Research On Inhibition Of Porcine Circovirus Replication In PK-15 Cells By Small Interfering RNAs

Porcine circovirus (PCV), a member of the Circoviridae family, is a small,non-enveloped, spherical single-stranded DNA virus. There are two genetypes of PCVs,namely PCV1 and PCV2. Both of them are presumed to be widespread in the swinepopulation. Though PCV1 is considered to be nonpathogenic, it can infect humanleukocytes in vitro, and is now considered to be a potential risk for viral transmissionwhen porcine tissues and organs are used for xenotransplantation. PCV2 is associatedwith postweaning multisystemic wasting syndrome, porcine dermatitis nephropathysyndrome, congenital tremors, late-term abortions and exudative epidermitis. Althoughthe impact of PCV infection on the pig industry is disastrous, unfortunately, there is noeffective commercial vaccine for PCV infection. RNA interference (RNAi) is asequence-specific RNA degradation mechanism mediated by small interfering RNA(siRNA), which represents a possible therapeutic application for the treatment of viralinfections. In this study, three siRNA expression plasmids (pS-RepA, pS-RepB,pS-RepC) were generated to target three different coding regions of the Rep gene ofPCV. These siRNAs were used to inhibit PCV replication in a porcine kidney cell line(PK-15).1. Inhibition of PCV2 Rep expression by siRNAsRep is one of important non-structural proteins in PCV, which are essential forPCV DNA replication. To inhibit Rep expression, three small interfering RNAs(siRNAs) expression plasmids (pS-RepA, pS-RepB, pS-RepC) were generated to targetthree different coding regions of the Rep gene, then they were separately co-transfectedinto PK-15 cells with Rep-EGFP fusion expression plasmid pCDRep-EGFP. Repexpression was evaluated by fluorescence microscope, flow cytometry assay, RT-PCRand Western blot. The results revealed that pS-RepA, pS-RepB and pS-RepC couldeffectively and specifically inhibit Rep expression in PK-15 cells. Among thoseeffective siRNAs, the inhibition effects of pS-RepA and pS-RepB were 20%—50%,and the inhibition effects of pS-RepC is 84.5%.2. Inhibition of PCV1 and PCV2 replication by siRNAsReal time-PCR, Western blotting, TCID₅₀ was used to measure the effects of thesesiRNAs on the infection and replication of PCV1 and PCV2. The results of theseexperiments showed that three siRNAs (pS-RepA, pS-RepB and pS-RepC) could alsolead to effective reduction of PCV1 and PCV2 Cap gene transcription and proteinexpression in PK-15 cell. Our study also showed that the replication of PCV1 and PCV2was inhibited by these siRNAs, the results are consistent to the previous data. pS-RepC which targets the middle region of Rep gene, proved to be the most efficient siRNA forinhibition of Rep expression and viral replication. Taken together, our study illustratesthat RNAi could be used as a feasible treatment for PCV infection.