整合进禽反转录病毒长末端重复序列的马立克氏病病毒的分离鉴定

应用鸭胚成纤维细胞(DEF)从曾免疫过CVI988/Rispens株疫苗的患马立克氏病(MD)肿瘤的三黄鸡中分离到一株马立克氏病病毒(MDV,命名为GXY2株。用禽肿瘤病聚合酶链式反应(PCR)鉴别诊断技术对患鸡的肿瘤组织病料及克隆纯化毒株的DEF培养物进行检测,结果均扩增到MDV-1强毒株的132-bpr特异性带和网状内皮组织增殖病病毒(REV)的长末端重复序列(LTR)。用基于抗MDV-1的gB蛋白单克隆抗体BA4、MEQ蛋白单克隆抗体3G12E6和抗REV的单克隆抗体11B118分别对毒株的培养物进行间接免疫荧光试验(IFA),结果样品只与抗MDV-1的单克隆抗体呈现阳性反应,而与抗REV的单克隆抗体呈现阴性反应。应用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,结果发现其序列与我们之前分离鉴定的MDV-1野强毒株G2和YL040920高度同源。研究的结果表明,分离株GXY2为整合有REVLTR片段的重组MDV强毒株。

Identification of a field isolate of Marek's disease virus integrated with retroviral long terminal repeat (LTR) sequence

A field isolate of Marek's disease virus (MDV) named GXY2 was obtained from a commercial Three-Yellow chicken flock, which had been vaccinated with CVI988 vaccine and experienced significant MD visceral tumors, by transfecting the monolayer of duck embryo fibroblast (DEF). A developed differential diagnosis technique based on a multiplex polymerase chain reaction (PCR) for avian neoplastic diseases was used to detect the tumor samples of birds and the DEFs transfected with the cloned viruses. The result showed that the specific 132-bpr sequence of the virulent MDV-1 and the long terminal repeat(LTR)sequence of reticuloendotheliosis virus (REV) were detected from all the samples. The DEFs infected with GXY2 were analyzed by an indirect immunofluorescent assay (IFA) using MDV-1 specific monoclonal antibodies (McAb) BA4 and 3G12E6, and 11B118 of REV-specific, respectively. The results showed that the samples had specific fluorescent staining with the anti-MDV-1 McAb, but not with the anti-REV McAb. The oncogene meq of the isolate was amplified by PCR technique, and then sequenced and compared with those of the other reference strains of MDV-1. The result also showed that the sequence was highly homologous with those of the previous isolated virulent viruses G2 and YL040920. Together, our results sugested that isolate GXY2 is a MDV-1 virulent strain integrated with LTR sequence of REV.