| Porcine circovirus type 2(PCV2) infection has been associated with post-weaning multisystemic wasting syndrome(PMWS),porcine dermatitis and nephropathy syndrome(PDNS),porcine respiratory disease complex and reproductive disorders.Since the first case was reported in Canada in 1991, PCV2 infections have been confirmed in many countries of the world.The pigs infected with PCV2 were in an immunosuppressive status which could trigger co-infection with other pathogens.It is not an easy task to get the epidemics under control or eradication because of wide spread of the virus.It is necessary to explore some novel approaches for better prevention and control of the disease. Within the scope of the development of new vaccines against PCV2 infection, subunit vaccines based on the baculovirus expression system should be a putative prophylactic tool.RNA interference(RNAi) is a recently developed technology in which double-stranded RNA(dsRNA) directs sequence-specific degradation of messenger RNA(mRNA) and serves as a potential therapeutic strategy for the inhibition of virulence-associated genes of some pathogens.In this study,10 isolates of PCV2 were obtained from the clinical cases infected with PWMS in Jiangsu Province.Complete genomes of these PCV2 strains were sequenced and analyzed.The cap gene of PCV2 strain XSC was expressed by the recombinant baculovirus reBac-Cap and its immunogenicity was evaluated in mice.Several small interference RNAs(siRNAs) specific to porcine circovirus type 2 were designed,synthesized and expressed by the plasmid vector. Their inhibition effect on the replication of PCV1 and PCV2 by these siRNAs was evaluated in both Dulac cells and mice.1.Molecular characteristics of ten isolates of PCV2 from Jiangsu ProvinceTen isolates of PCV2 were obtained from the clinical cases infected with PMWS by passaging in Dulac cells.Complete genomes of these PCV2 strains were amplified by over-lapping PCR and sequenced directly.The whole genomic sequences of all these isolates were spliced successfully and all of them were 1767 nucleotides long,which have been deposited in GenBank.Sequence analysis showed that these PCV2 isolates exhibited high level of nucleotide similarity and were classified into group 1 of PCV2 based on their genomic sequences.The phylogenetic tree analysis and multiple alignments of nucleotide sequences present that the isolate Suzhou 0511 and Yangzhou 0705 were in other cluster departed from other 8 isolates.All genomics of these isolates exhibited 98.3-99.5%nucleotide sequence identities and their cap genes were also in higher similarity.2.The immunogenicity of the PCV2 capsid protein expressed by the recombinant baculovirusThe capsid protein gene of PCV2 strain XSC was amplified by PCR and cloned into pGEM-T(?) easy vector,and subcloned into pFastBacTM1 donor plasmid,then inserted into baculovirus shuttle vector Bacmid by site-specific transposition.The recombinant baculovirus,designated as reBac-Cap,was constructed when the recombinant shuttle vector Bacmid-Cap was transfected into Sf9 cells.To evaluate the expression level of capsid protein gene,a viral stock with a high-titer up to 5×108 pfu/ml was obtained and determined by the plaque assay.Infected Sf9 cells with 105 pfu reBac-Cap were harvested at 72 h post infection,the expressed capsid protein with molecular weight of 28 kDa,the same size as the natural capsid protein,was identified to be expressed in cytoplasm in soluble,non-secreted form.Furthermore,the expression product was observed to react with the pig antisera specific to PCV2 virus.Sera samples collected from all control and vaccinated animals at 0,7,14,21,28,35,42 49,56 day post-innoculation(dpi) were assayed for anti-PCV2 capsid antibodies by IFA.The results indicated that the recombinant baculovirus reBac-Cap induced specific antibodies against the pathogenic PCV2 capsid antigen from 21dpi.The sera were detected by real-time PCR.The results indicated that the recombinant baculovirus reBac-Cap was experienced the highest replication inhibition rate of PCV2.3.Inhibition of the replication of the porcine circovirus type 2 in the Dulac cells by the siRNAs expressed by the plasmidsSequences of PCV capsid protein genes and replicase genes determined in this study or from the GenBank were submitted to the website of Ambion company for target sequences searching,and some candidate siRNA sequences were generated.The candidate siRNA sequences were blasted,those shared any identity with other known organism genes were exclued.Six siRNAs were chosen to be evaluated for their selected inhibition effect on the replication of PCVs,four of these were specific to the rep gene of PCVs,designated as SH1(S50-68),SH2(S554-572),SH3(S653-671),SH4(S624-642),and the others were specific to the cap gene of PCV2,designated as SH5(R1429-1411) and SH6(R1307-1289). Six DNA fragments with 67 nucleotides producing short hairpin RNA(shRNA) were designed based on the plasmid vector.The corresponding DNA fragments were synthesized,annealed and ligated to the downstream of the mouse originated U6 promoter of the RNAi-Ready pSIREN-RetroQ ZsGreen vector.As controls, the siRNAs specific to Luciferase