Summary Significance: Hepatitis B virus (HBV) infects ~260 million people worldwide and is the leading cause of liver disease and hepatocellular carcinoma (HCC) in the world. Despite an effective vaccine for infection prevention, chronic hepatitis B virus infection (CHB) is difficult to cure once established. The viral genetic material enters the infected cell's nucleus generating a stable covalently closed-circular DNA (cccDNA) molecule or a linear form that integrates into the host cell's chromosome (iDNA). Existing treatments have little effect on cccDNA and as a result have poor success rates. Treatment interruption often leads to rebound of HBV DNA and recurrent hepatitis. In addition, even with treatment, liver disease in CHB often progresses to decompensated cirrhosis requiring liver transplantation. This process is exacerbated by several viral co-infections, such as with HIV or hepatitis C virus (HCV). Thus, improved therapeutics for CHB cure are required. Innovation: Our proposal is innovative in three aspects. i) access to precious and unique samples of liver tissue; ii) using state-of-the-art experimental techniques for single cell analyses and contemporaneous blood biomarkers; iii) development of new multi-scale models of HBV infection informed by our rich data sets, which are designed for clinical usefulness. Approach: HBV has a complex lifecycle comprising intracellular and extracellular phases. Our team has developed tools to interrogate the intracellular phase of HBV at the single-hepatocyte level. We have made preliminary observations of HBV in single cells but have only recently begun to link the intracellular and extracellular phases of HBV. Thus, we are poised to uncover key links in HBV biology between blood and liver. Using an integrated approach, we propose developing models of HBV that characterize replication in the organism uniting our understanding of intracellular and extracellular phases of the virus. Our specific aims are: i) To develop integrated models of intracellular and extracellular HBV replication, measuring and modeling a comprehensive set of HBV-related biomarkers in the blood and at the single-cell level in the liver, in untreated and treated CHB; and ii) To develop models of HBV spatial spread and persistence in the liver, using single-cell viral sequencing of HBV to make spatial linkages between genetically related HBV variants in the liver. These data and models will allow inferences about local versus non-local HBV spread. Specifically, we will test if clonal proliferation of HBV infected hepatocytes may contribute to continued persistence of infected cells, which has important implications not only for cure research but also for HCC oncogenesis. Impact: As multiple novel compounds for HBV treatment are in development, the timely insights generated in this proposal will fill gaps in our understanding of HBV replication and pathogenesis. Previous efforts by us and others in modeling HCV and HIV were impo...