Luciferase Assay

A minigenome assay was used to detect luciferase activities as reported previously (Bortz et al., 2011). Briefly, the p-Luci plasmid (p-Luci, carrying an IAV reporter minigenome in which the firefly luciferase gene is flanked by the non-coding regions of the NS gene from IAV, a truncated PolI promoter and the hepatitis delta virus ribozyme) (Ma et al., 2015) was transfected into 293T cells with Polyfect Transfection Reagent (Qiagen) together with the four pcDNA3.1(+) expression plasmids containing PB2, PB1, PA, and NP genes (400 ng each) from CK/10, YB/7, QD/5, QD/1, or CZ/73, and 40 ng internal control Renilla plasmid (an internal control plasmid to standardize transfection efficiency that encodes the Renilla luciferase protein). Likewise, as for DF1 cells, the four pcDNA3.1(+) expression plasmids of PB2, PB1, PA, and NP genes (400 ng each) were mixed with paviPolIT-Luc plasmid (paviPolIT-Luc, containing the firefly luciferase gene flanked by the non-coding regions of the NP gene of IAV is inserted into a pPolI plasmid housing the 250-nucleotide sequence of the avian polymerase I promoter) (Song et al., 2011) (400 ng) and the Renilla plasmid (80 ng) were co-transfected into DF1 cells. At 24 or 48 h post-transfection, cell lysates were prepared with Dual-Luciferase Reporter Assay System (Promega) and the firefly and Renilla luciferase activities were measured using GloMax 96 microplate luminometer (Promega). The ratio of the firefly luciferase activity value and Renilla luciferase activity value to represent RNP activity of the virus. All results are the means with SD from three independent experiments.