Josan Chung

Viral clearance during hepatitis B virus (HBV) infection has been thought to reflect the destruction of infected hepatocytes by CD8(+) T lymphocytes. However, in this study, HBV DNA was shown to largely disappear from the liver and the blood of acutely infected chimpanzees long before the peak of T cell infiltration and most of the liver disease. These results demonstrate that noncytopathic antiviral mechanisms contribute to viral clearance during acute viral hepatitis by purging HBV replicative intermediates from the cytoplasm and covalently closed circular viral DNA from the nucleus of infected cells.

Hepatitis B virus (HBV) is a prototype for liver-specific pathogens in which the failure of the immune system to mount an effective response leads to chronic infection. Our understanding of the immune response to HBV is incomplete, largely due to the narrow host restriction of this pathogen and the limitations of existing experimental models. We have developed a murine model for studying human HBV replication, immunogenicity, and control. After transfection of hepatocytes in vivo with a replication-competent, over-length, linear HBV genome, viral antigens and replicative intermediates were synthesized and virus was secreted into the blood. Viral antigens disappeared from the blood as early as 7 days after transfection, coincident with the appearance of antiviral antibodies. HBV transcripts and replicative intermediates disappeared from the liver by day 15, after the appearance of antiviral CD8 + T cells. In contrast, the virus persisted for at least 81 days after transfection of NOD/Scid mice, which lack functional T cells, B cells, and natural killer (NK) cells. Thus, the outcome of hydrodynamic transfection of HBV depends on the host immune response, as it is during a natural infection. The methods we describe will allow the examination of viral dynamics in a tightly controlled in vivo system, the application of mutagenesis methods to the study of the HBV life cycle in vivo, and the dissection of the immune response to HBV using genetically modified mice whose immunoregulatory and immune effector functions have been deleted or overexpressed. In addition, this methodology represents a prototype for the study of other known and to-be-discovered liver-specific pathogens.

The virological and cellular consequences of persistent hepatitis C virus (HCV) infection have been elusive due to the absence of the requisite experimental systems. Here, we report the establishment and the characteristics of persistent in vitro infection of human hepatoma-derived cells by a recently described HCV genotype 2a infectious molecular clone. Persistent in vitro infection was characterized by the selection of viral variants that displayed accelerated expansion kinetics, higher peak titers, and increased buoyant densities. Sequencing analysis revealed the selection of a single adaptive mutation in the HCV E2 envelope protein that was largely responsible for the variant phenotype. In parallel, as the virus became more aggressive, cells that were resistant to infection emerged, displaying escape mechanisms operative at the level of viral entry, HCV RNA replication, or both. Collectively, these results reveal the existence of coevolutionary events during persistent HCV infection that favor survival of both virus and host.

To better define the mechanism(s) likely responsible for viral clearance during hepatitis B virus (HBV) infection, viral clearance was studied in a panel of immunodeficient mouse strains that were hydrodynamically transfected with a plasmid containing a replication-competent copy of the HBV genome. Neither B cells nor perforin were required to clear the viral DNA transcriptional template from the liver. In contrast, the template persisted for at least 60 days at high levels in NOD/Scid mice and at lower levels in the absence of CD4(+) and CD8(+) T cells, NK cells, Fas, IFN-gamma (IFN-gamma), IFN-alpha/beta receptor (IFN-alpha/betaR1), and TNF receptor 1 (TNFR1), indicating that each of these effectors was required to eliminate the transcriptional template from the liver. Interestingly, viral replication was ultimately terminated in all lineages except the NOD/Scid mice, suggesting the existence of redundant pathways that inhibit HBV replication. Finally, induction of a CD8(+) T cell response in these animals depended on the presence of CD4(+) T cells. These results are consistent with a model in which CD4(+) T cells serve as master regulators of the adaptive immune response to HBV; CD8(+) T cells are the key cellular effectors mediating HBV clearance from the liver, apparently by a Fas-dependent, perforin-independent process in which NK cells, IFN-gamma, TNFR1, and IFN-alpha/betaR play supporting roles. These results provide insight into the complexity of the systems involved in HBV clearance, and they suggest unique directions for analysis of the mechanism(s) responsible for HBV persistence.