FIG. 1.

Schematics of the removal of the polybasic cleavage site from the HA segment, of truncations of the NS segment, and of the virus rescue system. (A) Alteration of the nucleotide sequence of the HA gene of VN1203. The encoded polybasic amino acid sequence, RRRKK, was removed and replaced with the amino acid threonine. Additionally, adenosines were replaced with guanosines or cytosines wherever possible without disrupting the encoded amino acid sequence. Amino acids and nucleotides in the wild-type sequence that were changed are shown in red; introduced amino acids and nucleotides predicted to lower the pathogenicity are shown in green. (B) Schematic diagram of wild-type and truncated NS influenza virus gene segments. The NS gene segment was modified to create NS1 genes encoding 73, 99, and 126 aa, respectively. The nucleotide positions bordering the deletion in each case (nt 379 to 456, 298 to 456, and 219 to 456) are indicated above each NS segment in the schematic. In all truncated segments, stop codons were introduced in each reading frame in place of the deleted sequence. NS1 mutations did not affect the sequence of the NEP. (C) Schematic of the rescue of influenza A/Viet Nam/1203/04-based viruses incorporating truncated NS segments.