[Inhibition of porcine circovirus type 2 replication by the plasmid vector expressing siRNAs].
OBJECTIVE
To develop siRNA as a potential tool for control of porcine circovirus 2 (PCV2) infection.


METHODS
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 corresponding DNA fragments were synthesized, annealed and ligated into the downstream of the mouse originated U6 promoter 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 DH5a and positive clones were selected. The positive clones were sequenced 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%.


CONCLUSIONS
The siRNAs specific to PCV2 expressed by vectors should be potential in the control of the diseases related to PCV2 infection.
Wang
Haiyan
H
Key Laboratory of Animal Infectious Diseases of Ministry of Agriculture, Yangzhou University, Yangzhou 225009, China. wanghyyz@hotmail.com
Liu
Wenbo
W
Gao
Song
S
Liu
Xiufan
X
D000818:Animals; D017925:Circovirus / Q000187:drug effects / Q000472:pathogenicity* / Q000502:physiology; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D010957:Plasmids / Q000235:genetics*; D034622:RNA Interference / Q000187:drug effects; D034741:RNA, Small Interfering / Q000235:genetics / Q000494:pharmacology*; D012367:RNA, Viral; D014779:Virus Replication / Q000187:drug effects*
D034741:RNA, Small Interfering; D012367:RNA, Viral