Fig. 3.

IFN-β production by A549 cells infected either with MACV wt, r3MACV or SeV. A549 cells were infected for one hour with the MACV wt, r3MACV or SeV viruses at a MOI of 3, then the inoculum was removed and fresh culture medium was added. At indicated time points, a virus titers in the supernatants were determined by virus TCID₅₀, as described in Fig. 2; b production of IFN-β was detected in supernatant solutions using the VeriKine Human IFN BETA ELISA Kit (PBL assay sciences); and c intracellular RNA was extracted using the QIAmp Viral RNA Mini kit (Qiagen). RT-qPCR were performed using the QuantiTect Probe RT-PCR kit (Qiagen) in a 30 μl final volume with 5 μl of purified RNA and primers and probe at a final concentration of 400 nM and 200 nM, respectively. The assay was carried out using a CFX96 model (Bio-Rad) with a cycling profile of 50 °C for 30 min, 95 °C for 15 min, and 40 cycles at 94 °C for 15 s followed by 60°c for 1 min. The primers and probes (available on request) were designed using the Eurofins Genomics’ online tool (www.eurofinsgenomics.eu). Probes were 5′- and 3′-labelled with the fluorescent reporter dye 6-carboxyfluorescein (FAM) and the Black Hole Quencher (BHQ-1), respectively. All procedures were carried out following manufacturers’ instructions. This experiment was carried out in triplicate. Asterisks denote significant differences (P < 0.05, two-way ANOVA with the Bonferroni correction, ns: no significate, * ≤ 0.05, **** ≤ 0.0001)