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.
García-Sastre
A
A
Department of Microbiology, Mount Sinai School of Medicine, New York, NY.
Percy
N
N
Barclay
W
W
Palese
P
P
D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002460:Cell Line; D004686:Encephalomyocarditis virus / Q000235:genetics* / Q000276:immunology; D005814:Genes, Viral; D005822:Genetic Vectors; D009980:Influenza A virus / Q000235:genetics* / Q000276:immunology; D008969:Molecular Sequence Data; D009439:Neuraminidase / Q000235:genetics*; D016133:Polymerase Chain Reaction; D012313:RNA / Q000138:chemical synthesis / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics; D012261:Ribonucleoproteins / Q000235:genetics; D014162:Transfection; D014614:Vaccines, Synthetic; D014764:Viral Proteins / Q000235:genetics*; D015678:Viral Structural Proteins / Q000235:genetics*; D014779:Virus Replication
D012367:RNA, Viral; D012261:Ribonucleoproteins; D014614:Vaccines, Synthetic; D014764:Viral Proteins; D015678:Viral Structural Proteins; D012313:RNA; D009439:Neuraminidase