FIG 5.

Growth properties of recombinant viruses generated under different conditions in reverse genetics. (A) Representative plaque morphologies in ST cells of the viruses rescued under both original and 17 different conditions (shown in Table 2), as well as that of the D/OK-clone, are shown. The plaques were immunologically stained with mouse anti-D/OK polyclonal antibody. (B) Growth kinetics of recombinant OK-RG/14 and plaque-purified OK-RG/14p generated under transfection condition 14 were examined. OK-RG/14, OK-RG/14p, OK-RG, and the D/OK-clone were inoculated onto ST cells at an MOI of 0.01. Virus titers were determined at 12-h intervals postinfection by plaque assay and reported as the mean titer with standard deviation (n = 3). (C) Representative plaque morphologies of D/OK-EcoRI-RGs under transfection conditions 12 to 17 (as shown in Table 2) in ST cells. Three independent experiments (no. 1 to 3) were performed for the generation of D/OK-EcoRI-RG. (D) The HEF gene of D/OK-RG, D/OK-EcoRI-RG/14p, or D/OK-clone was amplified by RT-PCR with primers that yielded a full-length 2,049-bp fragment, followed by digestion with the restriction enzyme EcoRI. The presence of EcoRI sites in the PCR products yielded both 1,307- and 738-bp fragments.