title: Cloning of the canine RNA polymerase I promoter and establishment of reverse genetics for influenza A and B in MDCK cells.
abstract: BACKGROUND
  Recent incidents where highly pathogenic influenza A H5N1 viruses have spread from avian species into humans have prompted the development of cell-based production of influenza vaccines as an alternative to or replacement of current egg-based production. Madin-Darby canine kidney (MDCK) cells are the primary cell-substrate candidate for influenza virus production but an efficient system for the direct rescue of influenza virus from cloned influenza cDNAs in MDCK cells did not exist. The objective of this study was to develop a highly efficient method for direct rescue of influenza virus in MDCK cells.
  
  
  RESULTS
  The eight-plasmid DNA transfection system for the rescue of influenza virus from cloned influenza cDNAs was adapted such that virus can be generated directly from MDCK cells. This was accomplished by cloning the canine RNA polymerase I (pol I) promoter from MDCK cells and exchanging it for the human RNA pol I promoter in the eight plasmid rescue system. The adapted system retains bi-directional transcription of the viral cDNA template into both RNA pol I transcribed negative-sense viral RNA and RNA pol II transcribed positive-sense viral mRNA. The utility of this system was demonstrated by rescue in MDCK cells of 6:2 genetic reassortants composed of the six internal gene segments (PB1, PB2, PA, NP, M and NS) from either the cold-adapted (ca) influenza A vaccine strain (ca A/Ann Arbor/1/60) or the ca influenza B vaccine strain (ca B/Ann Arbor/1/66) and HA and NA gene segments from wild type influenza A and B strains. Representative 6:2 reassortants were generated for influenza A (H1N1, H3N2, H5N1, H6N1, H7N3 and H9N2) and for both the Victoria and Yamagata lineages of influenza B. The yield of infectious virus in the supernatant of transfected MDCK cells was 106 to 107 plaque forming units per ml by 5 to 7 days post-transfection.
  
  
  CONCLUSIONS
  This rescue system will enable efficient production of both influenza A and influenza B vaccines exclusively in MDCK cells and therefore provides a tool for influenza pandemic preparedness.
authors.affiliation: MedImmune, 297 North Bernardo Avenue, Mountain View, CA 94043, USA. wangz@medimmune.com
authors.forename: Zhaoti
authors.initials: Z
authors.lastname: Wang
authors.forename: Gregory M
authors.initials: GM
authors.lastname: Duke
mesh_terms: D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D004285:Dogs; D006801:Humans; D009980:Influenza A virus / Q000235:genetics*; D009981:Influenza B virus / Q000235:genetics*; D007252:Influenza Vaccines / Q000235:genetics / Q000276:immunology; D007251:Influenza, Human / Q000517:prevention & control; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D012318:RNA Polymerase I / Q000235:genetics*; D016415:Sequence Alignment; D014613:Vaccines, Attenuated / Q000235:genetics; D014779:Virus Replication
chemical_list: D007252:Influenza Vaccines; D014613:Vaccines, Attenuated; D012318:RNA Polymerase I