Study On Molecular Epidemiology And PCV2/PCV3 Cap Genetically Engineered Candidate Subunit Vaccines Of PCV2 And PCV3

Porcine circovirus(PCV)is a member of the genus Circovirus in the Circoviridae.Porcine circovirus type 2(PCV2)is the main pathogen of circovirus diseases(PCVD)and one of the main pathogens threatening the pig industry worldwide,causing huge economic losses to the pig industry;PCV3 was first reported in the United States in 2016.It is related to a variety of diseases in pigs and has been prevalent all over the world.The purpose of this study is to investigate the molecular epidemiology,genetic variation and evolution law of PCV2 and PCV3 in Jilin Province.The epidemic strains were isolated and identified,and their pathogenicity was studied.After the above investigation and identification,in view of the current epidemic strains,the research of PCV2 and PCV3 two subunit vaccines was carried out.The immune protective effect of the vaccine was finally evaluated by animal vaccination test to provide technical support for the prevention and control of PCV2 and PCV3 in Jilin Province.The results of this study are as follows:1.Establishment and application of dual fluorescent quantitative PCR(FQ-PCR)for PCV2and PCV3According to the PCV2 and PCV3 sequences published in Gen Bank,the conserved sequences were screened.Then specific primers and probes were designed to establish single FQ-PCR detection method of PCV2 and PCV3.Based on this method,the reaction system and reaction conditions were optimized,and the dual FQ-PCR methods of PCV2 and PCV3 were constructed.The results showed that the minimum detection concentration of the constructed dual FQ-PCR method was 1×10~1 copies/μL.The coefficient of variation between bathes and within batches was less than 2%,and there was no cross-reaction between PCV2 and PCV3,indicating that the sensitivity,reproducibility and specificity of this method were well.FQ-PCR was used to detect 137 tissue samples collected from Jilin Province from 2018 to 2019,which showed that the detection rates of PCV2,PCV3 and mixed infection of the two pathogens were 56.90%,25.5%and 20.44%.While the detection rates of PCR were 52.55%、23.36%and18.25%,indicating that the dual FQ-PCR of PCV2 and PCV3 established in this study could be used for the detection of clinical samples.2.Molecular epidemiological investigation and pathogenicity study of isolated PCV2 and PCV3 strainsTo determine the prevalence of PCV2 and PCV3 in Jilin Province,1065 samples were collected from 13 pig farms in different regions of Jilin Province between September 2018 and June 2021 for testing.The individual infection rates of PCV2 and PCV3 were 51.83%and33.99%;the mixed infection rate of both was 18.40%.16 PCV2 positive samples and 13 PCV3positive samples were selected for sequencing and genetic evolution analysis of their ORF2gene.The results showed that the nucleotide homology among 16 PCV2 sequenced strains was93.0%～99.7%,of which 3 strains were PCV2b genotype,and the other 13 strains were PCV2d genotype;The nucleotide homology among 13 PCV3 sequenced strains was 96.7%～99.5%,of which one strain was PCV3c genotype and the other 12 strains were PCV3b genotype.For the currently prevalent PCV2 and PCV3 genotypes,PCV2 isolated from PK-15 cells and PCV3isolated from porcine kidney epithelial cells to obtained PCV2 JL11 strain and PCV3 JL3 strain,with belongs to PCV2d genotype and PCV3c genotype respectively.In the pathogenicity study,the PCV2 JL11 strain caused a significant decrease in the daily weight gain of pigs,obvious viremia,and obvious pathological changes in the lungs and inguinal lymph nodes after challenge.The PCV3 JL3 strain caused a slight decrease in the daily weight gain of pigs after challenge,resulting in more obvious viremia.These results showed that the PCV2 JL11 strain and PCV3 JL3 strain were pathogenic to swine.3.Construction of recombinant Ac-PCV2-Cap,Ac-PCV3-Cap and Ac-PCV2/PCV3 Cap baculovirusTaking the Cap amino acids of epidemic strain PCV2 JL11 and PCV3 JL3 obtained in molecular epidemiology as reference,the codon was optimized for Sf9 cells,and baculovirus Ac-PCV2-Cap and Ac-PCV3-Cap expressing PCV2 Cap protein and PCV3 Cap protein were obtained with baculovirus pfastbac1 as vector,and baculovirus Ac-PCV2/PCV3 Cap expressing both PCV2 Cap protein and PCV3 Cap protein.Western blot and IFA results showed that PCV2Cap,PCV3 Cap and PCV2/PCV3 Cap proteins could be expressed in Sf9 cells,indicating that the proteins expressed by Ac-PCV2-Cap,Ac-PCV3-Cap and Ac-PCV2/PCV3 Cap had good reactogenicity.4.The Research of immunogenicity of duplex recombinant baculovirus subunit vaccine PCV2/PCV3 Cap in miceThe mice were immunized with ISA206 and GEL02 adjuvants,and GEL02 was identified as the adjuvant of the subunit vaccine by specific antibody detection and lymphocyte proliferation assay.The subunit vaccines were prepared with different concentrations of PCV2Cap protein and PCV3 Cap protein,and the antigen concentration of 30μg/m L was determined by antibody detection and lymphocyte proliferation assay.PCV2-Cap subunit vaccine,PCV3-Cap subunit vaccine,PCV2-Cap+PCV3-Cap subunit vaccine and PCV2/PCV3 Cap duplex subunit vaccine were prepared according to the optimized adjuvant and antigen concentrations,respectively,and mice were immunized.The results of specific antibody,neutralizing antibody,lymphocyte proliferation and cytokine showed that the four subunit vaccines induced good humoral and cellular immune responses in immunized mice.The results of the attack test showed that the four subunit vaccines produced good protection against PCV2 JL11 and PCV3JL3 strains,respectively,and were able to reduce viremia,decrease viral load in tissues and reduce pathological damage to organs.5.The Research of immunogenicity of duplex recombinant baculovirus subunit vaccine PCV2/PCV3 Cap in pigsThe prepared subunit vaccines PCV2-Cap,PCV3-Cap and diphasic subunit vaccine PCV2/PCV3 Cap were immunized to 28-day-old piglets respectively.The antibody test results showed that specific and neutralizing antibodies could be detected 21 days after the first vaccination,and the antibody potency was highest at 35 days.The results of lymphocyte proliferation response and lymphocyte subpopulation content of peripheral blood showed that each subunit vaccine could induce a high level of cellular immune response in piglets.The cytokine test results showed that each subunit vaccine group could induce a high level of IFN-γand IL-4,which caused Th1 and Th2 type immune responses.The results of post-immunization attack protection test showed that after PCV2 JL11and PCV3 JL3 attack respectively,the subunit vaccine PCV2-Cap,PCV3-Cap and PCV2/PCV3 Cap groups were able to reduce viremia,decrease viral load in tissues and reduce pathological damage to organs compared with the PBS group,indicating that vaccine immunization could resist the attack of PCV2 and PCV3and provided good protection for the test pigs.