Tailoring virulence of dengue virus in mammals and mosquitoes

描述（由申请人提供）：我们计划探索虫媒病毒基因组的特定序列空间的变化，所述虫媒病毒已经进化为在两种不同温度（27和37 ℃）下在两个分类群（哺乳动物和昆虫）的细胞中有效复制，并且具有微妙平衡的密码子对偏好（CPB），其适应哺乳动物和昆虫中的CPB差异。我们选择了登革热病毒（DENV）作为我们的研究对象，它是世界上节肢动物传播的人类疾病的主要原因。这是一个真正的多学科项目，汇集了来自研究登革热病毒发病机制和分子生物学的领先实验室的研究人员（加利福尼亚州伯克利），来自虫媒病毒实验室（奥尔巴尼，纽约）研究昆虫传播病毒的生物学，来自计算机科学系（迈阿密大学），专攻生物系统编码，来自分子遗传学和微生物学系（斯托尼布鲁克大学，纽约）以正链病毒的研究而闻名。借助计算机和专门定制的算法，我们将重新编码DENV的基因组，使其包含哺乳动物中不受欢迎但在昆虫中正常的密码子对的大片段（编码蛋白E，NS3或NS5）。我们预测这些基因组在哺乳动物细胞中的复制将受到严格的限制，但在昆虫细胞中将以野生型动力学进行复制。为了避免重新编码的病毒可能对环境造成生物危害的可能性（通过Ae。埃及，它们的天然载体），将在蚊子中测试重新编码的病毒的复制表型。重新编码的DENV基因组反过来也将在实验动物（小鼠和猴子）中测试其毒力和减毒作用。由于基本策略允许我们通过特异性下调病毒编码的蛋白质的表达来定制药剂的毒力，因此我们的项目提供了研究DENV组织培养细胞和实验动物的复制表型的多种可能性。与此同时，它可以产生可能适合于进一步开发为疫苗的候选菌株。然而，疫苗开发并不是本申请所追求的主要目标。

DESCRIPTION (provided by applicant): We plan to explore changes of the specific sequence space of genomes of arboviruses that have evolved to replicate efficiently in cells of two taxa (mammals and insects) at two different temperatures (27 and 37 C) and with a delicately balanced codon pair bias (CPB) accommodating CPB differences in mammals and insects. We have chosen for our studies dengue virus (DENV) that is the leading cause of arthropod-borne human diseases in the world. This is a truly multidisciplinary project that brings together investigators from a leading laboratory studying dengue virus pathogenesis and molecular biology (Berkeley, CA), from an Arbovirus Laboratory (Albany, NY) studying the biology of insect borne viruses, from a Department of Computer Sciences (University of Miami) specializing in encodings of biological system, and from a Department of Molecular Genetics and Microbiology (Stony Brook University, NY) known for work on plus stranded viruses. With the aid of computers and specifically tailored algorithms we will recode the genome of DENV such that it will contain large segments (encoding either the proteins E, NS3, or NS5) of codon pairs dis-favored in mammals but normal in insects. We predict that these genomes will encounter severe restrictions of replication in mammalian cells, but will replicate in insects cells with wild type kinetics. To avoid the possibility that the recoded viruses could be a biohazard for the environment (via Ae. aegypti, their natural vectors), the recoded viruses will be tested in mosquitoes for replication phenotypes. The recoded DENV genomes, in turn, will also be tested for their virulence and attenuation in experimental animals (mice and monkeys). Since the basic strategy allows us tailoring the virulence of the agent by specifically down-regulating the expression of virus- encoded proteins, our project offers a variety of possibilities to study replication phenotypes of DENV tissue culture cells and experimental animals. At the same time it may yield candidate strains that may be suitable for further development as vaccines. Vaccine development, however, is not the primary goal pursued in this application.