PRODUCTION OF ATTENUATED VACCINE CANDIDATE DENGUE VIRUSES FROM INFECTIOUS CDNA

登革病毒基因组是一种正链RNA分子， 11 kb。 纯化的基因组RNA在转染入 合适的细胞。 该项目的目标是克隆一个cDNA拷贝的 登革病毒2型新几内亚C株（DEN2 NGC）的基因组，使得 基因组的忠实的、传染性的复制可以通过体外 使用可商购的SP6 RNA聚合酶进行转录。 Creatio 将允许将定义的突变引入到感染性克隆中， 登革热基因组。 这种能力在许多类型的研究中都是有用的， 包括绘制导致宿主范围改变的突变， 神经嗜性菌株的DEN2，并试图减弱DEN2的可能 用作疫苗。 已培养DEN2 NGC的原种并制备基因组RNA。 引物 逆转录聚合酶链反应（RT PCR） 并合成 我们试图扩大相对较大的部分， 基因组，所以只有少数克隆片段必须组装，使一个 完整的副本。 使用未引发的R 反应. 整个基因组除了右边的120个核苷酸外， 被成功放大。 左侧5.3kb的片段在一个 这是一个片段，但还没有克隆。 右边的3kb也被扩增了 但不是克隆的。 下一步是克隆基因组的剩余部分， 化学合成右边的120个核苷酸， 从碎片中克隆出的全长克隆体 希望体外转录 这个克隆将产生感染性RNA。

The dengue virus genome is a positive-strand RNA molecule of approximately 11 kb. Purified genomic RNA is infectious upon transfection into appropriate cells. The goal of this project is to clone a cDNA copy of the genome of dengue virus type 2, New Guinea C strain (DEN2 NGC) such that a faithful, infectious copy of the genome can be made by in vitro transcription using the commercially available SP6 RNA polymerase. Creatio of an infectious clone will allow introduction of defined mutations into th dengue genome. Such a capability would be useful in many types of studies, including mapping mutations responsible for altered host range in neurotropic strains of DEN2, and attempting to attenuate DEN2 for possible use as a vaccine. Stocks of DEN2 NGC have been grown and genomic RNA prepared. Primers for reverse transcription/polymerase chain reaction (RT PCR) have been designed and synthesized. We have tried to amplify relatively large segments of the genome, so that only a few cloned pieces have to be assembled to make a full-length copy. The best PCR results have been obtained using unprimed R reactions. The entire genome except for the right-hand 120 nucleotides has been successfully amplified. The left-hand 5.3 kb was amplified in one piece, but is not yet cloned. The right-hand 3 kb also has been amplified but not cloned. The next steps are to clone the remainder of the genome, chemically synthesize the right-hand 120 nucleotides, and then assemble a full-length clone from the pieces. Hopefully, in vitro transcription of this clone will produce infectious RNA.