituted recombinant BoHV-4-A-PPRV-H-ΔTK after the corresponding bacterial artificial chromosome (BAC) DNA electroporation into bovine embryo kidney (BEK) cells expressing cre recombinase (Magnification, ×10). (D) Replication kinetics of BoHV-4-A-PPRV-H-ΔTK growth on BEK cells and compared with the parental BoHV-4-A isolate. The data presented are the mean ± standard errors of triplicate measurements (P > 0.05 for all time points as measured by Student’s t-test). (E) Western immunoblotting of cells, infected with BoHV-4-A-PPRV-H-ΔTK or the parental BoHV-4-A used as a negative control. The lanes were loaded with different amounts of total protein cell extract (5, 10, and 20 µg).