一株具有急性致瘤特性的马立克氏病病毒的分离与鉴定

Isolation and Identification of a Field Isolate of Marek's Disease Virus with Acute Oncogenicity

应用鸭胚成纤维细胞(DEF)从免疫过CVI988/Rispens疫苗株患马立克氏病(MD)的三黄鸡中分离到一株马立克氏病病毒(MDV)(命名为YL040920株)。从该分离株蚀斑克隆获得的9个克隆在蚀斑形成时间及其形态大小上均无明显差别,表明它较为单一;应用聚合酶链式反应(PCR)技术扩增并测定了毒株的致瘤相关基因meq的核苷酸序列,并与其他MDV-1参考毒株的序列进行比较分析,发现其序列具有MDV-1强毒株的特征;用基于抗MDV-1 MEQ蛋白的单克隆抗体3G12E6的免疫荧光试验(FA)对毒株的DEF培养物进行检测,发现有特异性的荧光定位于细胞核内;应用毒株感染霞烟鸡,最早在接种后(PI)21d即可诱发明显的内脏器官,在各器官肿瘤中以心脏、肝脏和皮肤的肿瘤发生率最高;用禽肿瘤病三重PCR鉴别诊断技术对毒株的DEF培养物以及感染鸡的内脏器官组织进行检测,均能扩增到MDV-1强毒株的特异性带,而网状内皮增生症病毒(REV)和禽白血病病毒(ALV)的检测则均为阴性。研究的结果表明,分离株YL040920为单一的MDV-1强毒株,无REV、ALV以及疫苗株CVI988/Rispens的混杂,并具有以心脏、肝脏和皮肤肿瘤为主的急性致瘤特性。

A local field isolate of MDV named YL040920 was isolated from a commercial Three-Yellow chicken flock, which had been vaccinated with CVI988/Rispens vaccine and experienced significant MD visceral tumors, by transfecting monolayer of duck embryo fibroblast （DEF） with the peripheral blood lymphocytes （PBLs） of the bird. Nine individual clones of the isolate obtained by plaque assay were identical in the time of plaque formation and shape of plaques that demonstrated the purity of the isolate. The oncogenicity-related gene meq of the isolate was amplified by polymerase chain reaction （PCR） technique, and then was sequenced and compared with that of the other reference strains of MDV-1. The isolate was found to have the sequence characteristics of virulent MDV-1. Specific fluorescent staining localized in the nuclei of the infected DEFs by an immunofluorescence assay （FA） using an anti-MEQ monoclonal antibody 3G12E6. Xiayan chicks were challenged with the isolate and obvious tumors were observed at about PI 21d, showing the acute oncogenicity of the isolate. And among all the organs, heart, liver and skin had the highest tumor occurrences. The results of t-test showed that in term of the mortality and tumor occurrence, chickens of general line were significantly higher than those of chickens of MD resistant line （P 〈 0.05）, however there were no significant difference between the vaccinated and unvaccinated chickens with HVT（P〉0.05）. A developed differential diagnosis technique based on a multiplex PCR for avian neoplastic diseases was used to detect the DNA prepared from the virus infected DEFs and tumor samples of the challenged birds, the characteristic patterns of electrophoresis of the virulent MDV-1 were observed from all the samples detected, but were negative for detection of reticuloendotheliosis virus （REV） and avian leucosis virus （ALV）. The results of the study demonstrated that isolate YL040920, proved to be a pure virulent virus of MDV-1 and free of CVI988 virus, reticuloendotheliosis virus （REV） and avian leucosis virus （ALV）, posscesses the ability to cause acute tumors which mainly ocurred in heart, liver and skin.