The Effect Of N-glycosylation Site Mutation Of PCV2 Cap Protein On Viral Replication And Pathogenicity And Establishment Of Colloidalgold Immuno-chromatography For Detection Of PCV2 Antibody

The series disease caused by Porcine circovirus type 2 has been considered as the newly discovered swine infectious disease to the pig industry.PCV2 can cause postweaning multisystemic wasting syndrome,porcine dermatitis and nephropathy syndrome, porcine respiratory disease complex,sow abortion and mortality syndrome, porcine proliferative and necrotizing pneumonia and congenital tremors. PCV2 can lead to sow reproductive failure and cause immunosuppression, which results in serious economic losses to the pig industry.Porcine circovirus type 2(PCV2), the essential causative agent of the postweaning multisystemic wasting syndrome(PMWS), can be divided into distinct genotypes. 32 PCV2 strains were isolated from Shandong Province between 2005-2013. Complete genome sequences were obtained in three replicas for each virus and the sequences were submitted to the NCBI.The ORF1-encoded amino acid sequences had 98.4%-100% identity among the 32 strains.There were 3potential glycosylation positions- 23aa-25aa(NPS), 256aa-258aa(NQT), 286aa-288aa(NAT) and there was no significant differences between the 3 sites. The ORF2-encoded amino acid sequences had88%-100% identity among the 32 strains and the potential glycosylation position of cap protein,143aa-145aa(NYS), had no variation.The phylogenetic analysis revealed that PCV2b/1C genetic lineage was now prevalent in swine populations in Shandong Province. It is suggested that selection pressure has made the PCV2 isolates more distant from the vaccine strains.Porcine circovirus type 2(PCV2), the essential causative agent of the postweaning multisystemic wasting syndrome(PMWS),has posed a grave threat to global swine industry in recent decades.Infectious molecular clone is the best way to get the purificated virus for pathogenesis study and inactivated vaccine produce.The purpose of this study was to compare and get the best way for PCV2 infectious molecular clone to rescue viruses.In this study, four PCV2 infectious molecular clone was constructed and transfected into PK-15 cells. IFA and Real-time PCR was used to detect the presence of PCV2. The IFA results revealed the precence of rescued viruses. The real-time PCR results showed that there were the most DNA copies with Ligation DNA transfection, the least DNA copies with pEASY-PCV2 transfection, and almost the same with psk-2PCV2 and psk-PCV2 transfection.This assay afford the best way to construct and get PCV2 infectious molecular clone for further research.To study the effects of PCV2 Cap protein glycosylation sites to viral replication and pathogenicity, the mutant infectious, PCV2 Cap protein N- glycosylation sites 143aa-145aa(NYS)mutation(N to D),were constructed used overlap PCR and infectious clone construction methods above and successfully rescued virus. The results showed that there was not significantly different on the mutation of glycosylation site on virus replication.6 weeks old BALB/c female mice were infected with the rescued 5 passages virus. The results showed that rescued virus contained mutation site influenced weight gain and could detected PCV2 specific antibody in the blood. The virus was detected in various organs of the infected mice. Pathological examination results showed that PCV2 have caused varying degrees of damage. There was not significantly different on antibody levels, the degree of damage between the mutation groups and without mutation groups,but with the cell fluid control group it was significantly different.Staphylococcal protein A(SPA)labeled with colloidal gold particles prepared by trisodium citrate reduction and the labeled SPA was coated onto glass fibrous membrane to prepare gold labeled pad.The purified Cap protein of porcine circovirus 2 and rabbit anti-SPA antibody were coated respectively to the test line and quality control line on nitrate cellulose membrane, so as to establish a colloidal gold immuno-chromatography for detection of antibodies against porcine circovirus 2. The results indicated that the assembled test card reacted exclusively with PCV 2 positive sera and the test sensitivity was up-to 100 fold dilutions. 86 clinical samples were tested both with the test cardand the commercial ELISA kid,resulting in positive rates of 65.12% and 70.93% respectively. The method was rapid,specific,simple in operation and could be extended at grass-roots.