Ivestigation On Molecular Epidemiology Of Porcine Circovirus Type 2 And Development Of An Indirect-ELISA Mothod Or Detecting Specific Antibody Of PCV2

Porcine cireovirus type 2(PCV2) is an important pathogen in swine,to further investigate its prevalence in Jiangsu province,a PCR method was established by using a pair of primers that derived from the published nucleotide sequence of the PCV2 genome,deposited in GenBank.In order to evaluate its specificity,the PCR detection of PCV2 was performed using the template DNAs from the reference virus,and the expected products of 573bp were only detected in PCV-2,whereas no specific band was detected in PCV1 or other virus such as Pseudorabies virus(PRV) and Porcine parvovirus(PPV).Spleen and lymph node samples were obtained from 157 hoggery in Yangzhou and Taizhou region from July to September 2006,and submitted to PCR detection.The data indicated that 111 of the 157 hoggery were in danger of PCV2 infection.In addition,PCV2 co-infected in inguinal glands and spleen was 41.4%,while 21.7%just in inguinal glands and only 7.6%in spleen.Furthermore,32 inguinal glands from the slaughterhouse in Yangzhou were also tested,and the data revealed that the PCV2 infection rate in clinical healthy swine was 71.88%.According to the published genomic sequence of PCV2,a pair of specific primers was designed to amplify the complete genome of PCV2 from tissues of swines with PMWS.Six complete genomes of PCV2,named TZ60607,TZ60804,YC60711,YZ60615,YZ60721,YZ70717,were amplified and cloned into pGEM-T easy vector respectively.The genomes of PCV2 isolates showed that all of them were 1767nt in length.The six sequences were compared with other published genomes of PCVs by DNAstar.The results showed that the homology of sequences among the six isolates were 96.0%～99.8%.The phylogenetic tree was constructed.The results revealed that the similarity,of complete genomic sequences between the six isolates and the isolated strain from Zhejiang Province(AY099498) was very high,and they composed a bigger branch.The six isolates were close to most isolated strains from China,but had a distant evolutionary relation from isolated strains from the USA(AF264041) and Canada(AF086836).The recombinant capsid protein of Porcine circovirus type 2(PCV2) expressed in Escherichia coli BL21(DE3) was used as antigen for developing an indirect-ELISA assay to detect PCV2 specific antibody.Conditions of the ELISA were optimized,and suggested that the optimistic concentration of the recombinant capsid protein is 6.4μg/mL.Different serum samples were detected for the antibody of PCV2,and it was found that the developed ELISA method is sensitive and specific to the positive serum of PCV2,while not reactive to the negative serum of PCV2 and the sera of other virus,such as Classical swine fever virus,Pseudorabies virus,Porcine parvovirus, Porcine reproductive and respiratory syndrome virus.In addition,the data of further tests also confirmed the ELISA method is repeatable within subject and stable between subject,it was shown that the ELISA was specific and suitable for large-scale sero-epidemiological investigation for PCV2 infection.