title: Development of an Influenza A Master Virus for Generating High-Growth Reassortants for A/Anhui/1/2013(H7N9) Vaccine Production in Qualified MDCK Cells.
abstract: In 2013, the first case of human infection with an avian influenza A virus (H7N9) was reported in China, and the human infection with this virus has continued as of 2016. At the request of the WHO, we have successfully developed candidate reassortant vaccine virus using A/Anhui/1/2013 and the high egg-growth master virus A/PR/8/1934. Recent plans regarding influenza vaccine production include using cell-cultured systems in Japan and several other countries. However, egg-based vaccine viruses are not always suitable for cell-cultured vaccine production due to potential issues with growth, protein yield and antigenic stability. Therefore, in this study, we have developed a high-growth master virus (hg-PR8) adapted to qualified NIID-MDCK cells that are competent for vaccine production. The virus hg-PR8 was obtained after 20 serial passages of A/Puerto Rico/8/1934 (PR8) in NIID-MDCK cells. The viral titer of hg-PR8 was 108.6 plaque-forming units per milliliter (PFU/mL). Seven amino acid substitutions were identified in the amino acid sequences of PB2, PB1, PA, NA, M and NS of hg-PR8 compared to the sequence of the original PR8 (org-PR8) strain. The growth capacities of the reassortant viruses, which possess heterologous internal genes from hg-PR8 or org-PR8, indicated that the amino acid changes in PB2 and NS2 similarly affected growth capacity in NIID-MDCK cells. To assess the suitability of hg-PR8 as a master virus, we generated 6:2 reassortant viruses possessing the HA and NA segments from A/Anhui/1/2013 (H7N9) and the remaining segments from hg-PR8. The virus titers of the reassortant strains were 107-108 PFU/mL. The antigenicity of the viruses was stable during ten passages of the viruses in NIID-MDCK cells. In comparison with the egg-based reassortant vaccine viruses with identical HA and NA segments, the hg-PR8-based viruses showed 1.5- to 2-fold higher protein yields in NIID-MDCK cells.
authors.affiliation: Influenza Virus Research Center, National Institute of Infectious Diseases, Musashi-murayama, Tokyo, Japan.
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Suzuki
authors.affiliation: Influenza Virus Research Center, National Institute of Infectious Diseases, Musashi-murayama, Tokyo, Japan.
authors.forename: Takato
authors.initials: T
authors.lastname: Odagiri
authors.affiliation: Influenza Virus Research Center, National Institute of Infectious Diseases, Musashi-murayama, Tokyo, Japan.
authors.forename: Masato
authors.initials: M
authors.lastname: Tashiro
authors.affiliation: Influenza Virus Research Center, National Institute of Infectious Diseases, Musashi-murayama, Tokyo, Japan.
authors.forename: Eri
authors.initials: E
authors.lastname: Nobusawa
mesh_terms: D000220:Adaptation, Biological; D019943:Amino Acid Substitution; D000818:Animals; D000956:Antigens, Viral / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D002460:Cell Line; D002478:Cells, Cultured; D004285:Dogs; D005814:Genes, Viral; D006031:Glycosylation; D006801:Humans; D064766:Influenza A Virus, H7N9 Subtype / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D007252:Influenza Vaccines / Q000276:immunology*; D007251:Influenza, Human / Q000276:immunology / Q000517:prevention & control; D009154:Mutation; D016865:Reassortant Viruses / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D014779:Virus Replication / Q000235:genetics
chemical_list: D000956:Antigens, Viral; D007252:Influenza Vaccines