FIG 10.

Generation and characterization of mCherry-expressing BIRFLU. (A) Multicycle growth kinetics. Viral titers (in FFU per milliliter) from culture supernatants of MDCK cells (6-well plates, 10⁶ cells/well, triplicates) infected with PR8 WT or BIRFLU expressing mCherry (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 and C) Reporter gene expression. NLuc activity was quantitated from the same culture supernatants (B), and mCherry expression was imaged using a fluorescence microscope (C). Representative images are shown. Bars, 100 μm; magnification, ×20. (D) Plaque phenotype. MDCK cells (6-well-plate format, 10⁶ cells/well) were infected with PR8 WT-mCherry and BIRFLU-mCherry, and viral plaques were evaluated at 3 days p.i. for mCherry fluorescence (top), NLuc immunostaining (middle), and crystal violet staining (bottom). White arrows indicate the colocalization of mCherry fluorescence (top), NLuc immunostaining (middle), and viral lysis plaques (bottom). (E) Analysis of protein expression. MDCK cells (6-well plates, 10⁶ cells/well) were infected with PR8 WT-mCherry or BIRFLU-mCherry at an MOI of 0.1 or mock infected (lane M). Protein expression was examined by Western blotting using specific antibodies for NS1, mCherry, NLuc, and NP. Actin was used as a loading control. Numbers on the left indicate the size of molecular markers for proteins (in kilodaltons).