Genetic Variation Analysis Of Porcine Circovirus Type 2 And Development Of Recombinant Baculovirus Vector Inactivated Vaccine

Porcine circovirus disease(PCVD)is a porcine immune disease caused by porcine circovirus type 2(PCV2).The clinical manifestations of PCVD include porcine weaning wasting syndrome(PMWS)and porcine dermatitis nephrotic syndrome(PDNS),congenital tremor(CT),sow reproduction disorder,enteritis,and porcine respiratory syndrome(PRDC),etc.According to the genotype classification principle of genetic distance p value?0.035 suggested by PCV Europe Union,PCV2 can be divided into five genotypes:PCV2a,PCV2b,PCV2c,PCV2d and PCV2e.PCV2a,PCV2b and PCV2d are global distribution.Before 2002,PCV2a was the main genotype,PCV2b gradually became the dominant genotype in 2003,and pcv2d,a new genotype detected in 2010,has been becoming the main popular genotype in Chinese and American.It has been reported that The vaccines based on PCV2a or PCV2b,which have been widely used throughout the world are effective on PCV2a and PCV2b even PCV2d,but the homologous vaccine can reduce the virus loading of the same genotype better than the heterologous vaccine.In the United States,the pig farms inoculated with based on PCV2a commercial vaccine showed clinical manifestations of PMWS,and the high load of PCV2 were detected in the pigs,which indicates that the compulsoriness to speed up the development of a new vaccine based on pcv2d.On the other hand,PCV2 isolate has low proliferation titers in cell culture and inactivated vaccine widely used in China has the potential biosafety risk of due to the difficulty of PCV2 inactivation,while the subunit vaccine has high safety,low immune stress,early antibody production and strong protection.PCV2 genetic engineering vaccine should be more encouraged in the future.In this study,PCR detection and sequenceing of diseased samples of PCVD in Jiangsu province will be carried out in recent four years.Genotype and genetic diversity of PCV2 will be analyzed based on large data of genotype and genetic diversity of PCV2 isolates.The inactivated vaccine of porcine circovirus type baculovirus will be developed based on PCV2d,which is more effective to prevent and control the concurrent PCVD.1.Analysis of genotype and genetic variation of porcine circovirus type 2 in Jiangsu during 2014-2017 yearsThe diseased pigs samples collected from Jiangsu province and its surrounding areas from 2014-2017 were detected by PCR and the full-length DNA of PCV2 isolates was sequenced and analyzed.In total,748 cases of PCV2 positive samples were detected in 1806 clinical samples,the positive rate was 41.42%.A total of 268 PCV2 full-length sequences were obtained,and sequence alignment showed that the full length of 16 isolate genomes were 1,768nt and classified as PCV2a,the full length of 245 strains was 1767nt,of which 84 were PCV2b and 161 were PCV2d,and another 7 strains had a full length of 1766nt due to the lack of "g" in 1039 position,but also belonged to PCV2d.The proportions of PCV2a,PCV2b and PCV2d were 6%,31.3%and 62.7%,respectively,and no PCV2c and PCV2e were detected.Homology analysis showed that the similarity of full-length sequence of each strain was 94.5-100%,and the homology was 96.9-100%within the same genotype.The homology of all PCV2b strains was highest,the dispersion degree of PCV2a was relatively obvious,PCV2d was divided into two subgroups,the homology of same subgroup was 99.5-100%,and the homology of the different subgroup was 98.1-98.5%.Among the different genotypes,the homology of PCV2a with PCV2b and PCV2d were 95.1-96.0%and 94.4-95.0%,respectively.the homology of PCV2b and PCV2d was 96.1-97.6%,which was significantly higher than that with PCV2a.The amino acid sequence of all strains orfl nucleotide and its coding protein was relatively high,and the nucleotide sequence homology was 97.1%-100%.Among the same genotypes,the difference was relatively obvious only in PCV2d.As the same with the full length sequence,orfl sequence of PCV2d could be divided into two subgroups,homology of the same subgroups was 99.1-100%and among different subgroups 97.2-98.0%.The highest homology among PCV2b strains was 99.5-100%and PCV2a strains 98.7-100%.The homology was 97.1-100%among different genotypes.The