Constructions Of A Reporter Cell Line For Classical Swine Fever Virus Infection And A Sensitive Cell Line For Porcine Circovirus 2 Infection

Classical swine fever is a highly contagious disease caused by classical swine fever virus and had been causing huge economic losses for pig industry in China.Although the traditional diagnostic tools such as virus isolation continue to be the ‘gold standards’,however, they are time consuming and labor intensive. Molecular technologies could be used as the first choice for detection of CSFV due to the high sensitivity, while they were vulnerable to false positive results.The virus-infecting reporter systems with highly detection sensitivity specific could used as analysis multiple sample, isolation and culturing virus, and drug susceptibility testing.We construct a CSFV-infected reporter system. In orderto develop a dark-to-bright fluorescent reporter system in the presentstudy, the conserved junction peptide of NS4A/4B was ligatedbetween EGFP and quenching peptide. In the reporter cells,chromophore formation of EGFP can be prevented by quenchingpeptide until the quenching peptide was specifically cleaved by CSFV NS3 protease during CSFV infection. The result demonstratedthat green fluorescence in the reporter cell can be observed onlyupon infection with CSFV and there was a strong correlationbetween the fluorescence intensity of the reporter cells and CSFVRNA replication in CSFV-infected cells. The best time to examinethe fluorescence in CSFV-infected cells was at 48 hpi under ourexperimental conditions. Both the reporter cells and SYBR Greenreal-time RT-PCR can positively detect CSFV at 1:5-106 dilution. The reporter system to culture CSFV have same ability with ST and PK-15 cell lines.Porcine circovirus type 2(PCV2) is an extremely slow-growing virus,and PCV2 infection and replication in cell culture yield very low viral titers.PCV DNA replication depends on cellular enzymes expressed during S phase growth and secondly that in the natural multiplication cycle, PCV DNA replication starts only, if cells have passed mitosis. Endogenous and exogenous IL-2 can stimulate epithelial cell proliferation to enhance PCV2 replication. In order to establish PCV2-sensitive cell lines, we cloned porcine IL-2 from lymphocyte, which were isolated from fresh blood. Then the cell line which can stable express IL-2 was established and the IL-2 can promote mitosis to enhance PCV2 replication. PK-15-IL-2 cell lines promote PCV2 replicationsignificantly, which the copy number of PCV2 was 1×1010/m L and TCID50 was 109-109.5/m L.In our study, we constructed CSFV-infected reporter system and PCV2-replication system, which had significant on the related research of virus and disease prevention and had broad application prospect in clinical testing and laboratory research.