| 1. On the basis of general analysis of the complete genomic sequence variations between both types of porcine circovirus (PCV), three primers were designed and a multiplex PCR assay was constructed for detection and differentiation of PCV. With the established method, five porcine cell lines and 127 tissue samples infected with respiratory diseases from Huadong regions including Jiangsu, Shanghai, Zhejiang, Shandong and Fujian province or city were tested for the presence of PCV. The results showed that among these 5 porcine cell lines, 4 were tested PCVI positive, one PK-15 cell line was positive for both PCV type 1 and 2. From these 127 tissue samples, we found that the pig herds affected with respiratory diseases from Shanghai, Jiangsu, Shandong and Zhejiang province or city were widely infected with PCV, and PCV2 was the most widely distributed in the herds, the infection rate is 27.3% ( 3/11 X 15% ( 3/20 X 23.1% ( 6/26 ) and 48.1% (13/27 ), respectively. The double infection of both PCV types was also very prevalent in the pigs, the infection rate is 18.1%(2/11), 20% (4/20 X 7.7% ( 2/26) and 19.2% ( 5/27 ), respectively. Only few were infected with PCV 1 in the herds.2. According to the published genomic sequences of porcine reproductive and respiratory virus syndrome virus (PRRSV) and porcine parvovirus (PPV), primers were designed and RT-PCR, PCR were set up for detection of PRRSV and PPV, respectively. With the established methods, 38 clinical samples positive for porcine circovirus type 2 (PCV2) were tested for the presence of PRRSV and PPV. The results demonstrated that the co-infection of PRRSV/PCV2, PPV/PCV2 and PRRSV/PPV/PCV2 were detected in 39.4% (15/38), 5.3% (2/38) and 7.9% (3/38), respectively. The data obtained in the present studyindicated that it is prevalent for the co-infection of PRRSV, PPV and PCV2.3. Five tissue samples postive for PCV2 tested by multiplex PCR were inoculated into PCV 1-free PK-15 cells, respectively. After four blind passsages, the propagation of PCV2 was identified by PCR, indirect immunofluorescent assay (IFA) and electron microscopy, respectively. The results showed that PCV2 specific DNAs (1154bp) were detected by multiplex PCR and PCV2 antigens were also detected in the nuclei by IFA in all the five inoculated PK-15 cells. Topical porcine circovirus particles (17-20nm in diameter) were observed by electron microscopy. The five PCV2 isolates identified in the present study were designated JiangsuPCV, ShanghaiPCV, ZhejianglPCV, Zhejiang2PCV and ShandongPCV respectively according to the different regions. The TCID50 of all the five isolates were tested by IFA. In addition, IFA assay was established with the PCV isolates and clinical serum samples were detected for the presence of PCV antibody. The results revealed that the infection of PCV is very prevalent in the pig herds.4. According to the published complete genomic sequences of PCV2, a pair of PCV2 specific primers were designed, and five PCV2 complete genomes were amplified by polymerase chain reaction (PCR). The PCR products were cloned into the EcoRI enzyme site of pcDNA3 vector, and five recombinant plasmids containing the corresponding complete genome of PCV2 isolate were obtained. The cloned PCR products were sequenced and analysed, and the complete genomes of the five PCV2 isolates were determined, all of them are 1768bp in length. The five PCV2 sequences were compared with other PCV isolates in the Genbank, the results showed that the five PCV2 isolates sequenced in the present study are closely related to other PCV2 isolates in the Genbank, displaying 94.5%-100% nucleotide sequence identities, and there exist only 69.7%-71.1% nucleotide identities with the PCVI isolates. Although the genomes of PCV2 isolates are generally stable among different isolates, PCV2 isolates from different geographic regions vary in the genomic sequences. Phylogenetic analysis revealed that ShanghaiPCV, ZhejianglPCV and JiangsuPCV in the present study are closely related to a USA isolate AF264039, and Sh...
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