Figure 3

Addition of protease inhibitors reduced influenza virus propagation in mouse and human lung cell cultures. Mouse (MLE15) or human (A549) cell lines were infected with the PR8 virus at a multiplicity of infection of 0.01 and the amount of the virus NP in the supernatant was determined by NP-specific ELISA. Pretreatment of cultured MLE15 cells with serial dilutions of the serine protease inhibitors AEBSF (A; 0.13-1 mM) or p AB (B; 1.5-0.09 mM) prior to infection resulted in a decrease of the released virus particles as measured by a decrease in the amount of the viral NP in the supernatants at 24 hours p.i. (n = 3 cell culture wells for each inhibitor concentration). The lowest NP levels were recorded at the highest inhibitor concentration. A similar effect was observed upon pretreatment of A549 (n = 3 cell culture wells at each concentration) with serial concentrations of a cocktail consisting of AEBSF and p AB (C; 125-31 μg/ml of both inhibitors) prior to infection with H7N7 virus at a MOI 0.01. Pretreatment of MLE15 cells (n = 3 cell culture wells at each concentration) with the serine protease inhibitor cocktail (125-31 μg/ml of both inhibitors) followed by incubation of cells with H7N7 for 1 hour and then collection of medium (D) revealed that wells treated with higher cocktail inhibitor concentrations had higher NP titers in the supernatant than wells treated with lower concentrations indicating inhibition of virus entry. Each data point represents the mean of duplicate measurements of the virus NP titer in 3 individual culture wells +/- 1 SD.