Derdeyn C A, Frey T K
Department of Biology, Georgia State University, Atlanta 30302-4010.
Virology. 1995 Jan 10;206(1):216-26. doi: 10.1016/s0042-6822(95)80036-0.
During serial undiluted passage of rubella virus (RUB) in Vero cells, two species of defective-interfering (DI) RNAs of approximately 7000 and 800 nucleotides (nts) in length were generated (Frey, T. K., and Hemphill, M. L., Virology 164, 22-29, 1988). In this study, these DI RNAs were characterized by molecular cloning, hybridization with probes of defined sequence, and primer extension. The 7000-nt DI RNA species were found to be authentic DI RNAs which contain a single 2500- to 2700-nt deletion in the structural protein open reading frame (ORF) region of the genome. The 800-nt RNAs were found to be subgenomic DI RNAs synthesized from the large DI RNA templates. Analysis of the extent of the deletions using a reverse-transcription-PCR protocol revealed that the 3' end of the deletions did not extend beyond the 3' terminal 244 nts of the genome. The 5' end of the deletions did not extend into the nonstructural protein ORF; however, DI RNAs in which the subgenomic start site was deleted were present. Following serial undiluted passage of seven independent stocks of RUB, this was the only pattern of DI RNAs generated. DI RNAs of 2000 to 3000 nt in length were the majority DI RNA species in a persistently infected line of Vero cells, showing that other types of RUB DI RNAs can be generated and selected. However, when supernatant from the persistently infected cells was passaged, the only DI RNAs present after two passages were 7000 nts in length, indicating that this species has a selective advantage over other types of DI RNAs during serial passage.
风疹病毒（RUB）在Vero细胞中进行连续未稀释传代时，产生了两种长度约为7000和800个核苷酸（nt）的缺陷干扰（DI）RNA（弗雷，T.K.，和亨菲尔，M.L.，《病毒学》164，22 - 29，1988）。在本研究中，通过分子克隆、与特定序列探针杂交以及引物延伸对这些DI RNA进行了表征。发现7000 nt的DI RNA种类是真实的DI RNA，其在基因组的结构蛋白开放阅读框（ORF）区域含有一个单一的2500至2700 nt的缺失。发现800 nt的RNA是由大的DI RNA模板合成的亚基因组DI RNA。使用逆转录 - PCR方案分析缺失程度表明，缺失的3'端未延伸至基因组3'末端的244 nt之外。缺失的5'端未延伸至非结构蛋白ORF；然而，存在亚基因组起始位点被删除的DI RNA。在RUB的七个独立毒株连续未稀释传代后，这是产生的DI RNA的唯一模式。长度为2000至3000 nt的DI RNA是Vero细胞持续感染系中的主要DI RNA种类，表明可以产生和选择其他类型的RUB DI RNA。然而，当对持续感染细胞的上清液进行传代时，传代两次后存在的唯一DI RNA长度为7000 nt，表明该种类在连续传代过程中比其他类型的DI RNA具有选择性优势。
