猪细小病毒四川株的分离鉴定及其一步生长曲线

Isolation and identification of porcine parvovirus strain SC-L and its one-step growth curve

为了研究猪细小病毒(PPV)的增殖规律,利用PK-15细胞从流产胎儿的肝、肠系膜淋巴结和肾样品中分离到1株病毒,经PCR检测、蚀斑纯化、病毒理化试验、微量中和试验和血凝试验证实该分离株为猪细小病毒,命名为SC-L。针对PPV的VP1和NS1基因设计特异性引物,建立PPV的荧光定量PCR方法。以SC-L分离株感染PK-15细胞,感染后每隔4h分别收集感染细胞与上清液,利用荧光定量PCR方法对不同样品中的病毒DNA进行定量分析,绘制PPV SC-L的一步生长曲线。PPV SC-L株感染PK-15细胞,细胞外病毒含量在第4～20小时缓慢增加,20h后呈对数增长,第56小时达到最大值,随后病毒含量迅速下降,在第68小时时趋于稳定;在细胞内感染4h检测到微量的病毒粒子,第4～24小时细胞内病毒缓慢增长,24h细胞内病毒粒子呈对数增长,第44小时达到最大量,随后胞内病毒粒子逐渐降低。表明PPV在细胞内增殖到一定数量后逐步释放到细胞外,且细胞外病毒最大量高于细胞内。

To study the proliferation characteristics of porcine parvovirus（PPV）,a strain of virus was isolated from the liver,kidney and mesenteric lymph node of the abortion piglet using PK-15 cells,and the isolated virus was proved to be PPV,named SC-L,through PCR detection,plaque purification,physical and chemical properties experiment,microneutralization test and hemagglutination test.According to the VP1 and NS1 genes of PPV,a pair of specific primers was designed,and real-time fluorescent quantitative PCR（FQ-PCR） was introduced to quantify PPV.The SC-L isolate was used to infect PK-15 cells,and the infected cells and supernatant were collected at different time points post-infection.FQ-PCR was used to analyze PPV DNA quantity in different samples,and one-step growth curves were then drawn.At 4-20 h post infection,PPV DNA of extracellular virus increased slowly,virus DNA employed a logarithmic growth after 20 h and reached the maximum at 56 h.Afterwards the virus content decreased rapidly and tended to be stable at 68 h.At 4 h post-infection,microdosage PPV DNA of intracellular virus was detected,the virus content of intracellular virus increase slowly at 4-24 h post-infection,virus DNA employed a logarithmic growth after 24 h and reached the maximum at 44 h.Afterwards the virus content decreased slowly.While PPV multiplied to a certain number in PK-15 cells,virion was released from the infected cells and the maximum extracellular virion was higher than that of intracellular one.