2.4. Luciferase Reporter Assay

The luciferase reporter gene assay was performed as previously described [9,10,22] with minor modifications. Briefly, 293T were cultured into 6-well plates and co-transfected with a mixture of the following: (1) 1 µg of each pMPccdB expression plasmid encoding individual vRNP complex components for the parent, reassortant, and variant strains; (2) the vRNP-expressing plasmids were mixed with 40 ng Renilla luciferase expression plasmid (pRL-SV40) for normalization; and (3) 200 ng of a firefly luciferase reporter plasmid pHW72-Luc expressing negative-sense firefly luciferase flanked by noncoding sequences (NCR) of NS segment under polymerase I promoter control. The plasmid DNA mixture was then transfected into 293T cells using Trans-IT2020 (2 µL/µg) for 8 h as previously described [21,23]. Subsequently, the transfection mixture was replaced with 1x Opti-MEM with 0.2% BSA and 1% pen/strep and kept in a humidified 5% CO₂ incubator at 37% for 24 h. The transfected cells were then harvested, washed once with 1x PBS, and then lysed using 200 L of “1x passive lysis buffer” (Promega, Madison, Wisconsin, USA). The Dual-Luciferase Reporter Assay System (Promega) and a Spark 10M multimode microplate reader were then used to quantify the Firefly/Renilla luciferases as Relative Luminometer Units (RLU, standardized to Renilla luciferase). RLU corresponds to the fold induction of polymerase activity.