Generating Recombinant Influenza Viruses

Viral RNAs of XZ299 and H9N2 mutant were extracted and cDNA was synthesized as previously described [22 (link)]. The HA (GenBank accession No: MN227199) and NA(Neuraminidase) (GenBank accession No: MN227201) genes from XZ299 and the HA gene from H9N2 mutant were amplified and cloned into the linear influenza vector pDP2002 by the ExnaseTM II, provided by ClonExpressTM II kit (Vazyme Biotech Co., Ltd., Nanjing, China), as previously described [23 (link)]. Two recombinant viruses designated as rgPR8-H9 166N and rgPR8-H9 166D were rescued by transfection in the cocultured 293T and MDCK cells as previously described [23 (link)]. Briefly, 1 μg of the HA plasmid derived from XZ299 and H9N2 mutant respectively, and 1 μg of NA, NP, PB1, PB2, PA, MP, and NS plasmid derived from PR8 (A/Puerto Rico/8/34 (H1N1)) each were first mixed in 250 μL of Opti-MEM medium and then mixed with 16 μL of TransIT^®-LT1 Transfection Reagent (Mirus Bio LLC, Madison, WI, USA). The mixture was incubated at room temperature for 45 min, and then 1 mL of Opti-MEM medium was added. The mixture was then inoculated onto the co-cultured 293T and MDCK cells. After 12 h post-transfection, the medium was changed with 2 mL of fresh Opti-MEM medium with 1μg/mL TPCK-Trypsin. At day 4 post-transfection, the rescued viruses in the supernatant of the transfected cells were collected and titrated in MDCK cells.