Inhibition of Hazara nairovirus replication by small interfering RNAs. Part 3

HAZV titration

Monolayers of Vero E6 cultured in 12-well microplates were infected with serial 10-fold dilutions of supernatant from infected cells. After 1 hr incubation at 37°C, 3.2% carboxymethylcellulose (CMC) sodium salt (VWR International Ltd, Poole, England) were added into each well. CMC overlay was removed five days post-infection and cells were fixed with 4% formaldehyde for 20 min at room temperature (RT) and permeabilized in 0.5% Triton X-100 (Sigma-Aldrich, St Quentin-Fallavier, France) for 5 min. Viral foci were detected by probing with mouse anti-HAZV hyperimmune ascitic fluid (1:2000) for 1 hr at 37°C, followed by horseradish peroxydase-conjugated goat anti-mouse IgG (1:2000, Interchim, Montluçon, France) at 37°C for 1 hr. The cell monolayer was then incubated with 0.7 mg/ml of 3,3′-diaminobenzidine (DAB) solution (Sigma-Aldrich, St Quentin-Fallavier, France) diluted in PBS 1X for 10 min at RT. Once clusters of infected cells were visible (dark stain), the reaction was stopped by removing the DAB solution followed by water washing. The foci were counted manually under the light microscope.

Design and synthesis of siRNAs

The sequences of HAZV L, M and S genomic segments [GenBank:DQ076419.1, DQ813514.1 and M86624.1, respectively] were used to design the siRNAs. Duplexes of 21-nucleotide siRNAs with short 3′ overhangs were synthesized by Qiagen (Courtaboeuf, France). For each viral mRNAs, four lyophilized siRNAs were produced (Table 1) and their sequence was subjected to a BLAST search against GenBank to minimize off-target effects. In the present study, a non targeting siRNA (siNT) showing no complementarities neither with HAZV mRNAs nor with any human, mouse or rat mRNAs was used as a negative control. The TOX siRNA (siTOX) (Dharmacon RNAi technologies, Lafayette, USA) was used to determine transfection efficacy (see below). Before use, all siRNAs were reconstituted in rehydration buffer to obtain 20 μM solutions according to the company’s instructions.

Twenty four hours before transfection, A549 cells were seeded in 24-well microplates at a density of 8 × 104 cells/well to achieve 60% confluent cell monolayers the day after. Various siRNAs concentrations (ranging from 0.01 to 100 nM) were complexed with the Lipofectamine 2000 transfection reagent (Invitrogen, Cergy Pontoise, France) in Opti-MEM I medium (Gibco, Invitrogen Corporation, Paisley, United Kingdom). The final volume of Lipofectamine 2000 was 1.5 μl/well. The transfection mixture was incubated for 20 min at RT to allow the formation of siRNA/transfection reagent complexes and 100 μl of the solution were added in each well. One day post-transfection, cells were gently washed twice with F12K medium and infected with HAZV at a MOI of 0.1. The inoculum was incubated for 1 hr. Cells were then cultivated in F12K medium supplemented with 0.4% FCS for 48 hrs. Infected cells supernatants were tittered as described above. The EC50 was calculated as the mean of two independent experiments using the GraphPadPrism version 4.00 software (GraphPad Software, San Diego California, USA) for non linear regression.

For the post infection treatment studies, 60% confluent A549 cell monolayers were infected with HAZV for 1 hr at a MOI of 0.01. At 1 hr, 8 hrs or 24 hrs post-infection, 100 μl of the transfection mixture (containing 100 nM of siRNA) were added. One day after transfection, cells were washed, and grown in F12K medium with 0.4% FCS for 48 hrs. The supernatant from infected cells were then harvested and tittered.

For each experiment, transfection efficiency was monitored by transfecting A549 cells with 100 nM of siTOX under the same experimental conditions as described above. Cells successfully transfected with siTOX undergo apoptosis and cell death within 24-48 hrs. After 3 days of incubation, siTOX-transfected cells were trypsinized and manually counted using a hematocytometer (Trypan blue exclusion assay). Transfection efficiency was calculated as the ratio between the number of viable siTOX-transfected cells versus non-transfected cells. In our experiments, transfection efficiency was routinely above 90%.