"Despite effective vaccines, hepatitis B virus (HBV) infection remains a major health problem with 2 billion people infected worldwide. Among them, 350 million are chronically infected, a major risk factor for development of hepatocellular carcinoma (HCC). A new and efficient treatment against chronic infection and HCC is strongly needed, and therefore it is important to understand HBV replication and persistence. In the absence of a good model, woodchuck infection with woodchuck hepatitis virus (WHV) is used as the preferred system to study disease and by homology, HBV nature. In the present proposal we intent to establish a robust WHV reverse genetic system to study the virus replication and pathogenesis. By utilizing this WHV reverse genetic system, we plan to introduce two new strategies to generate virus-attenuated vaccines that could infect and interfere with wild type virus replication. WHV core is a structural protein required for virus replication and growth. We are proposing to construct a core deficient recombinant WHV that could replicate in wild type WHV infected cells, interfering with wild type infection. A second strategy focuses on Hepatitis B virus X (HBx) and woodchuck hepatitis virus X (WHx) multifunctional proteins, which are required for optimal HBV and WHV replication and development of WHV derived HCC. We propose to create recombinant WHV with a mutant WHx protein that will interfere with full-length protein activities if they are co-expressed. As an initial approach to study virus replication, we will utilize woodchuck hepatocyte xenograft transplants in RAG-1 transgenic mice. Finally, woodchuck animal model will be challenged to test the efficacy of recombinant viruses replication and interference with chronic infection and suppress HBV derived HCC. Our ultimate goal is to develop novel effective molecular therapeutic tools that could be combined with other HBV/WHV antiviral strategies to suppress chronic HBV infection and HBV derived HCC."