Introduction of foreign sequences into the genome of influenza A virus

The ability to apply reverse genetics technologies to influenza virus now allows us to construct novel viruses containing heterologous sequences. We have engineered two neuraminidase (NA) genes of influenza A/WSN/33 virus containing additional sequences which were inserted downstream of the open reading frame of the NA. These NA genes, NA/EMC and NA/EMC-NS1, possess a 546 and a 917 nucleotide (nt) insertion respectively. Transfectant viruses were rescued following ribonucleoprotein (RNP) transfection of the engineered NA genes into influenza helper-virus-infected cells. The transfectant viruses maintained their artificially introduced sequences stably during three passages. The rescued virus containing the NA/EMC-NS1 gene produced one log fewer infectious virus particles in MDBK cells than did wild type A/WSN/33 virus. The growth characteristics in tissue culture of the virus containing the NA/EMC gene was indistinguishable from that of wild-type influenza virus. Based on these results we conclude that influenza A viruses can tolerate heterologous insertions of at least a kilobase in the NA gene.