Establishment And Evaluation Of PK-15Cell Stocks And Virus Seed Banks For Chimeric PCV1-2Inactivated Vaccine Production

Porcine circovirus type2(PCV2) is the primary causative agent and a recently recognized pathogen of postweaning multisystemic wasting syndrome (PMWS) and other associated diseases. PMWS is initially recognized in weaning piglets of a high-health-status Canadian herd in1991, since then PMWS has become an economically important disease in virtually all regions of the world where pigs are produced. Clinical signs of Porcine Circovirus-Associated Disease (PCVAD) include wasting with progressive weight loss, lethargy, diarrhea lymphadenopathy, and jaundice.At present, effective vaccines which can effectively control PCV2infection are researched at home and abroad. Some vaccines have been put into clinical experiment and evaluation of immune effect. The inactivated chimeric PCV1-2developed by our laboratory could induce protective immunity against PCV2infection effectively.The purpose of this study was to establish three cell banks used for production of the inactivated chimeric PCV1-2, and ensured safety of PK-15cells as substrates of virus cultivation. At the same time, virus seed banks was also established to provide the seeds for vaccine manufacture.1Establishment of cell stocks of PK-15cell line and evaluation of the cell line as candidate viral substrates for approval production of the inactivated chimeric PCV1-2vaccineEstablishment of three cell banks used for production of the inactivated chimeric PCV1-2ensures safety of PK-15cells as substrates of virus cultivation. A series of tests including karyotypic analysis test and neoplasia test etc. were carried out according to the Chinese Regulations for Biologics Production (2002edition). The result showed that the number of PK-15cell chromosome was2n=38. Chromosome models in foundation cell bank also lied in maximum limit generation cells, and compared to foundation cell bank, the chromosome pattern number of other cells was lower than10%and the karyotype was same. Respectively passaged to passage#45, PK-15cells in three cell stocks didn’t display oncogenicity in nude mice and were not contaminated with any bacteria, fungi, mycoplasma and exogenous viruses, and reached to the requirements of the Chinese Regulations for Biologics Production (2002edition). PK-15cells stored at my institute can be used as cell bank producing the inactivated chimeric PCV1-2vaccine.2Establishment and evaluation of PCV1-2seed banksPCV1-2seed banks for vaccine maufacturing were established in order to ensure quality and stabilization of the vaccine. Three virus seed lots including the primary seed lot, the master seed lot and the working seed lot were developed. Titers and specificity of the virus seeds were respectively examined by indirect immunofluorescence (IFA). A series of tests including bacteria, fungus, mycoplasma and exogenous virus test were carried out according to the Chinese Veterinary Pharmacopeia (2005, the third edition). PCV1-2seeds were not contaminated with any bacteria, fungi, mycoplasma and exogenous viruses. Virus titers reached to more than1044.0TCID50/mL. The results were in accordance with the requirements of regulations of biologics. PCV1-2seed banks established at my institute can be used as the pure seeds producing the inactivated chimeric PCV1-2vaccine.