Detection of HAZV nucleoprotein by Western blot

A549 cells, seeded in 6-well microplates at a density of 3.2 × 105 cells/well, were transfected with siRNA and infected with HAZV as described above. Protein extraction was performed 48 hrs post-infection as follow: confluent cells were washed twice in fresh phosphate-buffered saline 1X (PBS 1X) and lysed in buffer containing 20 mM Tris pH 7.5, 100 mM NaCl, 0.6% NP40, 0.5 mM EDTA and protease inhibitors cocktail (Complete EDTA-free, Roche Diagnostics GmbH, Mannheim, Germany) for 10 min on ice. After removal of cellular debris by centrifugation at 12,000 × g for 10 min, 20 μl of protein extracts were boiled for 5 min in Laemmli buffer and separated on a 10% SDS-PAGE. Proteins were then electrotransferred onto a polyvinylidene fluoride (PVDF) membrane (Bio-Rad Laboratories, Marne-la-Coquette, France). The PVDF membrane was saturated with 5% dry milk in PBS 1X containing 0.1% Tween-20 and incubated overnight at 4°C with mouse anti-HAZV hyperimmune ascitic fluid (1:250) or with mouse anti-GAPDH monoclonal antibody (1:1000, Ambion, Austin, TX, USA). Horseradish peroxydase-labeled goat anti-mouse IgG (1:10000, Interchim, Montluçon, France) was used as secondary antibody followed by chemoluminescent (ECL) revelation (Amersham GE Healthcare, Orsay, France).
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ELISA-based assay for interferon-β detection

A549 cells were cultured in 24-well microplates at a density of 8 × 104 cells/well and transfected as previously described with either 100 nM (i.e. 1.48 μg/ml) of siNT, siS1 and siS2 or 0.25 μg/ml of poly(I:C) dsRNA (Sigma-Aldrich, St Quentin-Fallavier, France). Cell culture supernatants were harvested 24 hrs post-transfection to detect human beta interferon (IFN-β). The cytokine measurement was performed using a sandwich enzyme-linked immunosorbent assay (ELISA) kit (PBL Biomedical Laboratories Tebu-Bio, Le Perray-en-Yvelines, France), according to the manufacturer’s instructions.
Antiviral assays with ribavirin and combination with siS1 or siS2

Confluent monolayers of A549 cells in 24-well plates were infected with HAZV at a MOI of 0.1. Cells were then cultivated with F12K medium supplemented with 0.4% FCS or treated with 0.4% FCS F12K medium containing serial dilutions of ribavirin (1-β-D-ribofuranosyl-1H-1,2,4-triazole-3-carboxamide) (Sigma-Aldrich, St Louis, Missouri, USA). Forty eight hours post-infection, the supernatant of each well was collected and virus titer was performed. The EC50 value for ribavirin was determined as the mean of two independent experiments.

The combination assay required 60% confluent monolayers of A549 cells in 24-well microplates transfected with siS1, siS2 or siNT at a concentration of 1 nM and 10 nM. One day post-transfection, cells were washed twice, infected with HAZV at a MOI of 0.1. One hour after infection, the inoculum was removed and transfected cells were cultured for 48 hrs in 0.4% FCS F12K medium containing 0, 25 or 50 μM of ribavirin. The cell supernatants were then tittered.
Statistical analysis

In this study, we compared the antiviral effect of selected siRNAs to the negative control (siNT) to detect significant variations using the Student’s t-test (P ≤ 0.05 was regarded as significant difference between the two groups of transfected/infected cells).