FIG 1.

Schematic overview of the steps to generate the degenerate H9 HA PCR product and rescue the H9 HA virus library. (A) The wild-type WF10 HA plasmid was split into 2 plasmids with designed primers (pDPAO1-767 and pDPAO738-1742). The HA PCR product (1 to 767) carrying the mouse RNA polymerase terminator sequence and the degenerate NNN codon at position 226 was generated from the pDPAO1-767 plasmid either with a specific primer with the NNN codon (nnn226) or with a mix of primers able to introduce all 20 amino acids (equi226). Another PCR product with the remaining HA (residues 738 to 1742) and human polymerase 1 promoter was generated from pDPAO738-1742. Using overlapping PCR, a full-length HA was obtained with the degenerate codon at position 226 flanked by the mouse RNA polymerase terminator sequence and the human pol 1 promoter. (B) Generation of virus library by PCR-based reverse genetics using the ₂₂₆HA PCR product and 7 plasmids carrying proteins from WF10 (H9N2) or PR8 (H1N1).