results showed that the ORF1 of PCV2 is relatively stable and the difference of each genotype is not obvious.Particularly,the difference between the two subgroups of PCV2d is larger than that between one of the subgroups and the PCV2b.The amino acid sequence coded by orfl of all strains showed a higher homology and the homology of all the strains was 99.0-100%,the variation was not obvious even between different genotypes.The homology of some strains belonging to between the same and different genotypes was 100%.It is interesting that only one amino acid showed distinct between the two PCV2d subgroups with relative obvious nucleotide sequence difference,which indicates that there are many meaningless mutations in thePCV2 ORF1 nucleotide sequence.Compared to or,the variation of or was very significant.the homology of orf2 nucleotide sequence of all strains was 88.3-100%.Among the different genotypes,the homology among PCV2b strains was the highest,98.9-100%.The homology of PCV2d was was 98.3-100%.The largest difference was PCV2a,with 93.1-100%.However,the homology of orf2 nucleotide sequence between the different genotype was significantly low,the homology between PCV2a and PCV2b was only 90.9-92.5%,and 88.3-90%between PCV2a and PCV2d,and the homology between PCV2b and PCV2d was slightly larger,which was 93.9-95.6%.The homology of ORF2 amino acids is basically consistent with its nucleotide.The results showed that there were PCV2a,PCV2b and PCV2d infection in the pig population in Jiangsu and its surrounding areas,and PCV2d was the dominent genotype,so the immune and control technology of PCV2d should be further strengthened.2.Construction and immunogenicity of recombinant baculovirus of PCV2 orf2 geneThe full-length DNA of PCV2d was amplified by PCR as a target fragment,and then cloned into pFastBac vector and transformed to DH10 E.coli,and then screened for three rounds of blue white spot.The recombinant plasmid was obtained,and transfected into sf9 white liquid cells.The recombinant baculovirus was serially passage and identified by PCR and IFA.Western blotting results showed that specific reconbinant cap protein expressed effectively with molecular weight of 28kD and the expression of cap protein of recombinant baculovirus inserted into double copies was significantly higher than that of single copy.The recombinant baculovirus was expressed high density in the high 5 cells,and the was inactivated.The healthy piglets aged 19-21 days were immunized with the recombinant protein vaccine(including 10?g/ml of recombinant protein)and the protective rate was 100%with PCV2d virulent strain challenge after 3 weeks.This sthdy shows thatPCV2d orf2 recombinant baculovirus is successfully constructed and has very good immunogenicity,which can be used as a vaccine candidate.3.Development of inactived vaccine of porcine circovirus recombinant baculovirus vectorThe recombinant baculovirus rB-cap strain which can express pcv2d cap protein efficiently can stabilize more than 20 generations,and the strain of rB-cap was established as the vaccine seed.P-propylalcohol was used as the inactivator,the water-soluble adjuvant G was screened,and the technical parameters and process of suspension production of sf9,high 5 cells and rB-cap were determined.Three batches of porcine circovirus recombinant baculovirus vaccine laboratory products were prepared.After the inspection,a series of laboratory tests were carried out according to the requirements of the regulations.Results:the single dose/high dose and repeated inoculation of piglets of 3 batches of vaccine did not cause any adverse reactions.17-21 days old piglets immunized with one intramuscular injection of 1 mL vaccine(rcapprotein?10?g/mL)could resist the challenge of virulent PCV2d.The duration of vaccine immunization could reach more than 4 months and maintain efficacy for more than one year stored at 4?.Compared with the similar products,the vaccine showed the same good results as the subunit vaccine produced by Boehringer-Ingelheim,which was better than the commercial whole virus inactivated vaccine.The results show that the porcine cirovovirus baculovirus vector inactivated vaccine(strain rB-cap)based on PCV2d is safe,effective and can be used for registration of veterinary biologics.