Construction Of The Infectious Molecular Clone Of Porcine Circovirus Type 2 Guizhou Strain With A Genetic Marker

Porcine circovirus type 2(PCV2) is a small non-enveloped virus with a single-stranded circular DNA genome of approximately 1.8 kb in size in the genus circovirus, circoviridae family. PCV2 is the primary causative agent of postweaning multisystemic wasting syndrome(PMWS) and porcine circovirus-associated diseases(PCVAD) which include wasting, enteritis, respiratory signs, increased mortality and reproductive failure that can cause a large amount of economically lose for global pig industry. It is an effective way to control PCV2 by vaccination. Additionally, because iDNA vaccines have many advantages compared with traditional vaccines. It was based on molecular clone technology and combines the advantages of DNA immunization and the ef?cacy of live attenuated vaccines. This study aim to construction and application of infectious clones of PCV2 based on pcDNA3.1(+) plasmid and infection of viruses in mice provided a solid foundation for the development of a potential tractable iDNA vaccine. And it may represent a promising vaccination strategy for PCVAD. The main research contents are as follows:1. Molecular Genetic Characterization of the Complete Genome of Porcine Circovirus type 2 GZ-RH1 StrainTo better understand the genome information of the PCV2 GZ-RH1 strain that had mutations at the ORF2-encoded Cap protein with additional one amino insertion at the C terminus. The structure and function of the gene, secondary and tertiary structure of protein, molecular phylogenetic evolution, etc. were studied by means of bioinformatics software. Genome sequence analysis indicated that all genome segments of PCV2 GZ-RH1 was conservative excepted ORF5,8,10,11. The predicted results of deduced protein indicate all of PCV2 GZ-RH1 proteins were hydrophilicity proteins excepted ORF4 and ORF9. The ORF9 had an signal peptide sequence. Compared potential phosphorylation sites of ORF1 and ORF2 with vaccine strains, respectively, the results showed that ORF1 was conservative, but ORF2 was of great variability. Moreover, beside all proteins of PCV2 GZ-RH1 had conserved protein famliy domains by prediction, many of possibly possesses functional domains or motifs were predicted in ORF1 and ORF2, such as NACHT motifs and Arginine-rich region. Phylogenic analysis indicated that the strain belonged to PCV2 b type. Selective analysis revealed that the deduced amino acid sequence of ORF2 contained a variety of positively selected positions and it had two hypervariable regions, which were located in the first 53 to 91 and 185 to 215. Codon bias analysis revealed that ORF2 was closest to insect cells in codon usage pattern and insect cells should be used as the most suitable expression system for PCV2 ORF2 gene.2. Construction of Porcine Circovirus Type 2 with Genetic Marker and the Preliminary Study of iDNA VaccinesThe objective of this study was to construct the infectious molecular clone with genetic marker of PCV2 GZ-RH1 strain, a Sal I restriction enzyme site was inserted into the PCV2 clone as a genetic marker by applying iDNA infectious clone technology. The mutant PCV2 was rescued by transfecting the infectious clone into PK-15 cells. The recombinant marker virus was stable on multiplication through 10 passages in PK-15 cells, with a maximum titer of 105.3 50% tissue culture infective dose(TCID50/mL). Six-week-old female Kunming mice(n = 20) were randomly divided into four groups with 5 mice in each group. Mice in the experiment group?were each injected intramuscularly with 100 ?g of the PCV2 iDNA candidate marker vaccine(PCV2 infectious clone plasmid DNA). The experiment group ? was injected intramuscularly with 0.1 ml of the rescued virus derived from the PCV2 infectious clone(104TCID50/ml). The experiment group III was injected intramuscularly with 100 ?g of empty pcDNA3.1 plasmid. The experiment group ?was injected intramuscularly with 0.1 ml MEM. Serum samples were collected from each animal at 7, 14, 21, 28, 35 and 42 days postinoculation(DPI) for viral nucleic acid and antibody analyses. Seroconversion to PCV2-specific antibody appeared in the majority of mice from the experiment group of I and II at 7 DPI, and the specific antibody level was steady for at least 42 days. Viraemia, beginning at 7 DPI and lasting 28 days, was detected in the majority of the pigs from the two inoculated groups. This study revealed that the infectious clones of PCV2 will be useful for further research investigating a potential tractable iDNA vaccine by reverse genetics technology for attenuated virulance.