Inhibition of porcine circovirus type 2 replication by the plasmid vector ex-pressing siRNAs

Abstract:

Abstract: [Objective] To develop siRNA as a potential tool for control of porcine circovirus 2 (PCV2) infection. [Meth-ods] We designed two short interfering RNAs (siRNAs) related to the replicase (rep) gene of porcine circovirus and one related to capsid (cap) gene of PCV2 basd on the genomic sequences of the viruses deposited in GenBank. The corre-sponding DNA fragments were synthesized, annealed and ligated into the downstream of the mouse originated U6 pro-moter of the RNAi-Ready pSIREN-RetroQ ZsGreen vector. As controls, the siRNAs specific to Luciferase contained in the vector kit and negative fragments with no similarities to any known species were also included. We transformed the recombinant plasmids into the host bacterium DH5α and positive clones were selected. The positive clones were se-quenced and five clones carried the correct inserts. They were designated as Retro-SH1，Retro-SH4，Retro-SH6，Retro-Luc and Retro-NC. To test whether the siRNAs specific to PCV expressed by the vector could inhibit the virus replication, we evaluated the inhibition effect on PCV2 replication both in Dulac cells and experimental mice using real-time PCR and immunohistochemistry. [Results] The results showed that the purified plasmids could strongly inhibit the replication of the PCV2 virus and the inhibition rate reached to 99% in cell culture. In the animal experiments, siRNAs expressed by the plasmids could inhibit the replication of the PCV2 virus and the inhibition rate varied from 26% to 99%. [Conclusion] The siRNAs specific to PCV2 expressed by vectors should be potential in the control of the diseases related to PCV2 in-fection.