FIG 3.

Characterization of rgRSVs carrying deletion and insertion in the flexible hinge region of the L protein. (A) Delayed GFP expression by rgRSV mutants in Vero cells. Confluent Vero cells were infected by each rgRSV at an MOI of 0.1, and GFP expression was monitored at the indicated time by fluorescence microscope. (B) Quantification of GFP-positive cells by flow cytometry. Confluent HEp-2 cells were infected by each rgRSV (MOI of 1.0), and at the indicated time points, cells were trypsinized and GFP-positive cells were quantified by flow cytometry. Data are the average from three independent experiments ± standard deviation. (C) Single-step growth curve of rgRSV mutants. HEp-2 cells in 12-well-plates were infected with each rgRSV at an MOI of 1.0. After adsorption for 1 h, the inocula were removed and the infected cells were washed 3 times with Opti-MEM medium. Fresh DMEM containing 2% FBS was added, and the cells were incubated at 37°C for various times. The supernatant and cells were harvested by three freeze-thaw cycles, followed by centrifugation at 1,500 × g at 4°C for 15 min at the indicated intervals. The viral titer was determined by TCID₅₀ assay in HEp-2 cells. The viral titers shown are the geometric mean titer (GMT) from three independent experiments ± standard deviation. NS, no significant difference (P > 0.05); *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.