Suspension Culture Of H5,H7 Subtype Avian Influenza Virus And Evaluation Of Its Effect On Bivalent Inactivated Vaccine

Avian Influenza（AI）is a zoonosis caused by the type A avian influenza virus（AIV）that seriously endangers the poultry industry and human public health.Vaccination is an important method to prevent the disease.The traditional influenza vaccine production adopts chicken embryo culture antigen which has many defects,a large number of chicken embryo are required,and there is a risk of contamination during the antigen cultivation process and the cost are high.The use of cell suspension culture technology can not only reduce vaccine production costs,but also has the advantages of high antigen titers and easy mass production.In this study,the H5 and H7 subtypes of Avian Influenza vaccine strains were screened by MDCK cell suspension culture.The process parameters for large-scale suspension culture of virus antigens were obtained,and bivalent inactivated vaccines for H5and H7 subtypes were prepared,and their immune effects were evaluated.The results are as follows.For serum-free suspension culture of MDCK cells,cell density,cell viability and specific cell growth rate were calculated at different time points,and the cell growth curve was plotted.It was found that the density of living cells peaked at 96 h after cells were passaged,which was approximately 14.3×10⁶cells/m L,after that,cell vacuoles,cell death,and cellular debris of varying degrees were observed,the density of living cells decreased continuously.During the entire cell growth cycle,the highest cell viability can reach 98%,and the maximum specific growth rate is about 0.89d^-1.After the cells were inoculated with the virus,the HA titer of the harvested antigen was tested to further investigate about the optimum amount of virus,the optimum temperature,the optimum pH,the optimal time for virus collection,and the optimal concentration of trypsin for virus propagation.The results showed that when the cell density reached 2x10⁶cells/m L,the MOI was 0.01,the culture temperature was 33°C,the pH was about 7.4,the TPCK-trypsin concentration was 5μg/m L,and the virus solution was harvested 72h after inoculation,the two viruses could reach the maximum HA titer.The two viruses were serially passaged on MDCK suspension cells for 20 generations under optimum conditions,and the HA titer of each generation was tested,The H5 subtype AIV（r D8 strain）between 6～8 log2,H7 subtype AIV（r GD76 strain）between 6～9 log2.The virus titer of each generation was tested separately,the r D8 strain can reach a maximum of6.77 log₁₀TCID₅₀,and the r GD76 strain can reach 7.24 log₁₀ TCID₅₀.The whole genome sequence of the viral fluid harvested after the passage training of the two viruses was compared with that of the primary virus and the results showed that the nucleotide sequence homology of each gene of r D8 strain is 99.3%or more,and the amino acid sequence homology above 99.8%,there is a mutation of HA protein at position 446（W446C）and there is a mutation of NA protein at position 415（R415G）,the remaining amino acid mutations occur on PB1 and PA.The nucleotide sequence homology of each gene of r GD76 strain was 99.0%or more,and the amino acid sequence homology was 99.8%or more,and amino acid mutations only occurred on PB1 and PB2.The 9th generation virus liquid was selected to prepare inactivate bivalent vaccines of oil emulsions after two strains were subcultured and immunized with 3 weeks old SPF chickens,blood sampling was performed 21 days after the immunization.Among them,HI antibody level of H5 subtype up to 9 log2,geometric mean was 8.0 log₂,and H7 subtype up to 10 log₂,geometric mean was 8.4 log₂.Inoculation test for immunized chickens with the test strains of H5 and H7 subtype vaccine,all the chickens in the control group died,and all the chickens in the immunization group were protected,it is proved that the bivalent vaccine prepared from the harvested antigen solution in this experiment has a good immune protective effect.