L 1 tosylamido 2 phenylethyl chloromethyl ketone tpck treated trypsin

The rgPR8 strain was generated by transfection using Hoffmann et al.'s 8 reverse genetics plasmids [8 (link)] as previously described, with some modifications. The recombinant viruses that possessed C4 in NA, M, and both vRNAs were generated by replacing pHW196-NA and/or pHW197-M with pHW196-NA-C4 and/or pHW197-M-C4 and were named rPR8-NA-prom, rPR8-M-prom, and rPR8-MN-prom (wtPR8), respectively. Briefly, 293T cells were cultured (1 × 10⁶ cells/well in 6-well plates) and transfected with 300 ng of each plasmid by using lipofectamine 2000 and plus reagents (Invitrogen) in a final volume of 1 mL of Opti-MEM (Invitrogen). After overnight incubation, 1 mL of fresh medium and 0.5 mg/mL of L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK)-treated trypsin (Sigma-Aldrich, USA) were added. After 24 h, the culture medium was harvested and 200 µL were injected into 10-day-old SPF ECEs via the allantoic cavity route. After incubating for 2 to 3 days, the allantoic fluid was harvested and tested with an hemagglutination test using 1% (v/v) chicken red blood cells according to the World Health Organization Manual on Animal Influenza Diagnosis and Surveillance. All experiments were performed after obtaining permission from the Seoul National University Institutional Biosafety Committee (SNUIBC) (approval No. SNUIBC-R150729-1).