Isolation And Identification Of Porcine Circovirus Type 2 In Guizhou Province And Genetic Variation Analysis Of The Isolated Strains

Porcine circovirus type 2(PCV2) is part of Circoviridae, Circovims, also a small non-enveloped virus with a single-stranded circular DNA genome of approximately 1.8 kb in size. It's widely distributed about Various diseases caused by PCV2. In view of the fact that it was difficult to have a single PCV2 infection. PCV2 was often associated with CSFV, PRRSV or PRV to forming a double or multiple infection. PCV2 strains and the above virus appeal to have identical biological properties such as cell tropism. It's too difficulty to obtain pure PCV2 from the clinical multiple infection disease, hindered the etiology of different PCV2 strain related research. However, the reverse genetics technology has Unique advantages in Virus isolation. The whole genome of PCV2 can be amplified and obtained from the samples by reverse genetics technology. Construction of infectious virus clone become the effective way to isolating pure PCV2 from the samples. This study applies reverse genetic operation platform, be aim to construction and application of infectious clones of PCV2 based on pcDNA3.1(+) plasmid. Saving for virus by kunming mice to isolating the virus from six samples of different sources. The molecular genetic characteristics of PCV2 isolates from different sources are compared and analyzed. On the one hand, this study can enrich the data of PCV2 molecular epidemiology in Guizhou province, providing a new strategy for the separation of PCV2.On the other hand, it can provide a technical reference for the construction of the PCV2 virus library in Guizhou Province, which provides a technical reference for the screening of excellent immunogenicity. It is of great significance to carry out the research on the prevention and control of PCV2 vaccine.1?Isolation and preliminary identification of six strains of porcine circovirus type 2 in Guizhou ProvinceBuilding the six strains' s Infectious clone plasmid called ZYMT2015, ASXX2015, MJBM2015, GYYY2015, CSGS2012 and GYXW2014 from the material such as miscarriage, typical dermatitis nephritis syndrome porcine blood samples, multiple infection of normal tissue of diseased samples and PCV2 antibody positive for serum samples detection by a reverse genetic operating platform has been set up in the laboratory. After the intraperitoneal injection of plasmid in Kunming mice to carry out the in vivo virus rescue, the serum of mice were inoculated into PK15 cells. After 3 times of cell passages, the isolated strains were detected and identified. IPMA identification results showed that, after 3 times of PK15 cells passages, the infected cells showed orange specific staining; Using primers and identification of the whole genome of cell culture fluid for specific PCR detection, and the results showed that could be amplified by the specific DNA fragment. Preliminary identification results show that the reverse genetic operating platform has been established successfully, and 6 strains of PCV2 were isolated from the samples of multiple virus infections. Strains of PCV2 ZYMT2015, genome size is 1767bp;strain of PCV2 ASXX2014, genome size is 1767bp; strain of PCV2 MJBM2015, genome size for 1766bp; strain of PCV2 GYYY2015, genome size for the complete; strain of PCV2 CSGS2012, genome size for 1766bp; PCV2 GYXW2014 strains gene in the group size is 1767 bp.2?Analysis on genetic variation of 6 strains of Guizhou isolate of Porcine Circovirus type 2Application of bioinformatics software on the above 6 strains of different sources, 31 strains of domestic and foreign PCV2 reference strains to understand the genetic variation characteristics of isolated strains. In particular, the genome sequence of the existing vaccine strain(LG strain, SH strain). Complete nucleotide sequence analysis results showed that with previously reported containing 11 open reading frame(ORF) of PMWS compared to the control strain(AF027217) and the genetic variation is mainly manifested in the ASXX2014 GYYY2015 contains 10 ORFs(were lack of ORF9 and ORF4), ZYMT2015 GYXW2014 were only contains nine open reading frame(ORF)(respectively were lack of ORF8 and orf11). The main variation of CSGS2012 strains showed the premature termination of ORF1 and the initiation codon of ORF3; The main variation of MJBM2015 is the later movement of the ORF1 initiation codon; Genotyping results showed that MJBM2015 and ASXX2015 strains were PCV2 b genotype, CSGS2012 strains and GYYY2015 strains were PCV2 a genotype, ZYMT2015 and GYXW2014 strains were PCV2 d genotype. The prediction results of the main structural proteins showed that ORF1, ORF2 and ORF3 encoding proteins were mixed type proteins. In addition to greater CSGS2012 and BJMJ2015 ORF1 encoding protein potential phosphate of sites and reference strain differences, between the residual strains in addition to the serine phosphorylation sites difference isbigger, threonine, tyrosine phosphorylation sites exactly the same. There was a certain degree of variation in the potential phosphorylation sites of ORF2 encoding proteins between the nine strains. The prediction results of the three stage structure of Cap protein showed that the similarity of 3jcj.1 and vaccine strain in SWISS database was more than 90%.