title: Generation of a Genetically Stable High-Fidelity Influenza Vaccine Strain.
abstract: Vaccination is considered the most effective preventive means for influenza control. The development of a master virus with high growth and genetic stability, which may be used for the preparation of vaccine viruses by gene reassortment, is crucial for the enhancement of vaccine performance and efficiency of production. Here, we describe the generation of a high-fidelity and high-growth influenza vaccine master virus strain with a single V43I amino acid change in the PB1 polymerase of the high-growth A/Puerto Rico/8/1934 (PR8) master virus. The PB1-V43I mutation was introduced to increase replication fidelity in order to design an H1N1 vaccine strain with a low error rate. The PR8-PB1-V43I virus exhibited good replication compared with that of the parent PR8 virus. In order to compare the efficiency of egg adaptation and the occurrence of gene mutations leading to antigenic alterations, we constructed 6:2 genetic reassortant viruses between the A(H1N1)pdm09 and the PR8-PB1-V43I viruses; hemagglutinin (HA) and neuraminidase (NA) were from the A(H1N1)pdm09 virus, and the other genes were from the PR8 virus. Mutations responsible for egg adaptation mutations occurred in the HA of the PB1-V43I reassortant virus during serial egg passages; however, in contrast, antigenic mutations were introduced into the HA gene of the 6:2 reassortant virus possessing the wild-type PB1. This study shows that the mutant PR8 virus possessing the PB1 polymerase with the V43I substitution may be utilized as a master virus for the generation of high-growth vaccine viruses with high polymerase fidelity, low error rates of gene replication, and reduced antigenic diversity during virus propagation in eggs for vaccine production.IMPORTANCEVaccination represents the most effective prophylactic option against influenza. The threat of emergence of influenza pandemics necessitates the ability to generate vaccine viruses rapidly. However, as the influenza virus exhibits a high mutation rate, vaccines must be updated to ensure a good match of the HA and NA antigens between the vaccine and the circulating strain. Here, we generated a genetically stable master virus of the A/Puerto Rico/8/1934 (H1N1) backbone encoding an engineered high-fidelity viral polymerase. Importantly, following the application of the high-fidelity PR8 backbone, no mutation resulting in antigenic change was introduced into the HA gene during propagation of the A(H1N1)pdm09 candidate vaccine virus. The low error rate of the present vaccine virus should decrease the risk of generating mutant viruses with increased virulence. Therefore, our findings are expected to be useful for the development of prepandemic vaccines and live attenuated vaccines with higher safety than that of the present candidate vaccines.
authors.affiliation: Department of Microbiology, Kawasaki Medical School, Okayama, Japan tadanaito@med.kawasaki-m.ac.jp knagata@md.tsukuba.ac.jp.
authors.forename: Tadasuke
authors.initials: T
authors.lastname: Naito
authors.affiliation: Laboratory of Virology, Institute of Microbial Chemistry, Tokyo, Japan.|Faculty of Medicine, University of Tsukuba, Ibaraki, Japan.|Japan Society for the Promotion of Science, Tokyo, Japan.
authors.forename: Kotaro
authors.initials: K
authors.lastname: Mori
authors.affiliation: Department of Microbiology, Kawasaki Medical School, Okayama, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Ushirogawa
authors.affiliation: Laboratory of Virology, Institute of Microbial Chemistry, Tokyo, Japan.
authors.forename: Naoki
authors.initials: N
authors.lastname: Takizawa
authors.affiliation: Influenza Virus Research Center, National Institute of Infectious Diseases, Tokyo, Japan.
authors.forename: Eri
authors.initials: E
authors.lastname: Nobusawa
authors.affiliation: Influenza Virus Research Center, National Institute of Infectious Diseases, Tokyo, Japan.
authors.forename: Takato
authors.initials: T
authors.lastname: Odagiri
authors.affiliation: Influenza Virus Research Center, National Institute of Infectious Diseases, Tokyo, Japan.
authors.forename: Masato
authors.initials: M
authors.lastname: Tashiro
authors.affiliation: Faculty of Medicine, University of Tsukuba, Ibaraki, Japan.|Center for Biotechnology, National Taiwan University, Taipei, Taiwan.
authors.forename: Ryosuke L
authors.initials: RL
authors.lastname: Ohniwa
authors.affiliation: Faculty of Medicine, University of Tsukuba, Ibaraki, Japan tadanaito@med.kawasaki-m.ac.jp knagata@md.tsukuba.ac.jp.
authors.forename: Kyosuke
authors.initials: K
authors.lastname: Nagata
authors.affiliation: Department of Microbiology, Kawasaki Medical School, Okayama, Japan.
authors.forename: Mineki
authors.initials: M
authors.lastname: Saito
mesh_terms: D019943:Amino Acid Substitution; D000956:Antigens, Viral / Q000235:genetics* / Q000276:immunology; D009980:Influenza A virus / Q000235:genetics / Q000254:growth & development*; D007252:Influenza Vaccines / Q000235:genetics / Q000276:immunology*; D017354:Point Mutation; D016865:Reassortant Viruses / Q000235:genetics / Q000254:growth & development*; D013678:Technology, Pharmaceutical / Q000379:methods; D014764:Viral Proteins / Q000235:genetics; D014773:Virology / Q000379:methods
chemical_list: D000956:Antigens, Viral; D007252:Influenza Vaccines; D014764:Viral Proteins; C077346:influenza virus polymerase basic protein 1
keywords: RNA polymerases; fidelity; influenza; influenza vaccines; reverse genetic analysis