Production of reverse genetics viruses.

Plasmids were linearized by MluI, purified, and precipitated by phenol-chloroform (ratio, 1:1) and isopropanol (same volume as the phenol-chloroform). Ten μg of linearized DNA template was added to the T7 RNA polymerase reaction mixture of the RiboMax large-scale RNA production system to generate RNA by in vitro RNA transcription. The transcription reaction was terminated by adding 10 U of RQ1 RNase-free DNase and incubated at 37°C for 30 min. Two μg of purified RNA was transfected into 4 × 10⁵ RD cells (ATCC CCL-136) in a 6-well plate using TransMessenger transfection reagent (Qiagen). Reverse genetics viruses were harvested when the cytopathic effect (CPE) was observed in more than 75% of the cells, as previously described (44).