Genetic Evolution Of Porcine Circovirus Type 2 And Development Of DNA Vaccines Containing CpG Motifs

Porcine circovirus (Porcine circovirus, PCV) is divided into two genotypes,non-pathogenic PCV1 and pathogenic PCV2. PCV2 can cause postweaning multisystemicwasting syndrome (PMWS), porcine dermatitis and nephropathy syndrome (PDNS), porcinerespiratory disease complex (PRDC), sow abortion and mortality syndrome (SAMs), porcineproliferative and necrotizing pneumonia (PNP) and congenital tremors (CT). So PCV2 canlead to sow reproductive failure and cause immunosuppression, which results in seriouseconomic losses to the pig industry. The disease has been considered as the newly discoveredswine infectious disease after the porcine reproductive and respiratory syndrome byveterinarians and pig farmers of the world.In recent years, PCV2 constantly mutates and the new strains continue to emerge, whichresults in a certain amount of pressure to the prevention and control of the disease. To betterunderstand the prevalence of the disease and the development of new genetically engineeredvaccine for the prevention and control of the disease, based on the epidemiologicalinvestigation, 3 strains of PCV2 were isolated and identified. The genome of 17 strains ofPCV2 was sequenced and the PCV2 genetic evolution was analyzed. The prevalence ofTorque teno virus (TTV) in the herd of Shandong Province was also surveyed in the sametime. The real-time PCR method and PCV2 mouse model were developed. The DNA vaccinecontaining the CpG motif was also developed.The main work is as follows:Based on the epidemiological survey of PCV2 in pig farms of Shandong Province, it wasfound that PCV2 infection rates showed an increasing trend, the antigen positive rate wasfrom 11.3% to 40.38% and the antibody positive rate was from 38.7% to 72.99% from 2005to 2011. The phenomenon of mixed infection was still very serious. The rate of superinfection of PCV2+CSFV was 33.81% and the rate of triple infection of PCV2+PRRSV+CSFV was4.32%, which were the largest proportion ones. 3 strains of PCV2 were isolated and identified.The biological identification was also conducted. Among the 3 strains, the SD strain of PCV2was passaged to 20 generations and the titer was stable with 106.0TCID50/mL. At present, thevirus was passaged to 75 generations, the titer was stable and the strain of PCV2 can be usedas inactivated vaccine candidate strains. The whole genome sequences of 17 strains of PCV2were completed. PCV2 in China has been in continuous variation, PCV2b is the epidemicstrain, the reorganization virus of PCV2a and PCV2b was also appeared with the geneticcharacteristics of ORF1 from PCV2a and ORF2 from PCV2b. The genotype of PCV2 had nosignificant correlation with the geographical area. The regular pattern of genetic evolution ofdomestic popular PCV2 was the type of PCV2a to recombinant virus of PCV2a and PCV2band then PCV2b type. The type of PCV2c was not found.The PCV2 mice infection model was also conducted. Based on the continued virusreplication in spleens and lymph nodes, PCV2 antibody generated in the serum and thesignificant pathological damage, it was concluded that PCV2 could infect Kunming mice.This study established the animal infection model of PCV2 infection in Kunming mice anddemonstrated that PCV2 could be transmissed in Kunming mice. This laid the theoreticalfoundation of PCV2 pathogenesis studies and evaluation of immune effects of vaccines.SYBR Green I real-time PCR detection of PCV2 was established. The establishedmethod was very specific and repeatable. The sensitivity was 10 copies. The reaction timecould be shorten to 1.5 hours in clinical testing. So this study provided a new method for theclinical detection of PCV2, and the local standards of Shandong Province: porcine circovirustype 2 fluorescent PCR technology (DB37 to/T 1827-2011) was developed, which would beused in Shandong Province.The PCV2 nucleocapsid protein gene of ORF2 was cloned and inserted into theeukaryotic expression vector pVAX1, the pVAX1-ORF2 was successfully constructed. 2 CpGmotifs having the immune-enhancing effects on the basis of preliminary animal experimentswere synthesized and successfully inserted in pVAX1-ORF2 eukaryotic expression vector,two kinds of DNA vaccine CpG-pVAX1-PCV2 ORF2 containing CpG were constructed. The two kinds of DNA vaccine were tested in pigs, the antibody titer and IL-2 production levelwere detected using ELISA kits and the cellular immune effects were determined by flowcytometry. The safety assessment of genetically modified PCV2 DNA vaccine was declaredand approved.The two kinds of DNA vaccine with CpG motifs were tested in pigs and the best ofCpG-pVAX1-ORF2 and immunization dose were screened. The screened DNA vaccine couldsignificantly increase the average daily gain of pigs and stimulate the pigs to produce goodcellular immune response and humoral immune responses and effectively prevent theproliferation of the virus in the body.The main innovation of this study:1) A pathogen epidemiological survey results show that the prevalence of porcinecircovirus in Shandong Province appeared an increasing trend in 2005-2011. The regularpattern of genetic evolution of domestic popular PCV2 was the type of PCV2a to recombinantvirus of PCV2a and PCV2b and then PCV2b type. The type of PCV2c was not found.2) SYBR Green I real-time PCR detection of PCV2 was established, which provided anew method for the clinical detection of PCV2. Porcine circovirus type 2 fluorescent PCRtechnology (DB37 to/T 1827-2011) was developed.3) The PCV2 mice infection model was established. It was found that PCV2 could infectKunming mice and be transmissed in Kunming mice.4) Two kinds of DNA vaccine CpG-pVAX1-PCV2 ORF2 containing CpG wereconstructed, which were able to induce a good humoral and cellular immune responses. Thesafety assessment of genetically modified products in the Ministry of Agriculture (agriculturemethamphetamine Office word 2010-T179) were approved and obtained. The new DNAvaccine provided a material basis for prevention and control of PCV2.