Fig. 3
Intra-cellular effect of binase on viral replication and transcription. MDCK-II cells were pre-incubated with binase-containing media for 4 h, washed twice with PBS++ and infected with either H1N1pdm09 or H3N2-Vict for 1 h (MOI = 1). Inoculum was then removed and cells were washed twice with PBS++. DMEM/BA media, with and without binase, were added to the cell monolayers for 8 h p.i.. The cells were washed twice with PBS++ and fresh infection medium without binase was added to the cells. The supernatants containing progeny virions were collected 12 and 24 h p.i. for H1N1pdm09 (a) or 24 h p.i. for H3N2-Vict (b) and titrated using foci assay. c Effect of binase on the activity of the viral polymerase activity of H1N1pdm09 as determined by primer extension analysis. MDCK-II cells were infected (MOI = 3) with H1N1pdm09. At 8 h p.i., vRNA, mRNA, cRNA, and 5S rRNA (loading control) levels were determined. d Infected MDCK-II cells were treated with binase (105 U/ml, white bars) for the indicated time points or left untreated (black bars) in triplicates. The effect of binase on viral NP production was analyzed by Western blotting using a NP-specific antibody and calculated by setting the percentage of untreated cells at 100%. NP-specific signals were quantified and normalized to beta-actin expression (loading control) and calculated relative to the values obtained from cells without binase treatment (arbitrarily set to 100%). Statistical analysis was performed using the Student’s t-test and two-way ANOVA, followed by Bonferroni post hoc test (* = p < 0.05, ** = p < 0.01, *** = p < 0.001)