Differentiation of a Vero cell adapted porcine epidemic diarrhea virus from Korean field strains by restriction fragment length polymorphism analysis of ORF 3.
Differentiation of a Vero cell adapted porcine epidemic diarrhea virus from Korean field strains by restriction fragment length polymorphism analysis of ORF 3.
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DOI：
10.1016/s0264-410x(03)00027-6
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发表时间：
2003-05-16
期刊：
影响因子：
5.5
通讯作者：
Park BK
中科院分区：
文献类型：
--
作者：
Song DS;Yang JS;Oh JS;Han JH;Park BK
在 Vero 细胞中分离出指定为 DR13 的猪流行性腹泻病毒 (PEDV)，并以 100 级连续传代。以传代水平的规则间隔滴定病毒。使用计算机软件程序对第 20、40、60、80 和 100 代病毒的开放阅读框 (ORF) 3 序列进行比对和比较。研究了限制性片段长度多态性（RFLP）分析用于区分该病毒与其他韩国现场毒株的适用性。通过聚合酶链式反应 (PCR) 和病毒滴定观察到，DR13 野外分离株成功适应了 Vero 细胞。 RFLP 分析分别从传代水平 75 和 90 确定了 HindIII 和 Xho II 切割位点的变化； ORF 3 的这些 RFLP 模式将 Vero 细胞适应病毒与其亲本毒株 DR13 以及所研究的其他 12 种 PEDV 毒株区分开来。在仔猪和怀孕母猪中测试了细胞适应的 DR13 的致病性和免疫原性。结果表明，细胞适应的 DR13 降低了猪的致病性并诱导了保护性免疫反应。 Vero 细胞高度适应病毒和野生型病毒之间的分化可能是适应细胞培养的标志，也是 PEDV 感染流行病学研究的宝贵工具。这项研究的结果支持细胞减毒病毒可以作为针对 PEDV 感染的候选标记疫苗。
A porcine epidemic diarrhea virus (PEDV) designated DR13 was isolated in Vero cells and serially passaged by level 100. The virus was titrated at regular intervals of the passage level. Open reading frame (ORF) 3 sequences of the virus at passage levels 20, 40, 60, 80, and 100 were aligned and compared using a computer software program. Suitability of the restriction fragment length polymorphism (RFLP) analysis for differentiating the virus from other Korean field strains was investigated. The DR13 field isolate was successively adapted in Vero cells as observed through polymerase chain reaction (PCR) and titration of the virus. RFLP analysis identified change in cleavage sites of HindIII and Xho II from passage levels 75 and 90, respectively; these RFLP patterns of ORF 3 differentiated the Vero cell-adapted virus from its parent strain, DR13, and 12 other strains of PEDV studied. The cell adapted DR13 was tested for its pathogenicity and immunogenicity in piglets and pregnant sows. The results indicated that cell adapted DR13 revealed reduced pathogenicity and induced protective immune response in pigs. Differentiation between highly Vero cell-adapted virus and wild-type virus could be the marker of adaptation to cell culture and a valuable tool for epidemiologic studies of PEDV infections. The results of this study supported that the cell attenuated virus could be applied as a marker vaccine candidate against PEDV infection.
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