猪圆环病毒2型细胞培养适应毒株的培育和鉴定

Cultivation and identification of porcine circovirus type 2(PCV2)cell culture-adapted strain

从临床表现为仔猪断奶后多系统衰竭综合征(PMWS)淋巴组织病料,经聚合酶链式反应(PCR)证实为猪圆环病毒2型(PCV2)感染,采用无污染的猪肾细胞系(PK15)分离培养,并连续传代培育成一株细胞培养适应毒,命名为PCV2/LG株。分离毒株经细胞培养,于第25代后毒价显著升高,于第35代毒价可达105.6TCID50/mL。采用免疫过氧化物酶单层细胞染色法(IPMA)、免疫电镜技术、分子克隆及核酸序列分析等鉴定表明,分离株感染细胞后病毒抗原主要分布在细胞核及细胞质中;病毒感染的阳性细胞呈散在分布,阳性细胞数可达50%以上;免疫电镜观察到与PCV2特异抗体结合形成的病毒免疫复合物呈实心小颗粒样粒子团,病毒粒子直径约为17nm;病毒抗原基因组由1768个核苷酸组成,与GenBank登录的8个PCV2基因组序列同源性达96.2%以上。用2mL的病毒细胞培养物(105.6TCID50/mL)接种30日龄PCV2抗体阴性仔猪3头,可引起典型PMWS临床症状。本研究为进一步开展该病毒的致病性、疫苗免疫、诊断及分子生物学等研究奠定了基础。

A porcine circovirus type 2 （PCV2） was isolated from a piglet lymph node material with postweaning multsystemic wasting syndrome （PMWS） using PCVl-free pig kidney cell line （PK15）. The isolate was cultivated by a series of passages in the cells, and a cell culture-adapted strain was obtained, which was termed as PCV2/LG. The virus titer of PCV2/LG increased markedly after the 25 th passage, and harvested 10^5.6TCID 50/mL at the 35th passage. The virus antigen distributed mainly in the infected cell nucleolus and cytoplasm, the antigen positive cells showed spot distribution and accounted for more than 50 % by immunoperoxidase monolayer assay （IPMA）. The immune complex formed by the virus particles with anti-PCV2 antibodies was observed by electron microscope, which showed multiple sold virus particles. The diameter of single particle was measured about 17 nm. The virus genome sequence was determined to be 1 768 nucleotides, more than 96.2 % identical rate in comparison with other eight PCV2 sequences of GenBank. Three of 30-days-old piglets were inoculated with PCV2/LG culture liquid with 2 mL dose of 10^5.6TCID 50/mL, these piglets showed typical PMWS. The study establishes a base for further research on the virus pathogenicity, vaccination, diagnosis and molecular biology.