Wang Zhaoti, Duke Gregory M
MedImmune, 297 North Bernardo Avenue, Mountain View, CA 94043, USA.
Virol J. 2007 Oct 23;4:102. doi: 10.1186/1743-422X-4-102.
Recent incidents where highly pathogenic influenza A H5N1 viruses have spread from avian species into humans have prompted the development of cell-based production of influenza vaccines as an alternative to or replacement of current egg-based production. Madin-Darby canine kidney (MDCK) cells are the primary cell-substrate candidate for influenza virus production but an efficient system for the direct rescue of influenza virus from cloned influenza cDNAs in MDCK cells did not exist. The objective of this study was to develop a highly efficient method for direct rescue of influenza virus in MDCK cells.
The eight-plasmid DNA transfection system for the rescue of influenza virus from cloned influenza cDNAs was adapted such that virus can be generated directly from MDCK cells. This was accomplished by cloning the canine RNA polymerase I (pol I) promoter from MDCK cells and exchanging it for the human RNA pol I promoter in the eight plasmid rescue system. The adapted system retains bi-directional transcription of the viral cDNA template into both RNA pol I transcribed negative-sense viral RNA and RNA pol II transcribed positive-sense viral mRNA. The utility of this system was demonstrated by rescue in MDCK cells of 6:2 genetic reassortants composed of the six internal gene segments (PB1, PB2, PA, NP, M and NS) from either the cold-adapted (ca) influenza A vaccine strain (ca A/Ann Arbor/1/60) or the ca influenza B vaccine strain (ca B/Ann Arbor/1/66) and HA and NA gene segments from wild type influenza A and B strains. Representative 6:2 reassortants were generated for influenza A (H1N1, H3N2, H5N1, H6N1, H7N3 and H9N2) and for both the Victoria and Yamagata lineages of influenza B. The yield of infectious virus in the supernatant of transfected MDCK cells was 106 to 107 plaque forming units per ml by 5 to 7 days post-transfection.
This rescue system will enable efficient production of both influenza A and influenza B vaccines exclusively in MDCK cells and therefore provides a tool for influenza pandemic preparedness.
近期高致病性甲型H5N1流感病毒从禽类传播至人类的事件，促使人们开发基于细胞的流感疫苗生产方法，以替代或取代当前基于鸡蛋的生产方式。犬肾传代细胞（MDCK）是生产流感病毒的主要细胞底物候选者，但当时不存在一种能在MDCK细胞中从克隆的流感cDNA直接拯救流感病毒的有效系统。本研究的目的是开发一种在MDCK细胞中直接拯救流感病毒的高效方法。
用于从克隆的流感cDNA拯救流感病毒的八质粒DNA转染系统经过改造，使得病毒能够直接从MDCK细胞产生。这是通过从MDCK细胞中克隆犬RNA聚合酶I（pol I）启动子，并将其替换八质粒拯救系统中的人RNA pol I启动子来实现的。改造后的系统保留了病毒cDNA模板向RNA pol I转录的负义病毒RNA和RNA pol II转录的正义病毒mRNA的双向转录。通过在MDCK细胞中拯救由冷适应（ca）甲型流感疫苗株（ca A/Ann Arbor/1/60）或ca乙型流感疫苗株（ca B/Ann Arbor/1/66）的六个内部基因片段（PB1、PB2、PA、NP、M和NS）以及野生型甲型和乙型流感病毒株的HA和NA基因片段组成的6:2基因重配体，证明了该系统的实用性。针对甲型流感（H1N1、H3N2、H5N1、H6N1、H7N3和H9N2）以及乙型流感的维多利亚和山形谱系产生了代表性的6:2重配体。转染后5至7天，转染的MDCK细胞上清液中感染性病毒的产量为每毫升10^6至10^7个空斑形成单位。
该拯救系统将能够仅在MDCK细胞中高效生产甲型和乙型流感疫苗，因此为流感大流行防范提供了一种工具。
