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Changchun, China

Xie Y.-L.,Jilin Yatai Group Co. | Zhou Q.-L.,Jilin Yatai Group Co. | Wang W.-J.,Jilin Yatai Group Co. | Yue Z.-Q.,Jilin Yatai Group Co. | And 6 more authors.
Chinese Journal of Biologicals | Year: 2015

Objective: To optimize the condition for culture of influenza H5N1 virus in Vero cells. Methods: Influenza virus was cultured in various maintenance media [M199, M199 + epidermal growth factor (EGF), MEM, MEM + EGF and serum-free medium (SFM)] and M199 medium containing various concentrations of trypsin (3, 5 and 7 μg/ml) respectively, of which the culture supernatants were collected and determined for hemagglutinin (HA) titer to optimize the concentrations of maintenance medium and trypsin. Vero cells were infected with influenza H5N1 virus-infected cell supernatant before and after freezing and thawing and subcultured, of which the supernatant was collected and determined for HA titer to evaluate the effect of freezing and thawing on vims propagation. Vero cells were inoculated with influenza H5N1 virus at a MOI of 0.01 and cultured under the optimized condition, of which the culture supernatant was collected and determined for HA titer and infectious titer to evaluate the stability of H5N1 virus after continuous subculture in Vero cells. Results: When the SFM containing 5 μg/ml of trypsin was used, and the harvest was infected to Vero cells directly and subcultured for two passages before the maintenance medium was substituted with M199 medium, the HA titer of influenza H5N1 virus reached the maximum. Both the HA and infectious titers of H5N1 virus were stable after subculture in Vero cells under the optimized condition. Conclusion: Influenza H5N1 virus was subcultured continuously and stably in Vero cells under the optimized condition, which laid a foundation of establishment of working seed lot for the preparation of pandemic influenza vaccine.

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