3′-U3区碱基突变不影响禽白血病病毒的体外复制能力

The Mutations of 3′-U3 Region Make No Difference to ALV's Replication Capacity in vitro

为探索近年蛋鸡分离株中出现规律性突变的J群禽白血病病毒(ALV-J)3′-U3区对病毒体外复制能力的影响,以ALV-J原型毒株HPRS-103为骨架,利用融合PCR技术构建含蛋鸡分离株SD09DP04的3′-U3区的嵌合病毒的cDNA克隆。嵌合病毒的感染性克隆与原型毒株HPRS-103的感染性克隆分别在脂质体介导下转染DF-1细胞,对收获的病毒分别用间接免疫荧光试验、禽白血病抗原和反转录酶试剂盒检测,结果表明成功拯救到2株病毒。对拯救的2株病毒,进行其3′-U3区启动子活性和增强子活性的检测,以及复制动力学比较分析结果显示,出现特异性规律性突变的U3区对于原毒株的体外复制能力无显著影响。所构建的3′-U3区的嵌合病毒的成功拯救对于进一步探索近年ALV-J流行毒株其3′-U3区出现的规律性突变的功能及意义奠定了基础。

In order to study the influence of the 3′-U3 region with regular mutations of epidemic J subgroup avian leukosis virus(ALV-J)strains isolated from egg-type flocks on its replication capacity in vitro,a full-length cDNA clone was constructed based on ALV-J prototype HPRS-103 strain infectious clone via replacing its 3′-U3 region by the corresponding part of egg-type isolate SD09DP04.Both the constructed chimeric infectious clone and the HPRS-103 infectious clone were transfected into DF1 cells by lipofectamine 2000,respectively.The two infectious clones were passaged to another generation.And the harvested viruses were respectively confirmed by indirect immunofluorescence(IFA),ALV-antigen test kit and reverse transcriptase activity test kit.Analyzing the 3′-U3region's promoter activity or enhancer activity and TCID50 growth curves showed that such U3 region with regular mutations had no evident effect on the virus' s replication capacity in vitro.The successful rescue of the chimeric virus plays the basic role in further exploration of the functions and the significance of the regular mutations in the 3′-U3 region in the epidemic ALV-J strains in recent years.