Research On Production Technology Of Highly Pathogenic Porcine Reproductive And Respiratory Syndrome Virus Vaccine

Highly pathogenic porcine reproductive and respiratory syndrome (HP-PRRS), also named "Highly pathogenic porcine blue ear disease", is a high deadly infectious swine disease which was triggered by mutative PRRS Virus. The main difference of HP-PRRS and PRRS is that the NSP2protein of HP-PRRSV has30amino acid deletions. The main clinical manifestations of HP-PRRS include high fever, high morbidity and high mortality. Since2006, the HP-PRRS has brought a great loss to the swine industry in China. Currently, there are no effective drugs against the disease. Its prevention and control mainly rely on vaccination.Live vaccine accounted for75%vaccines market in China, mainly because of its strong immunogenicity, short producing period, and good immune responses for a longer time. Virus titre and immunogenicity are main properties of live vaccine. Veterinary vaccine industry in China mainly uses bottle cultivation mode to produce vaccine, but disparity between different batches and unstable quality are common problems for vaccine production. To ensure the quality of the vaccine, manufacturers must optimize the vaccine production process. This study is mainly concerning the production process for highly pathogenic porcine reproductive and respiratory syndrome virus vaccine, through screening and establishing the seed bank, and optimizing the culture conditions of Marc-145cells and the microcarrier culture conditions of Marc-145cell. It can make a sold foundation for PRRSV vaccine production.The cell lines and master seeds with good adaptability were screened by cell passages, inoculation and potency test. We successfully established a seed cell bank, a seed virus bank, and obtained18strains of frozen seed cells and15strains of seed virus, which had high safety and pureness after identification. And the regression experiment showed that the advantages of the seed cells and the seed virus could be reproduced, whcih played a role in the screening and in the building banks. All of these laid good foundations for the optimization of production process.Through the contrast test and orthogonal experiment, we analyzed the effects of various factors on the culture systems of roller bottle. The experimental results showed that high-glucose DMEM, and8%-10%FBS in the growth media were more suitable for the growth of Marc-145cells, and moreover, they also indicated that virus propagated better with rotating speed lOr/h, temperature37.5℃,2%FBS, pH7.3. Appropriate adjustment would make the production process stable and the optimization effect obvious.Finaly, Marc-145cell culture and virus inoculation were successfully achieved using microcarrier culture technique. The best cell inoculation density was found to be2.6×105cells/ml. The results showed that the cell density through this way was2.1times higher than the roller bottle culture, and virus titers through this way was0.46times higher than roller bottle culture. This would provide data and information for the upgrading of large scale production technology for PRRSV vaccine.