Preparation Of High-Growth Influenza B Virus In Vero Cell By Reassortant Methods And Optimization Of Conditions For Serum-free Culture Of Vero Cells And Influenza Virus On Microcarriers

Influenza (the flu) is acute respiratory infectious disease caused by influenza virus. Influenza virus can break out and cause a spread in a short time, and can cause disease among people at all ages. Influenza vaccination is the most effective way of the prevention and control of influenza. Traditionally chicken embryo is used as substrate to produce influenza vaccine. The way has many disadvantages. It is difficult to supply a lot of chicken for the production of vaccine in a short time. Mammalian cells can produce vaccines with great advantage. The cell can grow faster and quality control is easier. Meanwhile the vaccine can be produced in large-scale. Vero cells are recommended as a good substrate by WHO for vaccine production. Serum-free medium is a synthetic culture media that is not necessary to add serum to maintain the growth and reproduction of cells in vitro. That overcame the potential pollution sources and avoided shortcomings to downstream separation and purification. In addition, microcarriers cell culture can increase the yield of cells and virus. That makes mass production possible. In this study, firstly we use the traditional method of influenza virus recombination to obtain a Vero cell-adapted strain of influenza virus. And serum-free medium is used for microcarriers culture of Vero cells and influenza virus in stirring flask. Through a series of optimization of culture conditions, the optimum conditions of serum-free medium for microcarrier culture of Vero cells and influenza virus were selected.Ⅰ. Preparation of High-Growth Influenza B Virus in Vero Cell by Resorting MethodCo-infect chick embryo and Vero cells with Vero cell-adapted strain of influenza virus B/Yunnan/02/2005Va(B) as the donor strain, and the vaccine strain from 2006 to 2008 B/Malaysia/2506/2004 recommended by WHO. With antiserum of B/Yunnan/02/2005Va(B), ressortant virus was selected in Vero cells and was obtained after 10 passages in Vero cells. Reassortant influenza virus was purified and propagated in Vero cells until the 8th passage. The virus type was identified by using hemagglutination inhibition test, immunofluorescence assay, one-way immune agar diffusion test and RT-PCR. Finally a Vero cell adapted B-type influenza virus strain B/Malaysia/2506/2004Va(B) was obtained.Ⅱ.Optimization of Conditions for Serum-free Culture of Vero Cells and Influenza B Virus on MicrocarriersInfluenza B virus was propagated in Vero cells on mirocarriers in serum-free medium in stirring flask at various cell seeding densities and different concentrations of mirocarriers. Vero cells were infected at various MOI, various pH values and TPCK-trypsin concentrations. Moreover, culture time for the virus and method of harvesting the virus were also to be considered. Based on the hemagglutination(HA) titer of virus. Results indicated that the best cell seeding densities for culture of Vero cells were 1～5×105/ml, and the optimal pH value and MOI were 7.2 and 1.0 respectively. After 3 days culture, the virus was harvested with cell ultrasonic method, and hemagglutination titer is 1:512. Thus the condition for serum-free culture of Vero cells and influenza virus on microcarrier was optimized. Inactivated vaccine was prepared and mice were immunized with vaccine. The difference of immunogenicity was not statistically significant, compared with parental strain.