Song D S, Yang J S, Oh J S, Han J H, Park B K
Department of Veterinary Medicine Virology Lab, College of Veterinary Medicine, Seoul National University, Seoul 151-742, Republic of Korea.
Vaccine. 2003 May 16;21(17-18):1833-42. doi: 10.1016/s0264-410x(03)00027-6.
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.
一株命名为DR13的猪流行性腹泻病毒（PEDV）在Vero细胞中分离出来，并连续传代至100代。在传代过程中定期对病毒进行滴定。使用计算机软件程序对传代水平为20、40、60、80和100时该病毒的开放阅读框（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田间毒株的适用性。通过聚合酶链反应（PCR）和病毒滴定观察到，DR13田间分离株在Vero细胞中连续传代适应。RFLP分析分别在传代水平75和90时鉴定出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以及研究的其他12株PEDV毒株区分开来。对细胞适应的DR13在仔猪和怀孕母猪中进行致病性和免疫原性测试。结果表明，细胞适应的DR13致病性降低，并在猪中诱导了保护性免疫反应。高度适应Vero细胞的病毒与野生型病毒之间的差异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。本研究结果支持细胞减毒病毒可作为抗PEDV感染的候选标记疫苗应用。
