PRODUCTION OF ATTENUATED VACCINE CANDIDATE DENGUE VIRUSES FROM INFECTIOUS CDNA

登革热病毒基因组是一种正链RNA分子 大约11 kb。纯化的基因组RNA在转染后具有感染性 转化为合适的细胞。这个项目的目标是克隆一个cdna拷贝。 登革2型病毒的基因组，这样一个忠实的，有感染力的 基因组RNA的复制可以通过体外转录来实现。创作 感染性克隆的克隆将允许将定义的突变引入 登革热基因组。这样的功能在许多类型的 研究，包括绘制导致宿主范围改变的突变图谱 在嗜神经的DEN2菌株中，并试图减弱DEN2的作用 有可能被用作疫苗。我们一直使用的策略是做好准备 基因组RNA，然后用逆转录/聚合酶链式反应 来制造大量的cDNA.我们试图放大相对较大的 基因组的片段，所以只有几个克隆片段必须 组装成一份完整的复制品。我们通常可以扩增3-4kb 但我们不能可靠地放大更大的片段。因此， 已成功地将整个基因组扩增并克隆成四个片段。 其中的两个片段很容易地结合在一起，形成了正确的半基因组。 到目前为止，获取左半基因组的努力被证明是不成功的； 到目前为止分离的所有这样的克隆都被重新排列了。我们目前正在 寻求几种替代战略来解决我们的问题：我们正在 试图制造正确的四分之三基因组，以便产生 一种无需克隆的两段体外连接全长cDNA 整个基因组；我们正试图以较低的速度克隆左半基因组 在大肠杆菌中构建拷贝数载体，然后在 同样的载体；我们正在用设计成 繁殖“困难”的DNA片段；我们正开始尝试克隆 酵母。有希望的是，这些方法中的一种或多种很快会成功。

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 such that a faithful, infectious copy of the genomic RNA can be made by in vitro transcription. Creation of an infectious clone will allow introduction of defined mutations into the 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. The strategy we have been using is to prepare genomic RNA and then use reverse transcription/polymerase chain reaction to make large amounts of cDNA. 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. We can routinely amplify 3-4 kb segments, but we can not reliably amplify larger pieces. Thus, the entire genome has been successfully amplified and cloned as four pieces. Two of the pieces were easily combined to make the right half-genome. Efforts to obtain the left half-genome have so far proved unsuccessful; all such clones isolated to date have been rearranged. We are currently pursuing several alternative strategies to solve our problems: we are attempting to make the right three-quarter-genome, in order to produce a full-length cDNA by two-piece in vitro ligation without having to clone the entire genome; we are trying to clone the left half-genome in a low copy-number vector in E. coli, and then assemble a full-length clone in the same vector; we are experimenting with E. coli strains designed to propagate "difficult" pieces of DNA; we are beginning to try cloning in yeast. Hopefully, one or more of these approaches will soon succeed.