Isolation Of Porcine Circovirus 2 In Henan Province And Study On The Immunogenicity Of Screened Strain On Mouse

Porcine circovirus type 2,a member of the Circoviridae family,is a small,non-enveloped,single-strand circular DNA virus.The whole genome is 1766-1 768bp.There are two main Open Reading Frames in the anti-sense orientation with ORF1 encodes replication proteins and ORF2 encodes capsid proteins.PCV2 has been an epidemic in domestic pigs and wild boars all around the world,and caused a series of PCV2 associated disease syndromes named Porcine Circovirus Associated Disease(PCVAD),such as Postweaning Multisystemic Wasting Syndrome(PMWS),Porcine Dermatitis and Nephropathy Syndrome(PDNS)and Reproductive disorders.This brought a dramatic impact on pig-raising industry.Vaccine immunity is still the most effective method to prevent PCV2 infections.In order to get an excellent PCV2 strain,samples suspiciously infected with PCV2 were collected from some regions in Henan province and detected by PCR.13 PCV2 positive samples were inoculated into non-PCV contaminated PK-15 cell and further identified by immunoperoxidase mono-layer assay.PCR products of complete genomes of PCV2 isolates were cloned and sequenced.The complete genome nucleotide sequences of these PCV2 isolates were compared with PCV2 isolates published previously in GenBank using DNAStar software.The length of the complete genomic sequences of the 13 PCV2 isolates was 1 767 or 1768 bp.Comparison analysis of the complete genomic sequencess showed that they were closely related to each other(94.7%-99.9%).Comparisons of the complete OFR2 nucleotide sequence revealed 89.7%-100%homology within the 13 isolates.Out of the 13 isolates,10 were subtype PCV2b-1C,2 were subtpe PCV2b-1A,only one was genotype PCV2a.None PCV2c was discovered.All the results indicated that PCV2b-1c is the predominant subtype in Henan.The 13 PCV2 strains were inoculated into PK-15 cell and proliferated.Virus titers were detected by IPMA and strain DF was obtained as candidate strain with the virus titer of 106 50 TCID50/0.1 mL。Then,the virus was inactivated with 0.3%formaldehyde under 37℃ for 36 h.The emulsion was prepared with inactivated virus and appropriate oil-adjuvant,and was evaluated by sterility test,physical characteristic and safety of the vaccine due to the standard of inactivated vaccine.To evaluate the immune efficacies of inactivated oil-adjuvant emulsion against porcine circovirus,60 KM mouses were divided into three groups.Group A,B,C is inoculated with the inactivated emulsion,commercial vaccine and emulsified 1640,respectively.All the mouses were inactivated twice(at the age of 4 week old and 8 week old)and challenged with the PCV2(virus titer:106 TCID50/0.1 mL)at 12-week-old.PCV2 specific antibody,cytokines(IL-10、IL-18、IFN-γ、TNF-α、GM-CSF)were detected.Mouses were excuted 4 weeks post challenge and virual DNA of different organs(heart,liver,spleen,lung,kindey)were detected by PCR.The results showed that PCV2 inactivated emulsion could induce the effective immune response in KM mouse after immunization,and could prevent KM mouse from PCV2 infections.