Development of Maintenance Medium Using for Influenza Virus Production and Analysis of Cellular Metabolic Characteristics

doi:10.13560/j.cnki.biotech.bull.1985.2019-0056

Abstract

Abstract: This study developed a maintenance medium supporting the influenza virus production to high yield based on MDCK cell culture and using Simplex Lattice design,and studied the cellular metabolic characteristics during the virus infection under the maintenance medium,meanwhile the cell growth medium was as a control group. The maintenance medium was composed of A and F medium in a ratio of 0.586∶0.432 by the statistical analysis,in which the maximum virus titer was 2^14.062 HAU/100 μL in the 2 L bioreactor,which was 59.55% higher than that of the control group. When the initial concentrations of glucose were the same,the cells in the maintenance medium group enhanced the glucose uptake and the glycolysis pathway,the consumption rate of serine,arginine and isoleucine that were conducive to virus proliferation significantly increased,and the production rate of alanine inhibiting virus proliferation greatly decreased,compared with the control group. Therefore,this work emphasized the importance of maintenance medium development;meanwhile the results provide a methodological reference and theoretical guidance for the development and optimization of other viral vaccine media.

Key words: influenza virus, maintenance medium, MDCK cells, metabolism

XIE Pan, YE Qian, LIU Xu-ping, TAN Wen-song. Development of Maintenance Medium Using for Influenza Virus Production and Analysis of Cellular Metabolic Characteristics[J]. Biotechnology Bulletin, 2019, 35(5): 133-139.

References

[1] Youil R, Su Q, Toner TJ, et al.Comparative study of influenza virus replication in Vero and MDCK cell lines[J]. Journal of Virological Methods, 2004, 120(1):23-31.
[2] Audsley JM, Tannock GA.Cell-based influenza vaccines:Progress to date[J]. Drugs, 2008, 68(11):1483-1491.
[3] Mabrouk T, Ellis RW.Influenza vaccine technologies and the use of the cell-culture process(cell-culture influenza vaccine)[J]. Dev in Biol, 2002, 110(1):125-134.
[4] Keen MJ, Rapson NT.Development of a serum-free culture medium for the large scale production of recombinant protein from a Chinese hamster ovary cell line[J]. Cytotechnol, 1995, 17(3):153-163.
[5] Rourou S, van der Ark A, van der Velden T, et al. Development of an animal-component free medium for vero cells culture[J]. Biotechnology Progress, 2010, 25(6):1752-1761.
[6] Merten OW.Development of serum-free media for cell growth and production of viruses/viral vaccines--safety issues of animal products used in serum-free media[J]. Dev in Biol, 2002, 111:233-257.
[7] Tapia F, et al.Production of high-titer human influenza A virus with adherent and suspension MDCK cells cultured in a single-use hollow fiber bioreactor[J]. Vaccine, 2014, 32(8):1003-1011.
[8] Ru AL, Jacob D, Transfiguracion J, et al.Scalable production of influenza virus in HEK-293 cells for efficient vaccine manufacturing[J]. Vaccine, 2010, 28(21):3661-3671.
[9] Lorizate M, Kräusslich HG.Role of lipids in virus replic-ation[J]. Cold Spring Harb Perspec Biol, 2011, 3(10):1-20.
[10] Lohr V, Rath A, Genzel Y, et al.New avian suspension cell lines provide production of influenza virus and MVA in serum-free media:Studies on growth, metabolism and virus propagation[J]. Vaccine, 2009, 27(36):4975-4982.
[11] Kalbfuss B, Knöchlein A, Kröber T, et al.Monitoring influenza virus content in vaccine production:Precise assays for the quantitation of hemagglutination and neuraminidase activity[J]. Biologicals, 2008, 36(3):145-161.
[12] Bock A, Schulze-Horsel J, Schwarzer J, et al.High-density microcarrier cell cultures for influenza virus production[J]. Biotechnology Progress, 2011, 27(1):241-250.
[13] Wang H, Guo S, Li Z, et al.Suspension culture process for H9N2 avian influenza virus(strain Re-2)[J]. Archives of Virology, 2017, 162(10):3051-3059.
[14] 李春艳, 肖晶, 李曦, 等. 微载体规模比培养MDCK细胞增殖H9N2亚型禽流感病毒的研究[J]. 中国人兽共患病学报, 2009(12):1149-1153.
[15] Fontaine KA, et al.Dengue virus induces and requires glycolysis for optimal replication[J]. J Virol, 2015, 89(4):2358-2366.
[16] Ritter JB, Wahl AS, Freund S, et al.Metabolic effects of influenza virus infection in cultured animal cells:Intra- and extracellular metabolite profiling[J]. BMC Syst Biol, 2010, 4(1):61-83.
[17] Huang D, Xia-Hou K, Liu XP, et al.Rational design of medium supplementation strategy for improved influenza viruses production based on analyzing nutritional requirements of MDCK Cells[J]. Vaccine, 2014, 32(52):7091-7097.
[18] Genzel Y, Behrendt I, et al.Metabolism of MDCK cells during cell growth and influenza virus production in large-scale microcarrier culture[J]. Vaccine, 2004, 22(17-18):2202-2208.
[19] Saito K, Nishijima M, Kuge O.Phosphatidylserine is involved in gene expression from Sindbis virus subgenomic promoter[J]. Biochem Biophys Res Commun, 2006, 345(2):878-885.
[20] Kaushik R, Ratner L.Role of human immunodeficiency virus type 1 matrix phosphorylation in an early postentry step of virus replication[J]. Journal of Virology, 2004, 78(5):2319-2326.
[21] Lohr V, Genzel Y, Behrendt I, et al.A new MDCK suspension line cultivated in a fully defined medium in stirred-tank and wave bioreactor[J]. Vaccine, 2010, 28(38):6256-6264.
[22] Chen P, Harcum SW.Effects of amino acid additions on ammon-ium stressed CHO cells[J]. J Biotechnol, 2005, 3:277-286.