FIG 3.

Growth kinetics and plaque morphology of BIRFLU. (A) Multicycle growth kinetics. Viral titers (in FFU per milliliter) in culture supernatants from MDCK cells (6-well plates, 10⁶ cells/well, triplicates) infected with PR8 WT and BIRFLU (MOI, 0.001) were determined by immunofocus assay at the indicated times postinfection. Data represent the means ± SD for triplicates. The dashed line denotes the limit of detection (200 FFU/ml). *, P < 0.05, using an unpaired two-tailed Student's t test. (B) NLuc expression. NLuc was evaluated in the same culture supernatants obtained from the experiment whose results are presented in panel A. RLU, relative light units. (C) Venus expression. MDCK cells (24-well-plate format, 2 × 10⁵ cells/well) infected (MOI, 0.001) with PR8 WT or BIRFLU were visualized at the indicated times (in hours) p.i. using a fluorescence microscope. Representative images are shown. Bars, 100 μm; magnification, ×20. (D) Plaque phenotype. Representative pictures of viral plaques in MDCK cells (6-well-plate format, 10⁶ cells/well) infected with PR8 WT and BIRFLU at 3 days p.i. are shown. Fluorescent Venus expression (top), NLuc immunostaining (middle), and crystal violet staining (bottom) are indicated. White arrows show the colocalization of Venus fluorescence (top), NLuc expression (middle), and virus lysis plaques stained with crystal violet (bottom).