Influenza Virus Propagation Protocol

We used purified human AGP (Sigma–Aldrich; catalog no.: G9885) and the A/Philippines/2/1982 (Phil82) strain of IAV as glycoprotein standards. Phil82 was expressed in chicken eggs and generously provided to us by Dr Kevan Hartshorn at Boston University School of Medicine. The A/Switzerland/9715293/2013 (H3N2) (SWZ13) viral samples, both WT and 5B8 mutant, were propagated in parallel in specific pathogen-free embryonated chicken eggs and MDCK CCL-34 cells (American Type Culture Collection). The protocol for generation of the influenza virus has been described elsewhere (12 (link)). The mutant HA sequence, herein referred to as strain 5B8, had three amino acid substitutions, Q132H, Y219S, and D225N, none of which disrupted any N-glycosylation sequons.