FIG 4.

Analysis of wild-type and codon-deoptimized viruses in vitro in MDCK cells. (A) Multicycle growth kinetics. Virus titers in tissue culture supernatants of MDCK cells infected (MOI, 0.001) with WT NS or codon-deoptimized (NS1cd, NEPcd, and NScd) viruses were analyzed at the indicated times postinfection (12, 24, 48, and 72 h) by immunofocus assay (FFU/ml) using the anti-NP MAb HT103. Data represent the means ± SDs of the results determined for triplicate wells. (B) Plaque phenotype. MDCK cells were infected with WT NS or codon-deoptimized (NS1cd, NEPcd, and NScd) viruses, and at 2 days postinfection, monolayers were stained with crystal violet. (C) Analysis of protein expression levels in infected cells by Western blotting. MDCK cells were mock infected (mock) or infected with WT NS or codon-deoptimized (NS1cd, NEPcd, and NScd) viruses at an MOI of 0.001, and NS1, NEP, and NP expression levels were analyzed (24 h postinfection) by Western blotting with specific antibodies. Actin was used as a loading control. The protein molecular sizes (in kDa) are indicated to the left. Western blots were quantified by densitometry using the software ImageJ (v1.46). The bands were normalized to the level of actin expression for protein quantitation and then to the level of viral NP expression for determination of viral infection. The expression of WT protein was considered 100% for comparison with the level of expression by the corresponding codon-deoptimized viruses.