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Abstract
Influenza is a serious public health problem that causes a contagious respiratory disease. Vaccination is the most effective strategy to reduce transmission and prevent influenza. In recent years, cell-based vaccines have been developed with continuous cell lines such as Madin-Darby canine kidney (MDCK) and Vero. However, wild-type influenza and egg-based vaccine seed viruses will not grow efficiently in these cell lines. Therefore, improvement of virus growth is strongly required for development of vaccine seed viruses and cell-based influenza vaccine production. The aim of our research is to develop novel MDCK cells supporting highly efficient propagation of influenza virus in order to expand the capacity of vaccine production. In this study, we screened a human siRNA library that involves 78 target molecules relating to three major type I interferon (IFN) pathways to identify genes that when knocked down by siRNA lead to enhanced production of influenza virus A/Puerto Rico/8/1934 in A549 cells. The siRNAs targeting 23 candidate genes were selected to undergo a second screening pass in MDCK cells. We examined the effects of knockdown of target genes on the viral production using newly designed siRNAs based on sequence analyses. Knockdown of the expression of a canine gene corresponding to human IRF7 by siRNA increased the efficiency of viral production in MDCK cells through an unknown process that includes the mechanisms other than inhibition of IFN-α/β induction. Furthermore, the viral yield greatly increased in MDCK cells stably transduced with the lentiviral vector for expression of short hairpin RNA against IRF7 compared with that in control MDCK cells. Therefore, we propose that modified MDCK cells with lower expression level of IRF7 could be useful not only for increasing the capacity of vaccine production but also facilitating the process of seed virus isolation from clinical specimens for manufacturing of vaccines.
Highlights
Influenza is a global public health issue that causes a serious illness with a high mortality rate
To identify key host factors in canine Madin Darby canine kidney (MDCK) cells, we used A549 human lung adenocarcinoma cell line for the first screening because human genome database includes much more information than canis lupus familiaris genome database and related resources including pre-designed siRNA library are available in human
The siRNAs for each target gene were individually transfected into A549 cells, and the cells were infected with influenza A/Puerto Rico/34/8 (PR8) at a MOI of 0.01
Summary
Influenza is a global public health issue that causes a serious illness with a high mortality rate. Two continuous cell lines have been approved by regulatory authorities to be used for the production of influenza vaccines: Madin Darby canine kidney (MDCK) cells and African green monkey kidneyderived Vero cells [1,2,3,4,5]. Human retina-derived cell line PER.C6 has been shown useful for propagation of influenza viruses [6]. These cell lines produce notable yields of a wide variety of influenza viruses, attempts to develop novel cell lines with greater potentials have been made for more rapid preparation of influenza vaccines. The siat7e-expressing cells that proliferate in suspension would facilitate influenza virus productions [5]
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