Multiple Gene Segments Control the Temperature Sensitivity and Attenuation Phenotypes of ca B/Ann Arbor/1/66

Abstract

ABSTRACT Cold-adapted ( ca ) B/Ann Arbor/1/66 is the influenza B virus strain master donor virus for FluMist, a live, attenuated, influenza virus vaccine licensed in 2003 in the United States. Each FluMist vaccine strain contains six gene segments of the master donor virus; these master donor gene segments control the vaccine's replication and attenuation. These gene segments also express characteristic biological traits in model systems. Unlike most virulent wild-type (wt) influenza B viruses, ca B/Ann Arbor/1/66 is temperature sensitive ( ts ) at 37°C and attenuated ( att ) in the ferret model. In order to define the minimal genetic components of these phenotypes, the amino acid sequences of the internal genes of ca B/Ann Arbor/1/66 were aligned to those of other influenza B viruses. These analyses revealed eight unique amino acids in three proteins: two in the polymerase subunit PA, two in the M1 matrix protein, and four in the nucleoprotein (NP). Using reverse genetics, these eight wt amino acids were engineered into a plasmid-derived recombinant of ca B/Ann Arbor/1/66, and these changes reverted both the ts and the att phenotypes. A detailed mutational analysis revealed that a combination of two sites in NP (A114 and H410) and one in PA (M431) controlled expression of ts , whereas these same changes plus two additional residues in M1 (Q159 and V183) controlled the att phenotype. Transferring this genetic signature to the divergent wt B/Yamanashi/166/98 strain conferred both the ts and the att phenotypes on the recombinant, demonstrating that this small, complex, genetic signature encoded the essential elements for these traits.