FIG 5.

Effect of the selected mutations in E1 on the replication of BVDV strain CP7. Top row (24h p.EP.): MDBK-B2 cells were first transfected by electroporation with the RNAs transcribed from the given plasmids. The corresponding plasmids are mentioned at the top. The plasmid BVDV-CP7 E1-2M contains the same mutations as pYM-39, and the 4M and 6M versions are equivalent to pYM-52 and pYM-53, respectively. One day after EP, the cells were fixed with 4% PFA and permeabilized with 0.05% Triton X-100. The viral protein NS3 was detected with the primary antibody Code4 and α-mouse FITC. Middle row: supernatant reinfection after electroporation. At 2 days after EP, the supernatant from the electroporated cells was added to fresh MDBK cells for reinfection. At 2 days after the reinfection, the SN reinfected cells were checked again for the presence of NS3 by indirect immunofluorescence. Bottom row: freeze/thaw extract reinfection after electroporation. At 2 days after EP, the supernatant of the electroporated cells was removed, the electroporated cells were lysed via three times complete freeze/thaw cycling, and cell extract was added to the fresh MDBK cells for a reinfection test. At 2 days after the reinfection, the cells were checked again for the presence of NS3 by indirect immunofluorescence.