gene within the vector and negative fragments for any organisms were also included.Recombinant plasmids were transformed into the host bacteria DH5αand positive clones were selected.Positive clones carried the expected insertions were identified by sequencing and designated as Retro-SH1,Retro-SH2,Retro-SH3,Retro-SH4,Retro-SH5,Retro-SH6,Retro-Luc and Retro-NC,respectively.To test whether siRNAs expressed by these six plasmids inhibit the PCV2 replication,we first examined their effect on the replication of the PCV2 virus in the Dulac cells.The plamids were introduced into the Dulac cells using SofastTM transfection reagent.Each samples had triplication.As controls,Retro-Luc and Retro-NC were similarly introduced into the same cells,followed by PCV2 virus infection.The cells were collected at 72 h after infection.Virus loads was detected by real-time PCR assay established in our lab.The results showed that virus loads strongly reduced in cells transfected by Retro-SH1,Retro-SH4 and Retro-SH6,while did not reduce in the cells transfected by other plasmids and the controls.These three siRNAs expressed plasmids were further evaluated in the following experiments.The kinetic effect of siRNAs expressed by plasmids on PCV2 replication in Dulac cells was determined according to the method mentioned above.Three kinds of siRNA expressing plasmids and their mixture were transfected into the cells,and then infected by PCV2 at 24 h after transfection.Collected cell cultures at 24 h,36 h,48 h,60 h,72 h,96 h,120 h after infection were determined for the copies of genomic DNA of PCV2 by real-time PCR.The results showed that there was strong inhibition effect on PCV2 replication from 60 h post its infection,and the effect lasted at least 48 h.As the dose of plasmids transfected,a positive dose-dependent effect was confirmed.500 ng of each plasmid was optimal.The inhibition effect of the Retro-SH4 was the strongest among three plasmids,and no synergism was observed in the cells transfected by the mixture of the three plasmids.The results were also confirmed by the FACS technique.When Dulac cells were infected with PCV2 XSC strain,then each of three plasmids and their mixture were transfected into above cells at different intervals.The results indicated that the siRNAs expressing plasmids should be tranfected as early as possible in order to get strong inhibitions.Transfection of the plasmids at 36 h after virus infection could not inhibit the ongoing replication of the PCV2 effectively.For ten of PCV2 field isolates,the siRNAs expressed by Retro-SH1, Retro-SH4,Retro-SH6 could inhibit the replication of all isolates in Dulae cells. For PCV1,the siRNAs expressed by Retro-SH1,Retro-SH4 specific to the rep gene of PCV exhibited inhibition effect on the replication of PCV1 in Dulac cells while Retro-SH6 did not.4.Inhibition of the replication of the porcine circovirus type 2 in mice by the siRNAs expressed by the plasmidsSeventy-two of 8-week-old BALB/c mice were divided randomly into 12 groups,6 mice for each.Mice in four groups received 10μg of Retro-SH1, Retro-SH4,Retro-SH6 and Retro-NC,respectively,another group were intramuscularly injected with the mixture of Retro-SH1,Retro-SH4 and Retro-SH6,mice in these five groups were challenged by oral administration and intraperitoneal injection with 105 TCID50 of PCV2.Mice in other five groups were oral administration and intraperitoneal injected with 105 TCID50 of PCV2 and then intramuscularly introduced by Retro-SH1,Retro-SH4,Retro-SH6 or their mixture and Retro-NC at 24 h after PCV2 infection.One group was treated as challenge control and the other as a blank control group.All of the mice were slaughted for sera and tissues collection at 5 d and 11 d post challenge.The sera were detected by real-time PCR and the tissues were examined by histopathological and immunohistochemical techniques.Both of the prophylactic and therapy experiments showed that siRNAs expressed by Retro-SH1, Retro-SH4,Retro-SH6 could offer some protection against PCV2 challenge, Retro-SH4 inoculation group experienced the highest replication inhibition rate up to 99.8%.No synergism was observed in the group transfected by the mixture of the three plasmids.In summary,10 isolates of PCV2 were obtained from the clinical cases infected with PMWS in Jiangsu Province.Genomic sequences of these isolates were in high level of homology.The cap gene of PCV2 was expressed by the recombinant baculovirus reBac-Cap and its immunogenicity was evaluated in mice. The mice immunized with the recombinant baculovirus could elicit specific antibody against PCV2.Six of siRNAs specific for PCV2 genomic fragments were designed and the plasmid-based system for generating unimolecular,hairpin siRNAs were constructed successfully and three of them(Retro-SH1,Retro-SH4, Retro-SH6) were selected.These siRNAs expressed by the plasmids could inhibit the replication of the PCVs in the Dulac cells and provide some protection against PCV2 infection in mice.These data indicated that siRNA technique should be a potential useful tool for developing novel strategy to prevent and control PCV2 infection. |
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