Multiple amino acid residues confer temperature sensitivity to human influenza virus vaccine strains (FluMist) derived from cold-adapted A/Ann Arbor/6/60

Virology. 2003 Feb 1;306(1):18-24. doi: 10.1016/s0042-6822(02)00035-1.


FluMist influenza A vaccine strains contain the PB1, PB2, PA, NP, M, and NS gene segments of ca A/AA/6/60, the master donor virus-A strain. These gene segments impart the characteristic cold-adapted (ca), attenuated (att), and temperature-sensitive (ts) phenotypes to the vaccine strains. A plasmid-based reverse genetics system was used to create a series of recombinant hybrids between the isogenic non-ts wt A/Ann Arbor/6/60 and MDV-A strains to characterize the genetic basis of the ts phenotype, a critical, genetically stable, biological trait that contributes to the attenuation and safety of FluMist vaccines. PB1, PB2, and NP derived from MDV-A each expressed determinants of temperature sensitivity and the combination of all three gene segments was synergistic, resulting in expression of the characteristic MDV-A ts phenotype. Site-directed mutagenesis analysis mapped the MDV-A ts phenotype to the following four major loci: PB1(1195) (K391E), PB1(1766) (E581G), PB2(821) (N265S), and NP(146) (D34G). In addition, PB1(2005) (A661T) also contributed to the ts phenotype. The identification of multiple genetic loci that control the MDV-A ts phenotype provides a molecular basis for the observed genetic stability of FluMist vaccines.

MeSH terms

  • Animals
  • COS Cells
  • Cell Line
  • Chick Embryo
  • Cold Temperature*
  • Humans
  • Influenza A virus / chemistry*
  • Influenza A virus / genetics
  • Influenza A virus / immunology
  • Influenza A virus / physiology*
  • Influenza Vaccines / chemistry*
  • Influenza Vaccines / genetics
  • Influenza, Human / virology
  • Nucleocapsid Proteins
  • Nucleoproteins / chemistry
  • Nucleoproteins / genetics
  • Nucleoproteins / metabolism
  • Plasmids
  • RNA Replicase
  • Recombination, Genetic*
  • Transfection
  • Vaccines, Attenuated / chemistry
  • Vaccines, Attenuated / genetics
  • Viral Core Proteins / chemistry
  • Viral Core Proteins / genetics
  • Viral Core Proteins / metabolism
  • Viral Plaque Assay
  • Viral Proteins / chemistry
  • Viral Proteins / genetics*
  • Viral Proteins / metabolism


  • Influenza Vaccines
  • Nucleocapsid Proteins
  • Nucleoproteins
  • PB2 protein, Influenzavirus A
  • Vaccines, Attenuated
  • Viral Core Proteins
  • Viral Proteins
  • influenza virus polymerase basic protein 1
  • RNA Replicase