FIG 2.
Inhibition of avian IAV replication in human macrophages and moDCs by pretransfection with cIAV siRNAs. Human primary macrophages or moDCs obtained from four different blood donors were separately mock transfected (control, UV IAV, or no-siRNA bars) or pretransfected with the indicated siRNA or DsiRNAs (10 nM) for 21 h. Cells were then infected with live or UV-irradiated H5N1 or H7N9 virus at an MOI of 1. Macrophages were washed twice with PBS at 1 h p.i. and then maintained in macrophage medium. Input virus was retained in moDC cultures (A) Cells from four different blood donors were subsequently collected at 7 and 24 h p.i. and were pooled; then IAV M1 RNA expression was determined by qRT-PCR from isolated total cellular RNA samples. The values were normalized against β-actin gene-specific mRNA, and relative IAV M1 RNA levels were calculated by the ΔΔCT method using untreated cellular RNA as a calibrator. The means (±SD) of three parallel analyses are shown. Data are representative of three individual experiments. Statistical significance was determined against results from samples of nontransfected cells (boxed bars). *; P < 0.05, **, P < 0.01. (B) Western blot analysis for the expression of viral PB1, NP, M1, and NS1 proteins and β-actin and GAPDH proteins in siRNA/DsiRNA transfected human macrophages and moDCs. Cells were collected at 24 h after avian H5N1 or H7N9 IAV infection, and whole-cell lysates were prepared. Cellular proteins (30 μg/lane) were separated by 10% SDS-PAGE, followed by electrophoretic transfer of the proteins onto polyvinylidene difluoride membranes and visualization of the transferred proteins by protein-specific antibodies, as indicated. The data of one representative experiment of three independent experiments is shown.