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.