Rescuing and Propagation of Viruses

Rescuing of chimeric and mutant viruses was performed by cotransfecting the full-length ICs with three supporting plasmids (NP, P, and L) into BSR T7/5 cell lines using Lipofectamine 3000 (Invitrogen, United States), as previously described (Hu et al., 2017). Briefly, a final concentration of 10% allantoic fluid was added to the BSR T7/5 cells at 12 h post-transfection. The rescued viruses were propagated by inoculating 400 μL of cell lysate into the allantoic cavity of 10-day-old SPF chicken embryos. Four days after inoculation, the infected allantoic fluids were harvested, and the rescued viruses were examined by HA assay using 1% chicken red blood cells. All generated viruses were passaged in 10-day-old SPF chicken embryos five times, and the sequences of the F and HN genes were confirmed by sequencing.