Liver Cancer Research – Prof Anna Kramvis
- Hepatitis Virus Diversity Research Programme, Department of Internal Medicine, University of the Witwatersrand
- Read profile of Prof Kramvis
- E-mail: Anna.Kramvis@wits.ac.za
Title of the project
Functional characterization of pre-S1/pre-S2 deletion mutants of Hepatitis B virus isolated from Southern African hepatocellular carcinoma patients.
Highlights of the project
Ethics clearance was obtained from Wits Human Research Ethics Committee [#M10226] to undertake this project. We have identified a number of deletion mutant strains to be used to construct our replication competent clones. Deletion mutants have been identified in HIV infected individuals. The wild-type (no deletion) replication competent clone has been constructed, sequenced and shown to be replication competent in tissue culture. The intracellular and extracellular expression of viral proteins in cells transfected with this clone have been monitored using enzyme linked immunosorbent assay and confocal microscopy. We have now designed a new strategy for constructing the deletion mutant clones.
Non-Scientific Progress Report
Hepatitis B virus (HBV) has been shown to be a major causative agent of liver cancer (hepatocellular carcinoma). Various mutants of this virus, containing deletions in the genome, have been isolated from liver cancer patient and individuals co-infected with HBV and HIV. The aim of the present study is to follow the replication of representative deletion mutants in comparison with the wild-type virus, in order to determine whether these mutants have characteristics that can lead to liver cancer.
HBV cannot infect cells in tissue culture and this has hindered the study of viral replication in tissue culture in the laboratory. As an alternative, one can construct artificial clones of the genome of the virus in the laboratory and introduce them into tissue culture cells and follow the replication of the virus. These constructs are known as “replication competent clones” because they are capable of producing virus in tissue culture. We have optimized a strategy in our laboratory to produce replication competent clones and have generated clones from virus without the deletions in the genome.
During the phase of the research (June 2011 to October 2011), we attempted to use this strategy to generate a replication competent clone of a deletion mutant of HBV. Two fragments of the viral genome were amplified and these were introduced into piece of DNA derived from bacteria to produce the replication competent clone. The fragments are joined together in a manner, which allows the genome to code for the components of the virus and to produce viral progeny when introduced into immortalized liver cancer cells in the laboratory. Unfortunately this strategy proved unsuccessful in generating a clone of the deletion mutant. We have now devised an alternative strategy and new Masters student, Ms Suzanne Nicholson, will continue with this project in 2012. We have also optimized the techniques to be used in order to follow the expression of viral proteins under the microscope. This will be tested for both the wild-type and deletion mutant clones.
- Makondo, E; Bell, T; Kramvis A: Genotyping and molecular characterization of hepatitus B virus (HBV) from Human Immunodeficiency Virus (HIV) Infected Individuals in Southern Africa (manuscript in preparation).
- Bell, T; Makondo, E; Martinson, N; Kramvis, A: “Hepatitis B Virus Infection in HIV-positive Adults from Rural South Africa: Occult or Overt – that is the question“, International Meeting on Molecular Biology of Hepatitis B Viruses, International Conference Hall, Humanities and Social Sciences Building, Academia Sinica Taipei, Taiwan. October 9-13, 2010
- Makondo, E; Bell, T; Kramvis, A: Genotyping and molecular characterization of hepatitis B virus (HBV) from human immunodeficiency virus infected South African patients – International Meeting on Molecular Biology of Hepatitis B Viruses, International Conference Hall, Humanities and Social Sciences Building, Academia Sinica Taipei, Taiwan. October 9-13, 2010
- Hepatitis B virus research
- Hepatocellular carcinoma research
- Liver cancer research