HCV diversity and resistance analyzed by DNA bar coding and pyrosequencing

描述（由申请人提供）：此申请概述了一个5年计划，旨在培养候选人从事研究丙型肝炎病毒（HCV）感染的发病机制和耐药性机制的学术生涯。他在耶鲁大学完成了内科住院医师培训，并在宾夕法尼亚大学获得了传染病临床奖学金。他在机械酶学方面有很强的背景，在天普大学Charles Grubmeyer博士的指导下获得了生物化学博士学位，目前在他的赞助人Frederic Bushman博士的实验室工作，使用DMA条形码和焦磷酸测序研究慢病毒载体在基因治疗患者中的功能。王博士建立了一个全面的研究和培训计划，使他能够获得独立研究HCV发病机制和耐药性问题的专业知识。病毒学领域的世界权威Bushman博士和HCV发病机制专家Kyong-Mi Chang博士将共同指导候选人的科学发展。一个由著名科学家和临床医生组成的顾问委员会将指导他的职业发展。HCV是美国慢性肝病的主要病因。FDA唯一批准的干扰素治疗通常无效，但耐药性的基本机制在很大程度上尚不清楚。几种靶向病毒编码酶的化合物目前正处于临床开发阶段。然而，它们的使用几乎肯定会因为体内高度多样化的病毒种群而变得复杂，这可能导致复杂的耐药途径。最近，基因组测序技术在病毒耐药性问题上的应用使得大量鉴定病毒变异成为可能。在已发表的工作中，我们利用DNA条形码和焦磷酸测序来鉴定HIV中罕见的耐药突变，并监测体内逆转录病毒整合位点的位置。我们将把这些强大的工具与生物信息学和各种分子技术结合起来，以促进对HCV进化和体内耐药性的理解。我们提出以下目标：1)建立HCV高效扩增和高通量焦磷酸测序方法；2)研究干扰素治疗对病毒种群的选择性压力；3)明确HCV人群结构。建议的深入实验室和教学培训将指导候选人成为病毒发病机制和耐药性的独立研究者。

DESCRIPTION (provided by applicant): This application outlines a 5 year plan to train the candidate for an academic career investigating the pathogenesis of hepatitis C virus (HCV) infection and mechanism of drug resistance. Dr. Wang has completed residency training in Internal Medicine at Yale University and a Clinical Fellowship in Infectious Diseases at University of Pennsylvania. He has a strong background in mechanistic enzymology, having received a Ph.D. in biochemistry under Dr. Charles Grubmeyer at Temple University, and is now working in the laboratory of his sponsor, Dr. Frederic Bushman, studying the function of lentiviral vectors in gene therapy patients using DMA bar coding and pyrosequencing. Dr. Wang has established a comprehensive research and training program so that he can gain the expertise to independently attack questions of HCV pathogenesis and resistance. Dr. Bushman, a world authority in the field of virology, and Dr. Kyong-Mi Chang, an expert in HCV pathogenesis, will jointly mentor the candidate's scientific development. An advisory board of prominent scientists and clinicians will mentor his career development. HCV is a leading cause of chronic liver disease in the U.S. The only FDA approved treatment using interferon is often ineffective, yet the basic mechanisms of resistance are largely unknown. Several compounds targeting viral-encoded enzymes are currently in clinical development. However, their use will almost certainly be complicated by the highly diverse viral populations in vivo, which may result in complex drug resistance pathways. Recently, the application of genome sequencing techniques to such problems in viral resistance has allowed mass identification of viral variants. In published work, we have utilized DNA bar coding and pyrosequencing to identify rare drug resistant mutations in HIV and to monitor locations of retroviral integration sites in vivo. We will combine these powerful tools with bioinformatics and a variety of molecular techniques to advance understanding of HCV evolution and resistance in vivo. We propose the following Aims: 1) establish methods for efficient amplification and high-throughput pyrosequencing of HCV; 2) investigate the selective pressures on viral populations resulting from interferon therapy; 3) define the population structure of HCV. The proposed in-depth laboratory and didactic training will guide the candidate in becoming an independent investigator in viral pathogenesis and resistance.