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Journal of Virology, September 2005, p. 11014-11021, Vol. 79, No. 17
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.17.11014-11021.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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

Erich Hoffmann,1 Kutubuddin Mahmood,1 Zhongying Chen,1 Chin-Fen Yang,1 Joshua Spaete,1 Harry B. Greenberg,1 M. Louise Herlocher,2 Hong Jin,1 and George Kemble1*

MedImmune Vaccines, 297 North Bernardo Ave., Mountain View, California 94043,1 Department of Epidemiology, 109 Observatory St., Ann Arbor, Michigan 481092

Received 3 August 2004/ Accepted 10 February 2005

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.

* Corresponding author. Mailing address: MedImmune Vaccines, Inc., 297 North Bernardo Ave., Mountain View, CA 94043. Phone: (650) 603-2356. Fax: (650) 603-3356. E-mail: kembleg{at}medimmune.com.

Journal of Virology, September 2005, p. 11014-11021, Vol. 79, No. 17
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.17.11014-11021.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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