Pcdna3.1 swineh1 flag

Influenza A Virus (IAV) subtypes, A/England/195/2009 (pH1N1) and the A/HK/1174/99 (H3N2), were obtained as described recently (32 (link), 33 (link)). H1N1 and H3N2 pseudotyped viral particles were produced using the plasmids obtained from Addgene (32 (link), 33 (link)). pcDNA3.1-swineH1-flag (H1 from swine H1N1 A/California/04/09) (Invitrogen), pcDNA3.1-swine N1-flag (N1 from swine H1N1 A/California/04/09), and pcDNA H3 (from A/Denmark/70/03 (H3N2) (Invitrogen) were purchased commercially. Plasmid pI.18-N2 [N2 from A/Texas/50/2012/(H3N2)] was provided by Dr Nigel Temperton (University of Kent). Anti-influenza viral antibodies and recombinant viral proteins were obtained from BEI Resources, National Institutes of Health, USA (32 (link), 33 (link)).

The IAV subtypes used in this study, including the pH1N1 (A/England/2009) and H3N2 (A/HK/1999), were provided by Wendy Barclay (Imperial College, London) and Leo Poon (University of Hong Kong), respectively. The plasmids used for the production of H1N1 and H3N2 pseudo-typed viral particles were obtained from Addgene; pHIV-Luciferase (Addgene plasmid #21375); psPAX2 (Addgene plasmid # 12260); and Vesicular Stomatitis Virus (VSV-G) (Addgene plasmid #8454). pcDNA3.1-swineH1-flag (H1 from swine H1N1 A/California/04/09) (Invitrogen), pcDNA3.1-swine N1-flag (N1 from swine H1N1 A/California/04/09), and pCDNA H3 (from A/Denmark/70/03 (H3N2) (Invitrogen) were obtained commercially. pI.18-N2 [N2 from A/Texas/50/2012/(H3N2)] plasmid was a gift from Nigel Temperton (University of Kent). Anti-influenza antibodies used were obtained from BEI Resources, NIAID, NIH, USA, and used as previously described (38 (link)); these include polyclonal anti-influenza Virus H3 HA, A/Hong Kong/1/1968 and monoclonal anti-influenza virus H1 HA, A/California /04/2009.

HEK293T cells were co-transfected with 20 µg of pcDNA3.1-swineH1-flag (H1 from swine H1N1 A/California/04/09) (Invitrogen), pcDNA3.1-swine N1-flag (N1 from swine H1N1 A/California/04/09) (Invitrogen), pHIV-Luciferase backbone (Addgene), which carries a modified proviral HIV-1 genome with env deleted and designed to express the firefly luciferase reporter, and psPAX2 (Addgene). psPAX2 is a second-generation lentiviral packaging plasmid and can be used with second or third generation lentiviral vectors and envelope expressing plasmid. VSV-G lentivirus was produced in a similar way as described above, without H1+N1 plasmids. Supernatant containing the released H1+N1 pseudotyped and VSV-G lentiviral particles were harvested at 24 and 48 h and centrifuged at 5,000 × g for 10 min to remove any debris, and concentrated via ultra-centrifugation. The transfected HEK293T cells were lysed using lysis buffer (50 mM Tris–HCl pH 7.5, 200 mM NaCl, 5 mM EDTA, 0.1% v/v Triton X-100). The filtered supernatant and the cell lysate were analyzed via western blotting and luciferase reporter activity assay.