Abstract: 【Objective】This study provided data support for assessing the effectiveness of cell-derived reassortant influenza virus H5 subtype inactivated vaccines.【Method】 A clearly defined MDCK cells was selected to be domesticated to suspend MDCK cells, and the reassortant avian influenza virus H5N1 Re-6 strain, Re-7 strain and Re-8 strain were proliferated in it. The proliferation differences of different strains in the adherent and suspended MDCK cells were compared. The reassortant avian influenza virus (H5 subtype) trivalent inactivated vaccine were prepared by suspended MDCK cells and by SPF chicken embryo, respectively, which were immuned in commercial chicken and commercial duck. Immune effects were compared between cell source and chicken embryo source by serological methods. 6 050 Hy-line brown laying hens were divided into 3 groups: 2 groups were immunized groups, 3 000 hens in each group, immunized 0.5 ml/head at 28 days of age and 80 days of age; 1 non-immunized control group, 50 hens were reared under the same conditions. The serums were collected from the layers at 49 days, 110 days, and 210 days (ie, 21 days, 82 days, and 6 months after the first immunization) to determine the reassortant avian influenza virus H5 subtype Re-6 strain, Re-7 strain and Re-8 strain, The HI antibody titer was monitored. 220 Changbai flying ducks were divided into 3 groups: 2 groups were immunized groups, 100 ducks in each group, immunized 0.5 ml/head at 10 days of age, immunized 1.0 ml/head at 24 days of age; 20 ducks in non-immunized control group, under the same conditions. The serums were collected at 24, 38, and 52 days (ie, 14 days, 28 days, and 42 days after the first immunization) to determine the reassortant avian influenza virus H5 subtype Re-6 strain, Re-7 strain and Re-8 strain, and the HI antibody titer was monitored. 【Result】A MDCK cell that could be grown in serum-free medium was obtained. Culture data, cell status in shake flasks and 5L bioreactors showed that the cell line was suitable for scale-up production. The cell density was cultured from 1.5×10⁶ cells/ml to 1.0×10⁷ cells/ml in 48-hours. Under the good condition, the cells had a high activity rate, and were individually suspended in the medium. The H5N1 reassortant avian influenza virus were produced in the suspension MDCK cells, Re-6 strain: HA titer 1﹕512, TCID50 10^7.67/mL, EID50 10^7.83/0.1mL; Re-7 strain: HA titer 1﹕512, TCID50 10^7.33/ml, EID50 10^7.17/0.1mL; Re-8 strain: HA titer 1﹕1024, TCID50 10^8.5/mL, EID50 10^8.38/0.1mL. The results was similar to that of the adherent MDCK cells. Cell-derived trivalent inactivated vaccine was used to immunize y-line brown laying hens after 21 days of the first immunization, and the average HI antibody titer of the avian influenza virus H5 subtype strain Re-6, Re-7 and Re-8 in serum was 1﹕446, 1﹕111, and 1﹕416. The average HI antibody titer of the three groups was 1﹕588, 1﹕362, and 1﹕776 at 30 days after secondary immunization, and they were 1﹕239, 1﹕128, and 1﹕223 at 6 months, maintaining high antibody levels. Changbai flying duck were immured, after 14 days of the first immunization, the average HI antibody titer of the avian influenza virus H5 subtype strain Re-6, Re-7 and Re-8 in serum was 1﹕30, 1﹕17, and 1﹕64. The average HI antibody titer of the three groups was 1﹕194, 1﹕91, and 1﹕137 at 28 days, and they were 1﹕416, 1﹕128, and 1﹕239 at 42 days. The experimental results of the above two groups were equivalent to those derived from chicken embryo.【Conclusion】A domesticated MDCK cell line that could be grown in serum-free medium, and were used as host cells to produce reassortant avian influenza virus. Its ability to proliferate reassortant avian influenza virus H5N1strain Re-6, Re-7 and Re-8 was strong. Viruses that were prepared as inactivated vaccines to immunize y-line brown laying hens and Changbai flying duck produced higher levels of antibodies. This study provided technical support for large-scale industrial production of the avian influenza vaccine.

Key words: MDCK cells, reassortant avian influenza virus, suspension culture, immune effect