Construction And Identification Of Porcine Circovirus Type2Replicons

Porcine circovirus (PCV) belongs to Circovirus genus, Circoviridae family. According to difference of pathogenicity, antigenicity and nucleotide sequences, porcine circovirus can be divided into two types:porcine circovirus type1(PCV1) and porcine circovirus type2(PCV2). It is generally believed that PCV1is nopathogenic, and PCV2can cause postweaning multisystemic wasting syndrome (PMWS), reproductive disorders, porcine proliferative and necrotizing pneumonia, porcine dermatitis and nephropathy syndrome (PDNS), congenital tremor (CT) and so on. PCVs spread all over the world, bringing huge economic losses to the pig industry.There is no effective treatment for the porcine cicovirus diseases which are mainly based on the vaccine prevention. Diffculties in culturing PCV2in vitro, low reproduction, and the unclear mechanism in replication challenge the prevention and treatment for porcine circovirus.In this study, four PCV2replicons and three non-replicons which containing EGFP or Renilla luciferase (Rluc) reporter gene were constructed. Two PCV2replicons based on Rluc have good activity in replication. These replicons can be a good tool for further research on PCV replication mechanism and can provide a tool for further screening of antiviral compounds. The main contents of this study are as follows:(1) Construction of PCV2replicons containing EFGP or Renilla luciferase reporter geneORF1gene was amplified from the plasmid pT-PCV2which contains the whole PCV2genome, and inserted into pIRES2-EGFP or pc-Rluc to generate pIRES2-ORF1-EGFP or pc-Rluc-2A-ORF1. CMV-polyA fragment was amplified from above plasmids by PCR using the primers containing the PCV2origin of replication (Ori), and ligated to pMD18-T to get PCV2replicons pT-Ori-ORF1-IRES-EGFP and pT-Ori-Rluc-2A-ORF1. CMV-polyA fragment without Ori was amplified and ligated to pMD18-T to generate the non-replicons pT-ORF1-IRES-EGFP and pT-Rluc-2A-ORF1.(2) Optimized the structure of PCV2repliconsPCV2replicons and non-replicons were transfected into293T cells to observe the expression of reporter gene. EGFP expression in pT-Ori-ORF1-IRES-EGFP and pT-ORF1-IRES-EGFP was found to be at same level. Rluc expression was extremely lower than that directly expressed in pcDNA3.1in both pT-Ori-Rluc-2A-ORF1and pT-Rluc-2A-ORF1. Therefore, these replicons were not effective. Two PCV2replicons were optimized by inserting Ori into pc-Rluc-2A-ORF1. To test the activity in replication, the optimized PCV2replicons were transfected. Rluc expression in pc-Ori-Rluc-2A-ORF1or pc-Rluc-2A-ORF1-Ori was significantly higher than that of pc-Rluc-2A-ORF1, moreover pc-Rluc-2A-ORF1-Ori was more effective in replication. Real-time PCR was used to detect copies of plasmids. Statistics showed that copies of pc-Ori-Rluc-2A-ORF1or pc-Rluc-2A-ORF1-Ori were significantly higher than that of pc-Rluc-2A-ORF1, so they were suitable replicons for PCV2.(3) Optimization of conditions for PCV2repliconsTo determine the best testing time, a certain dose of plasmids were transfected. Cells were collected at36,42, and48hours post-transfection for Renilla luciferase activity assay. The luminescence was found to be gradually increased from36hours to48hours post transfection. The luminescence from replicon transfected cells was obviously higher than the non-replicon transfected cells at different time points revealing48hours post transfection to be the most suitable one. To determine the best transfection dose, different doses of plasmids (1011,1010,109copies) were transfected. There were significant differences between replicon and non-replicon48h post-transfection. Interestingly, with the increase in transfection dose exceeding1011, it did not yield any significant increase in luminescence. The109copies plasmids luminescence was found susceptible at other conditions, so1010copies was chosen as an optimal transfection dose.(4) The establishment of drug screening method based on PCV2repliconsPCV2replicons were transfected with optimized conditions,4hours later, Ribavirin was added in a final concentration of100μM.48hours post-transfection, cells were collected for Renilla luciferase activity assay or Real-time PCR. The luminescence of pc-Ori-Rluc-2A-ORF1and pc-Rluc-2A-ORF1-Ori in group with Ribavirin decreased27.9%and16.7%respectively compared with the group without Ribavirin. The luminescence of the non-replicon had no significant differences. Copies of pc-Ori-Rluc-2A-ORF1and pc-Rluc-2A-ORF1-Ori in group with Ribavirin decreased80.6%and51.4%respectively compared with the group without Ribavirin, while the copies of the non-replicon had no significant differences.