Inhibition Of Plasmid-based SiRNA On The Replication Of Porcine Circovirus Type 2(PCV2) In Vivo And In Vitro

Porcine circovirus type 2-associated diseases (PCVAD) caused by porcine circovirus type 2 (PCV2) are one of the most important immunosuppressive diseases that hazards the global swine production. There are no satisfactory vaccines or drugs to prevent this disease yet. Therefore, the study of new biologics for the prevention and treatment of PCVAD are very important. RNA interference (RNAi) is one phenomenon of sequence-specific post-transcriptional gene silence caused by double-stranded small interfering RNA (siRNA). The RNAi technology develops rapidly in the field of treatment of infectious diseases, cancer, cardiovascular disease. In this study, PCV2-specific siRNA expression plasmids were constructed and inhibition on replication and pathogenicity of PCV2 was analyzed in vitro and in vivo.Ten pairs of PCV2-specific and one pairs of non-specific (scrambled DNA oligonucleotides, SCR) DNA oligonucleotide encoding shRNA were designed and synthesized following screening out 10 siRNA target sequence to the PCV2 rep and cap genes respectively. Eleven pairs of DNA oligonucleotides were annealed to make the double strand, respectively, and cloned into the downstream of the U6 promoter of pGensil-1 (siRNA expressing vector). Ten PCV2-specific siRNA expresstion plasmids (pGensil-C123, C297, C490, C563, C645, R148, R259, R295, R484, and R601) and one the control plasmid (pGensil-SCR) were constructed.The liposome-mediated siRNA expression plasmids were transfected into PK-15 cells and 20 h later, PCV2 of 500 TCID50 were inoculated. Expression of PCV2 DNA, mRNA and protein were detected by Real-time quantitative PCR, SDS-PAGE, Western-blot and immunoperoxidase monolayer assay (IPMA). The results showed that the PCV2-specific siRNA expression plasmids could inhibit the synthesis of PCV2 DNA, mRNA and the Rep and Cap protein in PK-15 cell in various degrees. Especially, the inhibitory effect of the siRNA which to the three targets of C297, C490 and R259 was significantly higher than that to other targets. Further studies showed that the replication of PCV2 in PK-15 can be inhibited significantly after plasmid transfection from16 h until 72 h; the concentration of the DNA was obviously lower when 1μg of siRNA expression plasmids were transfected than that when transfection doses were 0.25μg and 0.5μg; The inhibition on PCV2 increased remarkably when the plasmids expressing siRNAs to two or three different target sites transfected PK-15 cells at the same time. These results indicat that the inhibition of siRNA expressed by plasmids on PCV2 is of long-term effectiveness and dose dependence and siRNAs to different target sites present synergistic inhibitory effect on PCV2 replication.128 of 8-week-old BALB/c were randomly divided into 8 groups of 16 each. Group 1 (PC) infected PCV2 only was positive control and group 2 (NC) was non-infected negative control. Group 3 and 6 were blank plasmid and SCR control respectively. Group 4 and 5 were inoculated with the plasmids expressing siRNA to one target site and Group 7 and 8 to two target sites. The mice in the group 3 to 7 were inoculated with plasmids expressing siRNA 24 h before PCV2 infection while those in the group 8 were inoculated 24 h after PCV2 infection. 50μg of plasmids and 0.1 mL of PCV2 (105 TCID50/0.1 mL) per each mouse were inoculated intraperitonealy and intranasally. The body weight of the mice in each group inoculated with plasmids expressing siRNA was significantly higher than that of PC, empty plasmid and SCR control (P<0.05) 21 d after PCV2 infection. The groups which were inoculated with mixed plasmids expressing siRNAs to two target sites got higher weight than those with plasmids expressing siRNA to one target site (P>0.05). The antibody levels, detection rates of PCV2 nucleic acid in serum and the PCV2 load of the organization in the groups which inoculated with plasmid expressing siRNA were significantly lower than those in the PC, empty plasmid and SCR control group (P<0.01). Furthermore, the microscopic lesions that with the characteristics of lack of lymphocytes in lymph nodes and spleen, multinucleated giant cells infiltration tended to mild and then returnd to normal level 21 d later infection. The mixed plasmids expressing siRNA aiming different target site had the strogger inhibition on the proliferation and the pathogenicity of PCV2 than only one kind of plasmid expressing siRNA to one target site in mice. There was a lower viral load in tissues of mice inoculated with siRNA expression plasmid before than after PCV2 infection which indicates that siRNA expressed by plasmid when being used before PCV2 infection could perform stronger inhibition on PCV2. In addition, siRNA expression plasmid could exist stably for a long term (at least for 28 days) in mice tissues.In conclusion, this study demonstrated that PCV2-specific siRNA which based on the plasmid expressing can inhibit the replication and reduce the pathogenicity of PCV2 in vivo and in vitro. The siRNA aiming different target site appears different inhibition and various siRNAs express synergistic effect. The siRNA was expressed stably and long by plasmid in tissues and plays an inhibition on PCV2. These results provide a scientific basis for the further research and development of new biologics for preventing and treating PCVAD.