Generation of Recombinant Influenza Viruses

Virus rescues were performed as we described previously [2 (link),40 (link),52 (link)]. Briefly, co-cultures (1:1) of 293T and MDCK cells in 6-well plates were co-transfected with 1 μg of each of the ambisense plasmids (pDZ-PB2, −PB1, −PA_WT⁺ or PA_MT⁻, −HA, −NP, −NA, −M, and NS_WT⁺ or NS1_MT⁻) using Lipofectamine 2000 (LPF2000, Invitrogen). At 12 h post-transfection (p.t.), the transfection medium was replaced with DMEM containing 0.3% bovine serum albumin (BSA), 1% P-S-G, and 0.5 μg/mL of N-tosyl-l-phenylalanine chloromethyl ketone (TPCK)-treated trypsin (Sigma). At 3–4 days p.t., tissue culture supernatants (TCS) were collected, clarified, and used to infect fresh MDCK cells. At 3 to 4 days post-infection (p.i.), recombinant viruses were plaque purified and scaled up in MDCK cells. Stocks were titrated by immunofocus assay (Fluorescence Focus Units (FFU)/mL)) on MDCK cells [2 (link),52 (link)]. Virus stocks were confirmed by sequencing the PA and NS1 ORFs using purified total RNA (TRIzol reagent; Invitrogen) from infected MDCK cells according to the manufacturer’s specifications.