SARS Reverse Genetics

描述(申请人提供)：严重急性呼吸系统综合症是一种危及生命的人类疾病，其特征是老年人的死亡率超过50%。SARS冠状病毒含有一个约30Kb的单链正极性RNA基因组。SARS基因组全长感染性cDNA的可获得性不仅将提供对病毒的完全基因控制，还将允许合理设计活病毒作为候选疫苗。因此，我们认为，SARS反向遗传系统必须排在控制这种重要人类病原体的优先事项的前列。我们是美国唯一一个成功组装冠状病毒、小鼠肝炎病毒(MHV)和传染性胃肠炎病毒(TGEV)全长感染性cDNA的小组，并证明这些全长结构为研究冠状病毒复制和发病机制的遗传学提供了新的机会。在目标1中，我们将建立一个全长的SARS基因克隆，并通过生化分析和猕猴攻击实验比较拯救的分子克隆病毒和野生型病毒的表型。在目标2中，我们将开发用于表达载体和疫苗的高滴度SARS单击复制体。在目标3中，我们将筛选在小鼠细胞中复制的SARS宿主范围突变，利用反向遗传方法鉴定SARS跨物种传播的机制，并评估这些病毒在啮齿动物和非人类灵长类动物中的致病性。这项应用的目标是建立对SARS基因组的基因控制，并提供统一的试剂，供全国其他群体使用。

DESCRIPTION (provided by applicant): Severe acute respiratory syndrome is a life-threatening human illness characterized by mortality rates exceeding 50% in the elderly. The SARS coronavirus contains a approximately 30Kb single-stranded, positive polarity RNA genome. The availability of a full-length infectious cDNA of the SARS genome would not only provide complete genetic control over the virus, but allow for rational design of live viruses as candidate vaccines. Consequently, we believe that a SARS reverse genetic system must rank near the top of the priorities for controlling this important human pathogen. We are the only group in the US to have successfully assembled full-length infectious cDNAs of the coronaviruses, mouse hepatitis virus (MHV) and transmissible gastroenteritis virus (TGEV) and have demonstrated that these full length constructs provide novel opportunities for studying the genetics of coronavirus replication and pathogenesis. In aim 1, we will develop a full length SARS cDNA clone and compare the phenotype of rescued molecular cloned viruses with wildtype using biochemical assays and macaque challenge experiments. In Aim 2, we will develop high titer SARS single hit replicons for use as expression vectors and vaccines. In aim 3, we will select for SARS host range mutants that replicate in murine cells, identify the mechanism of SARS cross species transmission using reverse genetic approaches and evaluate the pathogenicity of these viruses in rodents and non human primates. The goal of this application is to establish genetic control over the SARS genome and provide uniform reagents that will be used by other groups throughout the country.