| Porcine circovirus (PCV) is classified in the genus Circovirus of the family Circoviridae. Its virion is non-enveloped and its genome is composed of a single-stranded, negative-strand, circular DNA. PCV1and PCV2are two genotypes of PCV. PCV1is non-pathogenic for pigs but PCV2is a major pathogen of porcine circovirus-associated diseases and is responsible for huge economic losses in the world pig industry. PCV2is mainly divided into five subtypes, including PCV2a, PCV2b,PCV2c PCV2d and PCV2e. In this paper, we studied the identification of PCV2subtypes, the methods of PCV2quantitative detection and the proliferative characteristics of PCV2a and PCV2b in PK15cells and mice.This study provided a technical support and theoretical basis for genetic variation, clinical diagnosis and proliferative characteristics of PCV2.The main contents of the paper are as follows:Experiment1:Study on the identification of PCV2subtypes in some regions of ChinaPCV2is epidemic in the provinces of China. In order to investigate the genetic variation of PCV2in Jiangsu, Zhejiang and Anhui province from2011to2012,28PCV2-positive samples were obtained by the PCV2detection primers from clinical samples and the genome sequences of them were obtained by the two pairs of PCV2full-length amplification primers and analysed respectively. The results showed that these PCV2strains could be classified into three clusters with the PCV2full-length of1767bp (25),1768bp(2),1766bp(1); The nucleotide homology among the28strains and compared with13reference PCV2strains in GenBank is94.2%-100%and93.9%-100%;There are three PCV2a strains, one PCV2b strain and24PCV2d strains, without PCV2c and PCV2e strain. This indicates that the main pandemic strain of PCV2is PCV2d currently.Experiment2:Establishment and application of two TaqMan real-time fluorescent quantitative PCR methods for the detection of porcine circovirus type2 According to the relatively conserved sequences of PCV2ORF1and ORF2, a pair of specific primers and TaqMan probe were designed for them respectively and a recombinant plasmid containing PCV2ORF1or ORF2gene was constructed as the standard sample. After optimization of the reaction system and the reaction conditions, the standard curves were plotted and two TaqMan fluorescent quantitative PCR methods were established for detection of PCV2. The Ct values of plotted standard curves showed good linearity to the log of copy number of template, with a R2value of0.99; the amplification efficiency was between90%and110%; they were all highly reproducible and had a coefficient of variation less than5%for intar-assay; The specificities are high.The sensitivity of ORF1method can be reached1.0×101copies/μL, The sensitivity of ORF2method can be reached1.0×102copies/μL. We detected some samples by the two methods respectively and compared the results.The results showed that the method based on ORF1is more accurate, specific due to the existence of P1in partial clinical samples and can be used for the rapid diagnosis and quantitative detection of PCV2.Experiment3:Study on the proliferative characteristics of two porcine circovirus type2isolates in PK15cellIn order to study the proliferative characteristics of two major subtypes of porcine circovirus type2(PCV2a and PCV2b) in vitro, we isolated the two subtypes in the lab and inoculated them into PCV-negative PK15cell which was passaged continuously, so as to study their proliferative characteristics. The cellular poison of Each generation was collected and detected by the fluorescent quantitative PCR; The TCID50of the10th,20th,30th,40th,50th and60th generation cellular poisons were tested using indirect immunofluorescence method, so as to compare the proliferative characteristics of the two subtypes in vitro.The results showed that, with the increasement of passage numbers, the infective titers of two subtypical viruses were significantly improved, but the proliferative rate of PCV2b is faster than that of PCV2a.Experiment4:Study on the proliferative characteristics of two porcine circovirus type2isolates in Balb/c miceIn order to study the proliferative characteristics of two major subtypes of porcine circovirus type2(PCV2a and PCV2b) in Balb/c mice, Balb/c mice were inoculated with the two subtypes of PCV2respectively, and then we studied the distribution of the virus in various organs and the level of virus and antibody in serum using fluorescent quantitative PCR and ELISA. The results showed that both the two subtypes were capable of proliferating in Balb/c mice, differently,the capacity of PCV2b was stronger than PCV2a.This test laid the foundation for in-depth study of PCV2pathogenesis. |
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