Study On A New Type Of Microcarrier Suspension Culture Of Classical Swine Fever Virus

Classical swine fever is a fever,acute and highly contagious infectious disease caused by classical swine fever virus(CSFV).With a high mortality rate,pigs of different ages,sexes and breeds can be infected by CSFV throughout the year.At present,there is no effective drug or treatment for this disease.Vaccination with high quality vaccine is still one of the most effective means for the prevention and control of classical swine fever(CSF).Therefore,the vaccine quality and preventive effect have become the key factors to determine the results of the epidemic prevention of CSF.From years of immunization and market application effect,the HCLV strain is excellent in safety and effectiveness,and has become the world recognized standard live strain of swine fever vaccine.Traditionally,rabbit-derived tissue vaccine and primary cell vaccine have been the main vaccines against classical swine fever,including rabbit-derived spleen lymph live vaccine and primary cell vaccine.Many shortcomings of traditional vaccines have become difficult problems in large-scale production of classical swine fever vaccine.With the successful development and application of ST cell-derived live CSFV vaccine,these technical problems will be solved easily.Because of the long-term universal application of roller bottle in large-scale cell culture,new problems affecting the quality of classical swine fever vaccine have also arisen,such as large differences between batches,poor quality stability,low antigen titer,poor immune effect,and large adverse reactions.Using developed independently new microcarriers and bioreactor technologies in this project,ST cells were cultured in large scale and high density so that stable and homogeneous CSFV antigen could be harvested.As a result,all kinds of difficulties in traditional technology process were fundamentally solved.Moreover,by optimizing the technical conditions of cell culture in the bioreactor,increasing the CSFV titer and stabilizing the virus production process,a set of perfect large-scale production technology of classical swine fever virus was established,which would lay a good technical foundation for the production of stable and efficient classical swine fever vaccine.Therefore,the content of this research mainly includes the following five parts.Part ?:Study on the characteristics of ST cell monolayer culture and CSFV roller culture technology.Through the study of the culture characteristics of ST cell line,we could know that the cell line grew fast and the cell state was good.The cell doubling time(DT)was 16.1 hours,and the density of ST cells was 7.6×10~5 cells/mL at 72hours,which showed a strong growth ability.After 48 hours of cell growing,CSFV seed cytotoxicity was inoculated at 4%(v/v)inoculation volume.The CSFV did not cause ST cells characteristic CPE.In terms of daily sugar consumption of cell,the level of sugar consumption in CSFV infected cells was slightly higher than that in normal cells.Virus harvesting started on the 5th day after inoculating CSFV,and lasted 5 times in total.Except for the first harvesting,the viral titers of other harvest were all 500,000 RID/mL.The results showed that CSFV could proliferate normally on the ST cell lines used in this study,which provided a good precondition for CSFV large-scale culture.Part ?:Study on the cell culture characteristics of microcarriers and selection of microcarriers.In this study,the biocompatibility of Cephodex and Cephodex D microcarriers and their adaptability to ST cells were investigated in terms of the percentage of cells attaching to microcarriers,anchorage time and maximum cell density on microcarriers.According to the effect of culturing ST cells at the stage of anchorage microbeads,the anchorage time of ST cells on Cephodex D microcarriers was 2 hours,and the anchorage rate was 92.7%.The cells on the surface of CephodexD microcarriers showed good spreading ability in a short time.ST cells on Cephodex carrier had an anchorage time of 6 hours and an anchorage rate of 62.3%.Moreover,the cellular spreading ductility on the Cephodex surface was poor.With the ST cell density on Cephodex D of 1.9×10~6 cells/mL and density on Cephodex of 1.3×10~6 cells/mL on 72 hours,the difference between two kinds of microcarriers was about 50%from the point of view of the maximum cell density.ST cells could survive for more than 7 days on Cephodex D microcarriers without replacing the culture medium,but less than 5 days on Cephodex microcarriers.A series of studies had shown that Cephodex D microcarrier was superior to Cephodex in biocompatibility and ST cell adaptability,which better adapted the technical requirements of large-scale CSFV culture.Part ?:Study on shaking flask culture of CSFV microcarrier and its amplification technology.In this study,the shaking flask culture method was used to study the technology of new microcarrier cell culture.Detailed conditions were explored,such as the usage and dosage of microcarrier,cell inoculation and methods on suspension culture of microcarrier,sugar consumption and viral titer.The microcarrier dosage of 4 g/L,the inoculation time of CSFV for 48 hours in cell culture and the amplification ratio of 1:5 were all preliminarily determined.The maximum cell culture density of 1.86×10~6 cells/mL can be achieved by the intermediate process feeding technique.The titer of CSFV could reach 750,000RID/mL by viral infection after ST cells growth of 48 hours,which was slightly higher than the roller bottle process.Scale-up culture at the ratio of 1:5 not only reduced the work load of seed preparation,but also raised the feasibility of the operating process,and ensured finally the cell culture scale.Part ?:Suspension culture of CSFV with new cell microcarriers in bioreactor and establishment of amplification technology.In this part,the large-scale suspension culture of ST cells was studied with bioreactor and CephodexD,on which based,a set of perfect production technology of CSFV was established.Referring to the technical data of shaking flask culture method,the scale-up process of suspension culture with microcarriers in bioreactor was completed from 3 L to 15 L.Trypsin two-step digestion method was used to complete the cells removing from microcarriers and the separation of cells each other.The maximum cell density was 2.93×10~6 cells/mL when ST cells were cultured for 72 hours in the bioreactor.After ST cells was cultured for48 hours,CSFV strains were inoculated at a volume ratio of 4%(v/v).From the 4th day after CSFV infection,the virus was harvested once every 3 days,and the end was the 5th harvesting.The viral titer was 1 million RID/mL,twice as much as that of the roller-bottler process.The whole process lasted 16 days,which was 5 days shorter than the roller-bottle process.The results showed that the suspension culture technology of the new microcarrier&bioreactor of CSFV not only greatly improved the production efficiency,but also improved the antigen quality.This also laid a good technical foundation for the industrialization production of high quality CSFV ST cell-derived live vaccine in the future.Part ?:Immunogenicity analysis and evaluation of immune effect.In this part,the CSFV produced by the new microcarrier and bioreactor technology was inoculated into the experimental pigs,whose blood was collected 14 days later.ELISA test showed that the serum of experimental pigs contained a high level of CSFV antibody.The antigen specificity of CSFV cultured in the reactor was studied by IFA method.In the vision of fluorescence microscope,the cytoplasm of ST cells infected with CSFV showed obvious granular or diffuse fluorescence,which was mainly concentrated in the cytoplasm.However,the non-infected ST cells had no fluorescence.These studies showed that CSFV cultured in the bioreactor had perfect immunogenicity and immunoreactivity.Meanwhile,the CSFV produced by the new technology was inoculated into the five experimental pigs and the five control pigs.After 14 days,challenge of CSFV was carried out.In the next 16 days,the immune pigs had no obvious clinical symptoms and no obvious fever reaction,and the protection rate was 100%.The control group had obvious clinical symptoms,fever and death.The results showed that CSFV antigen produced by the suspension culture technology of new microcarriers had perfect immune protection effect.