Isolation And Identification Of Porcine Circovirus 2 And Its Proliferation Features

Since porcine circoviru(sPCV) was found in 1974, it was considered to be the pathogen of PMWS, and also the main reason of PCVAD in 1997. The virus often infected with other pathogens and caused immunosuppression, which would increase the prevalence of pig disease. In recent years, it has been concerned more and more widespread. Currently, the PCV that was isolated in many regions, was some variations on genotype, and the prenalence were also not the same. To isolatte and identificate the PCV, then, made phylogenetic analysis and toxicity test would provide the material basis of the prevalence and variation.While PK-15 cells were widely used in recent studies, there were still some problems that the titer was usually can't meet our requirements. To discuss the host cells and culture conditions in vitro, woule provide theoretical basis of pathogenesis study and make a foundation for vaccines developing. Experimental results obtained as follows:(1)A PCV-2 strain was isolated from the PCV-2 positive samples and determined by conventional PCR, indirect immunofluorescent assay(IFA)and electron microscopic observation.(2)The TCID50 was determined by immunoperoxidase monolayer assay (IPMA), and the complete genomes of the isolated sreain was sequenced and analyed. The results were: TCID50 of the isolate was 10^-4.75, the homology of PCV-2 isolate with Canadia of PCV-2 isolate (DQ200735) was up to 99.4%.(3)Vaccinated the isolate virus into PK-15 cells, swine umbilical vein endothelial cells(SUVEC) and swine tracheal epithelial cell(sSTEC). Detection the titer of PCV-2 by PCR, real-time PCR and Indirect immunofluorescence ( IFA ) The best conditions were: 100g/LDMEM incubated in 37℃, 5%CO₂ in PK-15 cells for 72～96h. Finally, the replicative intermediate(RI) was detected in all the three cells, preliminary determine that PCV-2 may reproduce in SUVEC and STEC.(4)Vaccinated PCV-2 in PK-15 cells ,then, added IFN-γand NH4Cl.We found that the titer could be greatly improved by using 75 mmol/L NH4Cl and 500 U/mL IFN-γ, and no matter they were added before or after virus vaccinated, the results were the same. In conclusion, a PCV-2 strain was isolated from samples. The TCID50 of the isolate was 10^-4.75. The homology of PCV-2 isolate with a PCV-2 Canadia isolate(DQ200735)was up to 99.4%. And PK-15 cells were the b est host cells, the titer could be greatly improved by using 75 mmol/L NH4Cl and 500 U/mL IFN-γ.And all of these would provide the theoretical basis of pathogenesis studies.