Specific pathogen free eggs

IAV/Puerto Rico/8/34 (PR/8) was purchased from ATCC (Catalog #VR‐95, Manassas, VA, USA). IAV/WSN/1933 (WSN), IAV/Oklahoma/3052/09 (pdm/OK) and A/Oklahoma/309/2006 (H3N2) were kindly provided by Dr. Gillian Air, University of Oklahoma Health Sciences Center. Virus stocks were propagated in specific‐pathogen free eggs (Charles River Laboratories, Houston, TX, USA) as previously described.¹⁰ The viral stocks were aliquoted and stored in screw cap tubes at −80°C. The titres of the viral stocks were measured by plaque assay.

The generation of H1N1-GFP (A/Puerto Rico/8/1934) was described earlier (55 (link)). H5N1-GFP (A/Vietnam/1203/2004; low pathogenic without the multibasic site in HA), which contains a GFP reporter in the NS segment, was generated following a similar protocol (55 (link)). H5N1 (2:6) (A/Vietnam/1203/2004; low pathogenic without the multibasic site in HA), which contains the 6 internal genes from the PR8 strain, was rescued using standard reverse genetics techniques (55 (link), 56 (link)). Briefly, 0.5 μg of each of the six pDZ plasmids representing PB2, PB1, PA, NP, NS, and M from A/Puerto Rico/8/1934 (PR8) and two pPol-I plasmids representing the HA (low pathogenic) and NA segments of H5N1 were transfected into a cell mixture containing 293T-MDCK using Lipofectamine 2000 (Invitrogen). After 48 h, 200 μl of the rescue supernatants was used to infect fresh MDCK cells seeded in 6-well plates. The successful rescue of recombinant viruses was confirmed by performing a hemagglutination assay with chicken red blood cells. After plaque purification, the recombinant viruses were amplified in 10-day-old specific-pathogen-free eggs (Charles River). Viral titers were determined by plaque assay in MDCK cells using standard techniques.

Human lung epithelial (A549) and human embryonic kidney (HEK293) cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S). Madin-Darby canine kidney (MDCK) cells were cultured in MEM supplemented with 10% FBS and 1% P/S. Dr. Adolfo Garcia-Sastre at the Icahn School of Medicine at Mount Sinai, NY kindly provided the IAV strains A/Puerto Rico/8/1934 (H1N1), low pathogenic version of A/Vietnam/1203/2004 (H5N1), and A/Hong Kong/1/1968 (H3N2). IAV strains were grown in 10-day old specific pathogen-free eggs (Charles River) and titered by standard plaque assay on MDCK cells, using 2.4% Avicel RC-581 (a gift from FMC BioPolmer, Philadelphia, PA). Two days post infection plaques were quantified via crystal violet staining. Vesicular stomatitis virus expressing GFP (VSV) was kindly provided by Dr. Glenn Barber at the University of Miami, FL [79 (link)]. VSV viruses were grown in Vero cells and titered by plaque assay with 1% methylcellulose (Sigma).