Infectivity of lymphoid cell-derived woodchuck hepatitis virus in an in vitro experimental system

Lew, Yuan-Yee (1999) Infectivity of lymphoid cell-derived woodchuck hepatitis virus in an in vitro experimental system. Masters thesis, Memorial University of Newfoundland.

[English] PDF (Migrated (PDF/A Conversion) from original format: (application/pdf)) - Accepted Version Available under License - The author retains copyright ownership and moral rights in this thesis. Neither the thesis nor substantial extracts from it may be printed or otherwise reproduced without the author's permission. Download (31MB) [English] PDF - Accepted Version Available under License - The author retains copyright ownership and moral rights in this thesis. Neither the thesis nor substantial extracts from it may be printed or otherwise reproduced without the author's permission. (Original Version)

Abstract

Hepatitis B virus (HBV), the prototypic virus of the Hepadnaviridae family, induces chronic liver disease in approximately 5% of the global population and is a cause of incurable hepatocellular carcinoma (HCC). The woodchuck infected with woodchuck hepatitis virus (WHV) has been validated as the most valuable natural model of HBV infection, especially in the absence of practical in vitro systems for HBV investigations. Although HBV and WHV are primarily hepatotropic, they also infect the host lymphatic system. The significance of this extrahepatic infection in the establishment and progression of liver disease is unknown. Therefore, it would be highly advantageous to develop an in vitro cell culture system in order to investigate the interaction of hepadnavirus derived from lymphoid cells with host hepatocytes. This interaction should not be complicated by host immune surveillance directed against the virus. The main objectives of this study were to design and establish an in vitro experimental system for the propagation of WHV in hepatocytes and lymphoid cells and to investigate the ability of virus derived from lymphatic cells and serum to replicate in cultured woodchuck hepatocytes. In this work, both cultured woodchuck hepatocytes were shown to be susceptible to WHV infection and conditions for short-term maintenance of WHV in cultured lymphoid cells were established. In addition, highly sensitive methods for the detection of WHV genes, replicative forms of the genome and virus antigens in in vitro infected woodchuck cells were adopted and validated. -- The obtained results provide, for the first time, direct in vitro evidence that WHV released by naturally infected lymphoid cells is infectious to the host hepatocytes. The data indicate that lymphoid cells in the spleen constitute a site where infectious virus replicates with higher efficiency than in circulating lymphoid cells. Furthermore, it was shown that WHV originating from lymphoid cells could be passaged serially through cultured hepatocytes and remain infective to a virus- naive woodchuck. These experiments demonstrated that the virus continues to be biologically active in both in vitro and in vivo conditions. Finally, preliminary experiments indicated that replication of WHV in the hepatocyte culture system could be suppressed by the presence of antibodies to the N-terminal domain of the WHV large envelope protein or by a synthetic analogue derived from the putative WHV cell binding site 1 (CBS1). This finding suggests that WHV CBS1 plays an important role in virus-hepatocyte interaction. -- The woodchuck hepatocyte culture system developed in this work will serve as an important tool to study the early events in WHV infection and replication, the effects of the cellular microenvironment on infection and genetic variation of the virus, and for evaluation of novel anti-hepadnaviral agents.

Actions (login required)

View Item