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Journal of Virology, November 2008, p. 10502-10509, Vol. 82, No. 21
0022-538X/08/$08.00+0 doi:10.1128/JVI.00970-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Growth Determinants for H5N1 Influenza Vaccine Seed Viruses in MDCK Cells
Shin Murakami,1
Taisuke Horimoto,1,3*
Le Quynh Mai,4
Chairul A. Nidom,5
Hualan Chen,6
Yukiko Muramoto,1,3
Shinya Yamada,1,3
Ayaka Iwasa,1,3
Kiyoko Iwatsuki-Horimoto,1,3
Masayuki Shimojima,1,3
Akira Iwata,7 and
Yoshihiro Kawaoka1,2,3,8*
Division of Virology, Department of Microbiology and Immunology,1 International Research Center for Infectious Diseases, Institute of Medical Science, University of Tokyo, Tokyo, Japan,2 Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency, Saitama, Japan,3 National Institute of Hygiene and Epidemiology, Hanoi, Vietnam,4 Avian Influenza Laboratory, Tropical Disease Centre, Airlangga University, Surabaya, Indonesia,5 Animal Influenza Laboratory of the Ministry of Agriculture and National Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, People's Republic of China,6 Nippon Institute for Biological Science, Tokyo, Japan,7 Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin, Madison, Wisconsin8
Received 9 May 2008/ Accepted 20 August 2008
H5N1 influenza A viruses are exacting a growing human toll, with more than 240 fatal cases to date. In the event of an influenza pandemic caused by these viruses, embryonated chicken eggs, which are the approved substrate for human inactivated-vaccine production, will likely be in short supply because chickens will be killed by these viruses or culled to limit the worldwide spread of the infection. The Madin-Darby canine kidney (MDCK) cell line is a promising alternative candidate substrate because it supports efficient growth of influenza viruses compared to other cell lines. Here, we addressed the molecular determinants for growth of an H5N1 vaccine seed virus in MDCK cells, revealing the critical responsibility of the Tyr residue at position 360 of PB2, the considerable requirement for functional balance between hemagglutinin (HA) and neuraminidase (NA), and the partial responsibility of the Glu residue at position 55 of NS1. Based on these findings, we produced a PR8/H5N1 reassortant, optimized for this cell line, that derives all of its genes for its internal proteins from the PR8(UW) strain except for the NS gene, which derives from the PR8(Cambridge) strain; its N1 NA gene, which has a long stalk and derives from an early H5N1 strain; and its HA gene, which has an avirulent-type cleavage site sequence and is derived from a circulating H5N1 virus. Our findings demonstrate the importance and feasibility of a cell culture-based approach to producing seed viruses for inactivated H5N1 vaccines that grow robustly and in a timely, cost-efficient manner as an alternative to egg-based vaccine production.
* Corresponding author. Mailing address: Division of Virology, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan. Phone: 81-3-5449-5281. Fax: 81-3-5449-5408. E-mail for T. Horimoto: horimoto{at}ims.u-tokyo.ac.jp. E-mail for Y. Kawaoka: kawaoka{at}ims.u-tokyo.ac.jp
Published ahead of print on 3 September 2008.
Journal of Virology, November 2008, p. 10502-10509, Vol. 82, No. 21
0022-538X/08/$08.00+0 doi:10.1128/JVI.00970-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
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