Influenza viral RNA was extracted using a RNeasy Kit (Qiagen, CA). The full-length NA genes (numbering is based on an alignment of NAs from the following reference strains: A/Perth/16/2009 (H3N2) and B/Yamanashi/166/1998 [20 ]) of influenza A(H3N2) and B viruses were amplified by one-step RT-PCR with primers previously described for the NA segments of influenza A [21 (link)] and B [22 (link)] viruses. PCR products were gel-purified (QIAquick Gel Extraction Kit, Qiagen) and then digested with BsaI [21 (link)] or BsmBI [22 (link)] restriction enzymes (New England Biolabs, Boston, MA). The plasmid vectors were digested with BsmBI, and the NA segments were ligated with T4 DNA ligase (New England Biolabs, Boston, MA) into pHW2000 (for influenza A NAs) or pAD3000 (for influenza B NAs).
Plasmids were transformed into One Shot chemically competent Escherichia coli cells (Invitrogen, Carlsbad, CA), and positive clones were inoculated into Terrific Broth medium containing ampicillin. The positive clones were purified using HiSpeed Plasmid Maxi Kits (Qiagen, CA), and the plasmid DNA concentrations were determined by both UV spectrophotometry at 260 nm and analysis on agarose gels. The constructed plasmids were sequenced to ensure their identity with the field strains.
Plasmids were transformed into One Shot chemically competent Escherichia coli cells (Invitrogen, Carlsbad, CA), and positive clones were inoculated into Terrific Broth medium containing ampicillin. The positive clones were purified using HiSpeed Plasmid Maxi Kits (Qiagen, CA), and the plasmid DNA concentrations were determined by both UV spectrophotometry at 260 nm and analysis on agarose gels. The constructed plasmids were sequenced to ensure their identity with the field strains.