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特定序列空间的变化，虫媒病毒已经进化到在两种不同温度（27和37℃）下在两个分类群（哺乳动物和昆虫）的细胞中高效复制，并具有微妙平衡的密码子对偏性（CPB），以适应哺乳动物和昆虫的CPB差异。我们选择登革热病毒（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（加州伯克利）、研究虫媒病毒生物学的虫媒病毒实验室（纽约州奥尔巴尼）、专攻生物系统编码的计算机科学系（迈阿密大学）和分子遗传学和微生物学系（石溪大学）的研究人员。纽约)以研究正链病毒而闻名。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，使其包含在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（编码蛋白质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，但将在具有野生型动力学的昆虫细胞中复制。为了避免被编码的病毒对环境造成生物危害的可能性(通过Ae。埃及伊蚊（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。反过来，重新编码的DENV基因组也将在实验动物（小鼠和猴子）中测试其毒力和衰减。由于基本策略允许我们通过特异性下调病毒编码蛋白的表达来定制代理的毒力，我们的项目为研究DENV组织培养细胞和实验动物的复制表型提供了多种可能性。同时，它可能产生候选菌株，这些菌株可能适合作为疫苗进一步开发。然而，疫苗开发并不是这一应用所追求的主要目标。

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.