Hepatocytes are a normally self-renewing population of cells with a very low turnover in a healthy liver. Hepatocyte life-times are thought to exceed six months. Hepadnaviruses such as HBV infect hepatocytes, which then shed large amounts of progeny virus into the blood stream. Virus production however, does not appear to harm hepatocytes. In addition, infection of hepatocytes is not immediately recognized by the immune system, which allows time for the virus to spread to the entire hepatocyte population. Infection of individual hepatocytes is maintained by ~30 copies of a non-replicating form of viral DNA which resides in the nucleus and is the template for viral mRNA synthesis. These infected cells eventually become targets of the host immune response. Once a strong immune response is mounted, the infection is cleared, typically over the next few weeks. Hepatocytes in the recovered liver are largely derived from hepatocytes that had been infected. How the viral nuclear DNA is destroyed and uninfected hepatocytes are derived from infected hepatocytes is yet unresolved.

In regard to this issue, three models have been proposed:

1. Cytokines produced during the inflammatory response to the infection induce the destruction of the nuclear DNA as well as viral DNA replication intermediates, which are found in the cytoplasm of infected hepatocytes.
2. Cytokines induce destruction of replicating viral DNA, but not the nuclear viral DNA. The nuclear viral DNA is lost during division of hepatocytes that divide to replace others killed by antiviral cytotoxic T lymphocytes (CTLs).
3. Cytokines induce destruction of replicating DNA, but nuclear DNA survives mitosis, so that uninfected cells are only created by passage through multiple rounds of cell division.

Model 3 has been eliminated by recently completed experiments in woodchucks. Experiments to distinguish models 2 and 3 are now planned.

Focus of virus free hepatocytes in a woodchuck liver chronically infected with WHV

Persistent infection by hepatitis B virus (HBV) leads to chronic liver disease, cirrhosis and hepatocellular carcinoma (HCC). We are investigating the hypothesis that HCC, and other hepatic lesions associated with chronic HBV infection, initiate with the emergence of mutated hepatocytes that have lost the ability to support HBV replication. These mutant hepatocytes would have a survival advantage because, unlike normal hepatocytes, they would no longer be targets of the antiviral immune response. This survival advantage would cause them to expand clonally to gradually replace normal hepatocytes. Two issues are currently being addressed: 1) Does immune evasion result in clonal expansion of hepatocytes during chronic infection of woodchucks with woodchuck hepatitis virus (WHV) and chimpanzees and humans with HBV (Figure: Focus of virus free hepatocytes in a woodchuck liver chronically infected with WHV); 2) Is this clonal expansion responsible for the observed decline in infected hepatocytes and the increased risk of liver cancer during chronic infection.

Our studies in woodchucks previously showed that the chronically infected woodchuck liver contains, after a few years, >100,000 

Clonal expansion of hepatocytes in a chimpanzee chronically infected with HBV

clones of greater than 1000 hepatocytes, suggesting a strong selection for clonal expansion of selected hepatocytes after WHV infection. Recent studies, in collaboration with R. Lanford and A. Jilbert, indicate a similar size distribution of clones in chronically infected chimpanzees (Figure: Clonal expansion of hepatocytes in a chimpanzee chronically infected with human HBV), despite a 10-fold or greater duration of infection in the chimpanzees than in the woodchucks. This apparently lower rate of progression of clonal expansion may correlate with the fact that, unlike WHV in woodchucks and HBV in humans, HBV causes a milder liver disease that only rarely progresses to HCC in chimpanzees. Studies using archived human liver tissue provided as part of collaborations initiated with Dr. C. Liu (Dept. of Pathology, University of Florida) and Dr. Matthew Yeh (Dept. of Pathology, University of Washington) have yielded similar results.  Large clones of hepatocytes are found in chronically infected human liver, even in the absence of cirrhosis, which is known to be associated with clonal hepatocyte expansion.