Applications of infectious cDNA technology to RNA virus

感染性cDNA技术在RNA病毒中的应用

• 批准号: 6678955
• 负责人: B FALGOUT
• 金额: --
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6678955
• 关键词: Primates; RNA virus; attenuated microorganism; biological models; complementary DNA; dengue virus; encephalitis virus; genetic manipulation; immunity; immunologic substance development /preparation; molecular cloning; nucleic acid sequence; polymerase chain reaction; protein engineering; recombinant virus; tissue /cell culture; vaccine development; vector vaccine; virulence; virus genetics; virus infection mechanism

Summary: In a collaboration with Steve Whitehead and Brian Murphy at the NIH we had introduced a 30 nt deletion - which they had shown was attenuating for a DEN4 infectious clone - in the 3' non-coding region of our DEN1 WP infectious clone. The recovered DEN1 mutant virus grew more slowly than its parent, and made a smaller plaque. This virus has now been shown to be immunogenic and attenuated in monkeys. These results have been submitted for publication to the Journal of Virology. In collaboration with E Kelly, we had sequenced and made an infectious clone of a DEN2 PDK50 vaccine candidate and its virulent parent. Clone-derived viruses were comparable to their parents for growth kinetics in tissue culture cells. The cloned DEN2 PDK50 was manufactured under GMP by K. Eckels and his collaborators, and this virus was tested in monkeys for attenuation and immunogenicity; this study should be completed soon. We had also made an infectious clone of the Army's candidate live attenuated Japanese encephalitis virus (JEV) vaccine, a vero cell adapted version of a PDK cell derivative of the Chinese JEV live vaccine strain SA14 14-2. Clone-derived virus grew with the same kinetics as its parent in cell cultures. This virus was tested in mice and has been shown to be immunogenic. Finally, with help from Bangti Zhao I have been trying to make an infectious clone of DEN3 in the pRS424 yeast-E. coli shuttle vector. I was able to make a full-length DEN3 clone in yeast, but all attempts to recover this clone in E coli failed; the only plasmids which can be recovered have suffered large deletions of the DEN3 sequences. Subsequently, I introduced the mutF mutation into the 3' end of the DEN3 clone in yeast. Both the WT and mutF yeast clones were used in PCR to amplify the full 10.7 kb genome, using Expand polymerase. Full-length run-off RNA transcripts were made from these PCR products. Electroporation of these transcripts into cells resulted in the recovery of WT and mutF DEN3 viruses. Partial sequence analysis has shown that the mutF mutation has been retained, but that these viruses are not identical in the structural gene region, differing at two amino acids out of the approximately 1 kb sequenced. Preliminary analysis of the phenotype shows that DEN3 mutF makes a very small plaque, is severly growth restricted on LLCMK2 cells, and does not grow at all on mosquito C6/36 cells. This result is complicated to interpret, since there are an as yet unknown number of adventitious differences between the WT and mutF viruses, due to the use of full-length PCR in their derivation. Plans are underway to sequence both viral genomes, after repeating the phenotypic analysis. Also, work is underway to try to stabilize the DEN3 clone in E coli: I plan to introduce insertions at various locations in the left half of the genome; based on previous work in DEN2, I expect this to allow the clone to grow in E. coli. Subsequently, these insertions can be removed by restriction digestion prior to RNA transcription to make infectious RNA.

摘要：在与美国国立卫生研究院的Steve Whitehead和Brian Murphy的合作中，我们在我们的DEN1 WP感染性克隆的3‘非编码区引入了30个核苷酸的缺失-他们已经证明这对于DEN4感染性克隆是弱化的。恢复的DEN1突变病毒的生长速度比其亲本慢，并形成了较小的空斑。这种病毒现在已被证明在猴子身上具有免疫原性和减毒作用。这些结果已提交给《病毒学杂志》发表。 在与E Kelly的合作下，我们已经对DEN2PDK50候选疫苗及其强毒亲本进行了测序和感染性克隆。克隆病毒在组织培养细胞中的生长动力学与其亲本相似。克隆化的DEN2PDK50是由K.Ekerels和他的合作者在GMP下制造的，该病毒在猴子身上进行了减毒和免疫原性测试；这项研究应该很快就会完成。 我们还制作了陆军候选乙脑减毒活疫苗的感染性克隆，该疫苗是中国乙脑病毒活疫苗株SA14 14-2的PDK细胞衍生物的Vero细胞适应版本。克隆衍生病毒在细胞培养中的生长动力学与其亲本相同。这种病毒在小鼠身上进行了测试，并已被证明具有免疫原性。 最后，在赵邦迪的帮助下，我一直试图在pRS424酵母-E中获得具有感染性的DEN3克隆。Coli穿梭载体。我能够在酵母中克隆出全长的DEN3，但在大肠杆菌中恢复这个克隆的所有尝试都失败了；唯一可以恢复的质粒遭受了DEN3序列的大量缺失。随后，我在酵母中的DEN3克隆的3‘端引入了突变F突变。用WT和MutF酵母克隆进行聚合酶扩增，扩增出全长10.7kb的基因组。从这些聚合酶链式反应产物中获得全长的径流RNA转录本。将这些转录物电穿孔到细胞中，可以恢复WT和MutF DEN3病毒。部分序列分析表明，这些病毒的muT F突变被保留下来，但在结构基因区域并不相同，在大约1kb的序列中有两个氨基酸不同。初步的表型分析表明，DEN3muF在LLCMK2细胞上生长受到严重限制，在蚊虫C6/36细胞上完全不生长，形成一个很小的空斑。这一结果很难解释，因为由于在它们的派生过程中使用了全长PCR，在WT和MutF病毒之间存在着未知数量的不定差异。在重复表型分析后，正在计划对两种病毒的基因组进行测序。此外，正在进行尝试稳定DEN3克隆在大肠杆菌中的工作：我计划在基因组左半部分的不同位置引入插入；根据之前在DEN2中的工作，我预计这将使克隆能够在大肠杆菌中生长。随后，在RNA转录之前，这些插入片段可以通过限制性内切酶被移除，从而产生具有感染性的RNA。