Development Of Thermo-stable Live Vaccine For Highly Pathogenic Porcine Reproductive And Respiratory Syndrome

Highly pathogenic porcine reproductive and respiratory syndrome(HP-PRRS)is one of the most important diseases affecting the swine industry in China.Immunization with attenuating live virus vaccine is a main method to control PRRS.Nowadays,the domestic attenuating live virus vaccine of HP-PRRS is generally produced by rotary bottle technology and combined with common lyophilized protective agent.To study the JXA1-R strain of HP-PRRSV.researches on virus suspension culture technology and thermo-tolerance protector agent are carried out and the attenuating live virus vaccine of the HP-PRRS was developed(named as HP-PRRS live vaccine).Research of HP-PRRSV JXA1-R strain culture on Marc-145 cell-line was carried out with BC-7L bioreactor and the progress parameters including concentration of the micro-carrier,the quantity of the cell feeds,the initial parameter of the agitator,the growing parameter of the agitator and the method of culture media feeding all are optimized.The peak quantity of the suspension cell of Marc-145 may reach 2～3×106cells/ml under the technology of cell micro-carrier suspension culture.Parameters including the inoculating period of the virus feeds of the HP-PRRSV JXA1-R strain,the dose of the virus feeds,the maintaining serum,the parameter of dissolved oxygen and the temperature of the virus reduplication are optimized and the culture technology of HP-PRRSV JXA 1-R strain is conformed.The tilters of antigen acquired are all higher than 108.0TCID50/ml under the following technology of 0.1 MOI of virus feeds,maintaining medium with 2%new born calf serum,40%dissolved oxygen parameter,culture temperature at 37 centi-degree and harvest period from 70 to 74 hours after the virus inoculation.The magnification and semi-works production the technology of the HP-PRRSV suspension culture are carried out and the results showed that the JXA1-R strain virus of HP-PRRSV may reduplicate at a high quantity in BSISL-42L and BC-100L bioreactors.The tilters of three patches of antigen acquired from the BC-100L bioreactor are all higher than 108.0TCID50/ml;Three patches of semi-products are produced according to the formula B of thermo-tolerance protector agent and combination The fourth kind of freezing curve and all related tests are qualified.The results of safety and efficacy tests with three patches of HP-PRRSV live vaccine showed that they are all safe and may provide efficient protection for the piglets of 14 and 28 days-old which challenged post 28 days.4-6 weeks pigs are administered with HP-PRRSV live vaccine and 28 days later challenged with HP-PRRSV strain which isolated between 2010-2012.The results showed that the HP-PRRSV live vaccine provided effective protection.Three patches of the HP-PRRSV live vaccine were preserved in 2-8 centi-degree for 21monthes and then all related quality tests are carried out.The results showed that they are all qualified and the preservation period of the vaccine is determined as 2-8 centi-degree for 18 month.The difference on safety and efficacy between the vaccine produced in the research and those in market are compared.The results showed that no apparent difference was found.Studies of the influence of the HP-PRRSV live vaccine on classical swine fever live virus vaccine are carried out,the results showed that no outstanding difference was found on the generation and level of antibody against classical swine fever virus in the overdose immunization and single dose immunization group compared with blank group.Thus,the technology of suspension culture and thermo-tolerance freezing of HP-PRRSV is developed in this research and a safe,effective and quality controlled HP-PRRSV live vaccine combined with thermo-tolerance protector agent is produced which may be widely used in HP-PRRS disease control in China.