title: Mutations to PB2 and NP proteins of an avian influenza virus combine to confer efficient growth in primary human respiratory cells.
abstract: Influenza pandemics occur when influenza A viruses (IAV) adapted to other host species enter humans and spread through the population. Pandemics are relatively rare due to host restriction of IAV: strains adapted to nonhuman species do not readily infect, replicate in, or transmit among humans. IAV can overcome host restriction through reassortment or adaptive evolution, and these are mechanisms by which pandemic strains arise in nature. To identify mutations that facilitate growth of avian IAV in humans, we have adapted influenza A/duck/Alberta/35/1976 (H1N1) (dk/AB/76) virus to a high-growth phenotype in differentiated human tracheo-bronchial epithelial (HTBE) cells. Following 10 serial passages of three independent lineages, the bulk populations showed similar growth in HTBE cells to that of a human seasonal virus. The coding changes present in six clonal isolates were determined. The majority of changes were located in the polymerase complex and nucleoprotein (NP), and all isolates carried mutations in the PB2 627 domain and regions of NP thought to interact with PB2. Using reverse genetics, the impact on growth and polymerase activity of individual and paired mutations in PB2 and NP was evaluated. The results indicate that coupling of the mammalian-adaptive mutation PB2 E627K or Q591K to selected mutations in NP further augments the growth of the corresponding viruses. In addition, minimal combinations of three (PB2 Q236H, E627K, and NP N309K) or two (PB2 Q591K and NP S50G) mutations were sufficient to recapitulate the efficient growth in HTBE cells of dk/AB/76 viruses isolated after 10 passages in this substrate.
  
  
  OBJECTIVE
  Influenza A viruses adapted to birds do not typically grow well in humans. However, as has been seen recently with H5N1 and H7N9 subtype viruses, productive and virulent infection of humans with avian influenza viruses can occur. The ability of avian influenza viruses to adapt to new host species is a consequence of their high mutation rate that supports their zoonotic potential. Understanding of the adaptation of avian viruses to mammals strengthens public health efforts aimed at controlling influenza. In particular, it is critical to know how readily and through mutation to which functional components avian influenza viruses gain the ability to grow efficiently in humans. Our data show that as few as three mutations, in the PB2 and NP proteins, support robust growth of a low-pathogenic, H1N1 duck isolate in primary human respiratory cells.
authors.affiliation: Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, Georgia, USA.
authors.forename: Shamika
authors.initials: S
authors.lastname: Danzy
authors.affiliation: Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, Georgia, USA.
authors.forename: Lydia R
authors.initials: LR
authors.lastname: Studdard
authors.affiliation: Department of Microbiology, University of Chicago, Chicago, Illinois, USA.
authors.forename: Balaji
authors.initials: B
authors.lastname: Manicassamy
authors.affiliation: Public Health Research Institute and Regional Biocontainment Laboratory, New Jersey Medical School, Rutgers, The State University of New Jersey, Newark, New Jersey, USA.
authors.forename: Alicia
authors.initials: A
authors.lastname: Solorzano
authors.affiliation: Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, Georgia, USA.
authors.forename: Nicolle
authors.initials: N
authors.lastname: Marshall
authors.affiliation: Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, New York, USA Department of Medicine, Division of Infectious Diseases, Icahn School of Medicine at Mount Sinai, New York, New York, USA Global Health and Emerging Pathogens Institute, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
authors.forename: Adolfo
authors.initials: A
authors.lastname: García-Sastre
authors.affiliation: Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, Georgia, USA.
authors.forename: John
authors.initials: J
authors.lastname: Steel
authors.affiliation: Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, Georgia, USA anice.lowen@emory.edu.
authors.forename: Anice C
authors.initials: AC
authors.lastname: Lowen
mesh_terms: D000220:Adaptation, Biological; D000818:Animals; D002460:Cell Line; D004252:DNA Mutational Analysis; D004372:Ducks; D004847:Epithelial Cells / Q000821:virology*; D006801:Humans; D053118:Influenza A Virus, H1N1 Subtype / Q000235:genetics* / Q000254:growth & development* / Q000502:physiology; D005585:Influenza in Birds / Q000821:virology; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D019590:Nucleocapsid Proteins; D016601:RNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics* / Q000378:metabolism; D011995:Recombination, Genetic; D059386:Reverse Genetics; D012692:Serial Passage; D014758:Viral Core Proteins / Q000235:genetics* / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: D050505:Mutant Proteins; C048842:NP protein, Influenza A virus; D019590:Nucleocapsid Proteins; C057027:PB2 protein, Influenzavirus A; D016601:RNA-Binding Proteins; D014758:Viral Core Proteins; D014764:Viral Proteins; D012324:RNA-Dependent RNA Polymerase