key: cord-0947495-wk49ds8y authors: Harada, Yuichi; Takahashi, Hitoshi; Trusheim, Heidi; Roth, Bernhard; Mizuta, Katsumi; Hirata‐Saito, Asumi; Ogane, Teruko; Odagiri, Takato; Tashiro, Masato; Yamamoto, Norio title: Comparison of suspension MDCK cells, adherent MDCK cells, and LLC‐MK2 cells for selective isolation of influenza viruses to be used as vaccine seeds date: 2019-10-25 journal: Influenza Other Respir Viruses DOI: 10.1111/irv.12694 sha: 98aca16ac6513c25abcf54bc1dbe7b6fe65e9c49 doc_id: 947495 cord_uid: wk49ds8y BACKGROUND: Cell‐based influenza vaccines can solve the problem of the frequent occurrence of egg adaptation–associated antigenic changes observed in egg‐based vaccines. Seed viruses for cell‐based vaccines can be prepared from clinical specimens by cell culture; however, clinical samples risk harboring respiratory viruses other than influenza virus. Therefore, it is necessary to investigate the patterns of co‐infection in clinical samples and explore whether cell culture technology can selectively propagate influenza viruses from samples containing other respiratory viruses. METHODS: A total of 341 clinical specimens were collected from patients with influenza or influenza‐like illness and analyzed by ResPlex II assay to detect 18 respiratory viruses. The patterns of co‐infection were statistically analyzed with Fisher's exact test. The samples with double or triple infections were passaged in suspension MDCK cells (MDCK‐S), adherent MDCK cells (MDCK‐A), and LLC‐MK2D cells. Cell‐passaged samples were analyzed by ResPlex II assay again to investigate whether each cell line could amplify influenza viruses and eliminate other respiratory viruses. RESULTS: Double infections were detected in 8.5% and triple infections in 0.9% of the collected clinical specimens. We identified four pairs of viruses with significant correlation. For all samples with double and triple infection, MDCK‐S and MDCK‐A could selectively propagate influenza viruses, while eliminating all contaminating viruses. In contrast, LLC‐MK2D showed lower isolation efficiency for influenza virus and higher isolation efficiency for coxsackievirus/echovirus than MDCK‐S and MDCK‐A. CONCLUSIONS: Both MDCK‐S and MDCK‐A are considered suitable for the preparation of influenza vaccine seed viruses without adventitious agents or egg‐adaptation mutations. Influenza virus is highly transmissible and causes mild to severe illness, including high fever, headache, myalgia, and pneumonia. Annual influenza epidemics worldwide cause approximately three to five million cases of severe illness and 290 000-650 000 deaths every year, resulting in a great social impact. 1 The economic burden of influenza has been estimated to be $47. The MDCK cell line was established from the kidney of a healthy cocker spaniel dog in 1958 and has a long history in the studies of influenza viruses. The conventional MDCK cell with adherent growth (MDCK-A) is a good candidate for the preparation of vaccine seed viruses, since it supports efficient growth of human influenza viruses. 10, 11 The MDCK33016PF suspension cell line, designated as MDCK-S in this paper, was first developed and utilized to produce seasonal influenza vaccines. [11] [12] [13] Suspension cells are superior to adherent cells owing to the following advantages: simpler culture process without micro-carrier beads, lower cost, and higher virus yield. Therefore, MDCK-S could be a suitable substrate for influenza vaccine seed preparation. LLC-MK2, established from the kidney of a healthy rhesus monkey, has also been used to propagate a variety of viruses, including the influenza virus. 14-16 LLC-MK2D, which is a sub-line of LLC-MK2, was proven to be non-tumorigenic in nude mice and free of specific adventitious agents. This cell line could be a promising candidate for the preparation of vaccine seed viruses, since its safety is confirmed and it can be used in practical applications with little delay. In this study, we analyzed the pattern of co-infection of respiratory viruses in clinical specimens and evaluated the ability of MDCK-S, MDCK-A, and LLC-MK2D cells to propagate influenza viruses while eliminating other respiratory viruses. To examine if there were specific patterns of correlation between the detected viruses, all virus pairs in Table 1 were evaluated with (Table 4) . CVEV, RHV, RSVA, RSVB, OC43, and NL63 were In this study, using the ResPlex II assay, we analyzed the specific pat- its low ability to multiply influenza viruses and to eliminate the contaminating viruses. One of the limitations of this study is that the sample size of the multiple infection groups was small. Larger sample sizes will make it possible to identify more correlations among viruses and to test The use of MDCK-S, MDCK-A, and other cell lines could contribute to global public health through the rapid production of safe and effective cell-based vaccines. We thank the staff at the National Institute of Infectious Diseases, Estimates of global seasonal influenza-associated respiratory mortality: a modelling study Annual estimates of the burden of seasonal influenza in the United States: a tool for strengthening influenza surveillance and preparedness. 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