Isolation And Identification Of Porcine Circovirus Type 2 In Henan Province And Cloning Of Proliferation-Sensitive PK-15 Cellline

Porcine circovirus type 2, a member of the Circoviridae family, is a small, non-enveloped, single-strand circular DNA virus. The full length of gene is 1767-1768bp. There are two main ORFs in the anti-sense orientation. ORF1 encodes replication proteins; ORF2 encodes capsid proteins, which contain immunodominant antigenic epitopes. PCV2 has been an epidemic in domestic pigs and wild boars around the world, and caused a series of PCV2 related 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 about a dramatic impact on pig-raising industry. Vaccine immunity is the most effective method to prevent PCV2 infections. Considering the losses for the whole pig-raising industry brought by PCV2, studies on all aspects of PCV2 are looming ahead, which will have great significance on PCVAD prevention、controlling and treatment.To understand the pathogenesis and molecular biological characteristics of porcine circovirus (PCV2), Polymerase chain reaction (PCR) was used to detect the DNA of suspected PCV2 lymph nodes, lungs and spleens of borderline cases from HZ-09hou and Jiaozuo city of Henan Provence, then the PCR positive samples were used to isolate PCV2, the PCV2 were identified by PCR and indirect fluorescent antibody test. PCR products of complete genomes of two PCV2 isolates were cloned and sequenced. The complete genome nucleotide sequences of these PCV2 isolates were compared with those of PCV2 isolates published previously in GenBank using DNAStar software. The lengths of the complete genomic sequences of two PCV2 isolates were all 1767bp. Comparison analysis of the complete genomic sequences of two PCV2 isolates indicated that they were closely related to each other (94.2%) and to other PCV2 strains in GenBank (92.6%-99.9%).There was 74.6% and 77.7% sequences identity among PCV2 and PCV1 isolates gene respectively. All the results showed that the virus isolates were PCV2, HZ-09 isolate was genotype PCV-2a, JIAOZ-12 isolate was genotype PCV-2b.The virus titer was tested by indirect immunofluorescence assay (IFA). Optimized the reactive conditions, then added different concentrations of D-glucosamine into maintenance media and determined the best stimulation concentration of D-glucosamine by IFA, finally determined the best time to harvest the virus. Result:first antibody was diluted in proportion of 1:800; IG-g-FITC was diluted in proportion of 1:1000. The number of positive cells was the highest using maintenance media to stimulate and cultivate cells with the concentration of 2 mM/L for 72 hours under 37℃. Optimization of culture conditions laid a good foundation for further study.In order to get the higher titer of PCV2, experiment 3 cloned the PK15 cells lines which were adapt to PCV2 by serial dilution method, and obtained six PK15 cloned strains marked as L1,L5,L6,L8,L12 and L15. Vaccinated equal amounts of PCV2 with PK15 blast cells and these cloned cells, and then detected the positive cell number by IFA. Result:fluorescence number of L8 cloned cells was the largest. L8 cells were sub cultured and grew stably within twenty generations. Vaccinated PK15 blast cells and L8 with equal amounts of PCV2 and harvested virus suspension, and then determined the TCID50, the results were 104.75/0.1ml and 106.25/0.1ml respectively. Thus it could be seen that L8 had higher sensitivity for PCV2.