Influenza A virus minigenome assay

A minigenome assay based on the firefly and Renilla luciferase systems was performed as described previously [34,40]. The plasmids, pCAGGS (pCA)-PA, pCA-PB1, pCA-PB2, and pCA-NP harbored the influenza virus PA, PB1, PB2, and NP genes, respectively, driven by the cytomegalovirus enhancer fused to the chicken beta-actin promoter. pHH21-PolI/NP(0)Fluc(0) expressed the minus RNA strand of the firefly luciferase driven by the human RNA polymerase I promoter; this can be converted to the plus strand (mRNA) by influenza virus RNA-dependent RNA polymerase (RdRp). The pRL-TK-Rluc vector (Promega, CA, USA) expressing the Renilla luciferase gene driven by the herpes simplex viral thymidine kinase promoter was used as an internal control. MDCK cells (5 × 10⁴) were transfected with 0.12 μg each of pCA-PA, -PB1, -PB2, and -NP, pHH21-PolI/NP(0)Fluc(0), and pRL-TK-Rluc. At 6 h post-transfection, the cells were treated with the Y30 extract (final 6.25%) at 37°C in the presence of 5% CO₂. Water (final 6.25%) or ribavirin (final 25 μM) were used as the negative or positive control, respectively. After 24 h of incubation, firefly and Renilla luciferase activities in the transfected MDCK cells were measured using the Dual-Glo luciferase assay system (Promega) according to the manufacturer’s instructions. Luciferase activity was expressed as the relative light unit (RLU), where the activity of the water-treated cells was considered 100%.