A Chicken Model to Study Hepatitis E Virus Pathogenesis

描述（由申请人提供）：戊型肝炎病毒（HEV）是人类戊型肝炎的病原体，在许多发展中国家是一个重要的公共卫生问题，在美国和其他工业化国家也是地方病。据报告，受感染孕妇的死亡率高达30%。由于缺乏实用的动物模型和足够的体外细胞培养系统，对HEV的复制和致病机制知之甚少，并且仍然没有针对HEV的疫苗。在为期4年的R 01奖的支持下，我们已经产生了大量的重要数据，包括构建禽HEV株的感染性cDNA克隆和建立独特的小型同源鸡HEV模型。由于我们已经完成了所有拟议的工作，并且由于该4年R 01于2006年6月14日结束（目前为1年无成本延长期），因此我们提交了两次修订（A2）的竞争性更新，以继续我们对这种重要但研究非常不足的人类病原体的长期目标。核心假设是，禽戊型肝炎病毒和鸡是一个有用的模型系统，以描绘戊型肝炎病毒的复制和发病机制。本研究的长期目标是以鸡和禽HEV为模型系统，通过禽HEV感染性cDNA克隆，确定HEV复制和致病机制，并鉴定在这些过程中起重要作用的HEV基因和遗传决定簇。两次修订的竞争更新有三个具体目的：（1）比较非致病性禽戊型肝炎病毒株与致病性禽戊型肝炎病毒株在30周龄产蛋鸡中的致病性：（2）以禽戊型肝炎病毒感染性cDNA克隆和鸡为模型，鉴定戊型肝炎病毒致弱的遗传因子;（3）了解小ORF 3蛋白在HEV复制和/或发病机制中的作用。这将通过使用标准技术来完成，包括动物实验、细胞培养、PCR、克隆和测序、诱变、分子生物学、生物化学、病毒学、血清学和病理学方法。继续支持这个项目，将使我们能够充分利用过去资助期产生的宝贵试剂和工具，利用独特的小型同源动物模型，对戊型肝炎病毒基因的结构和功能关系进行更深入的研究。本研究的数据将有助于针对这种重要的人类病原体设计有效的预防和控制措施。 对戊型肝炎病毒基本生物学和致病机理的认识不足，严重阻碍了戊型肝炎疫苗的研制。从该项目中获得的信息将有助于了解HEV复制和发病机制，并有助于制定有效的预防和控制策略，以对抗这种重要但研究非常不足的人类病原体。

DESCRIPTION (provided by applicant): Hepatitis E virus (HEV), the causative agent of human hepatitis E, is an important public health problem in many developing countries, and is also endemic in the United States and other industrialized countries. The mortality rate is reportedly up to 30% in infected pregnant women. Due to the lack of a practical animal model and a sufficient in vitro cell culture system, the mechanisms of HEV replication and pathogenesis are poorly understood and a vaccine for HEV is still not available. Supported by a 4-year R01 award, we have generated enormous amounts of important data including the construction of an infectious cDNA clone of an avian strain of HEV and establishment of a unique small homologous chicken model for HEV. Since we have completed all proposed work and since this 4-year R01 ended on June 14, 2006 (currently on a 1- year no-cost extension), we thus submit this twice-amended (A2) competing renewal to continue our long-term objectives on this important but extremely understudied human pathogen. The central hypothesis is that avian HEV and chickens is a useful model system to delineate the mechanisms of HEV replication and pathogenesis. The long-term objectives are to define the mechanisms of HEV replication and pathogenesis by using avian HEV and chickens as a model system and by using avian HEV infectious cDNA clone to identify HEV genes and genetic determinants that are functionally important for these processes. There are 3 specific aims in this twice-amended competing renewal: (1).Comparative pathogenesis between an apparently non- pathogenic strain of avian HEV and the pathogenic avian HEV strain in laying hens of 30 weeks of age; (2).Identification of genetic determinants for HEV attenuation by using avian HEV infectious cDNA clone and chickens as a model; and (3).Understanding the roles of the small ORF3 protein in HEV replication and/or pathogenesis. This will be accomplished by using standard techniques including animal experiments, cell cultures, PCR, cloning and sequencing, mutagenesis, molecular biology, biochemical, virological, serological and pathological methods. Continuing support of this project will enable us to fully utilize the precious reagents and tools generated from the past funding period to launch more in-depth studies on the structural and functional relationship of HEV genes using a unique small homologous animal model. The data from this study will help design effective preventive and control measures against this important human pathogen. The lack of knowledge on HEV basic biology and pathogenesis has greatly hindered the development of a vaccine against HEV. The information gained from this project will help understand the mechanisms of HEV replication and pathogenesis, and help devise effective preventive and control strategies against this important but extremely understudied human pathogen.