title: Construction of replication competent plasmids of hepatitis B virus subgenotypes A1, A2 and D3 with authentic endogenous promoters.
abstract: Hepatitis B virus (HBV) is hyperendemic to southern Africa, with genotype A of HBV being the predominant genotype, and subgenotype A1 prevailing. Infection with this subgenotype is associated with rapid disease progression, and high frequency of hepatocellular carcinoma development. The objectives of our study was to construct recombinant 1.28 mer replication competent HBV DNA plasmids of subgenotypes A1, A2 and D3 containing authentic endogenous HBV promoters and to follow their replication in vitro after transfection of Huh7 cells. We found that subgenotype D3 replicated at a lower level, as measured by HBsAg and HBV DNA levels, when compared to cells transfected with genotype A. There was no difference in the intracellular and extracellular HBsAg between cells transfected with subgenotypes A1 or A2. Cells transfected with subgenotype A1 had higher levels of intracellular replicative intermediates and HBcAg, and lower extracellular expression of HBeAg from days 1 to 3, when compared to cells transfected with subgenotype A2. In conclusion, the generation of these replication competent clones is an important step in the functional characterization of subgenotypes of HBV circulating in Africa and their comparison to strains circulating in other geographical regions of the world.
authors.affiliation: Hepatitis Virus Diversity Research Programme, Department of Internal Medicine, School of Clinical Medicine, Faculty of Health Sciences, University of the Witwatersrand, 7 York Road, Parktown, Johannesburg 2193, South Africa.
authors.forename: Nimisha Harshadrai
authors.initials: NH
authors.lastname: Bhoola
authors.affiliation: Department of Virology, Heinrich-Pette Institute, Leibniz-Institute for Experimental Virology and Immunology, University of Hamburg, 52 Martini Street, Hamburg 20251, Germany.
authors.forename: Kerstin
authors.initials: K
authors.lastname: Reumann
authors.affiliation: Department of Medicine, Groote Schuur Hospital, Main Road, Observatory, Cape Town 7925, South Africa.
authors.forename: Michael C
authors.initials: MC
authors.lastname: Kew
authors.affiliation: Department of Virology, Heinrich-Pette Institute, Leibniz-Institute for Experimental Virology and Immunology, University of Hamburg, 52 Martini Street, Hamburg 20251, Germany.
authors.forename: Hans
authors.initials: H
authors.lastname: Will
authors.affiliation: Hepatitis Virus Diversity Research Programme, Department of Internal Medicine, School of Clinical Medicine, Faculty of Health Sciences, University of the Witwatersrand, 7 York Road, Parktown, Johannesburg 2193, South Africa. Electronic address: Anna.Kramvis@wits.ac.za.
authors.forename: Anna
authors.initials: A
authors.lastname: Kramvis
mesh_terms: D002460:Cell Line; D005838:Genotype; D006511:Hepatitis B Antigens / Q000032:analysis; D006515:Hepatitis B virus / Q000235:genetics* / Q000502:physiology*; D022781:Hepatocytes / Q000821:virology; D006801:Humans; D010957:Plasmids; D011401:Promoter Regions, Genetic; D014162:Transfection; D014779:Virus Replication
chemical_list: D006511:Hepatitis B Antigens
keywords: Huh7 cells; In vitro replication and protein expression; Transfection