Functional characterisation of pre S1/pre S2 deletion mutants of hepatitis B virus isolated from Southern Africans
Date
2016
Authors
Wolhuter, Suzanne
Journal Title
Journal ISSN
Volume Title
Publisher
Abstract
Both HBV and HIV are hyperendemic in sub-Saharan Africa, and there is a
correspondingly high incidence of hepatocellular carcinoma (HCC) in this region,
contributing to a high burden of disease. In regions where HBV genotypes B and C
prevail, a strong relationship exists between the rapid and more likely development of
HCC and infection with HBV containing deletions in the preS region. Similar preS
deletion mutants have been detected in southern African and Indian HCC patients
infected with subgenotype A1. Disturbingly in a cohort study conducted in Mpumalanga,
South Africa by our team, equivalent deletion-mutants were detected in 5 treatmentnaïve
HBV-HIV coinfected patients. Thus, the aim of this study was to characterize the
quasispecies of HBV in these patients and to construct plasmids containing preS
deletion-mutants in a subgenotype A1 backbone, in order to functionally characterize
them in vitro. Such studies may determine how these preS deletions contribute to the
working model of hepatocarcinogenesis and explain the high hepatocarcinogenic
potential of subgenotype A1.
The quasispecies populations of HBV deletion mutants isolated from four HIV-positive
patients were analysed phylogenetically, using both Neighbor-Joining and Bayesian
methods. It was found that the preS deletion mutants represented the majority
population, as 70% or more of clones in each of the four patients were sequentially
highly similar to the respective parental strains. In addition to the major populations in
each patient, minor populations were identified in the quasispecies.
The overlength subgenotype A1 wild-type replication competent plasmid, was
successfully altered to remove an unwanted XbaI site. This altered construct served as a
positive control to test whether the removal of this restriction site would affect
replication and viral protein expression of this construct compared to the original
plasmid. This plasmid was then successfully used as the backbone to construct three
overlength deletion mutant constructs. Using a new strategy a 784 bp fragment flanking
preS deletions from each of 4 patients, was inserted into the subgenotype A1 backbone.
Three fragments were derived from isolates with preS deletion mutants and one
fragment, without the deletion, was isolated from a patient with occult HBV infection.
The resulting plasmids together with the appropriate control plasmids were used to
transfect Huh7 cells in culture. Viral replication was followed at days 1, 3 and 5 using
enzyme linked immunosorbent assay (ELISA) for hepatitis B surface antigen (HBsAg) and
Hepatitis B e antigen (HBeAg). All deletion mutants were shown to express HBsAg and
HBeAg at levels comparable to the controls, with the highest levels on day 3. There
were no significant differences in the expression of HBeAg in Huh7 transfected cells
between the deletion-mutant constructs and the positive controls. The HBV viral loads
(VL) were measured at the same time points, using real-time quantitative PCR (qPCR).
Both the extracellular (supernatant) and intracellular (lysate) compartment of the Huh7
cells were tested. Furthermore, to clarify whether encapsidated virus was being secreted,
an immunocapture technique on the supernatant was employed prior to DNA extraction
and qPCR. In a similar manner to the ELISA experiments, qPCR measurement of VL
showed that despite the replacement of the 784 bp fragment with a fragment containing
deletion mutations, all constructs were producing detectable amounts of HBV DNA
suggesting that viral replication was occurring. Analogous to the HBsAg results, the
highest VL was seen in all constructs on day 3 post transfection in both the supernatant
and lysate experiments. Cell-associated (lysate compartment) VL was not particularly
increased when compared to the VL measured in the supernatant, which does not
indicate an inability to secrete the mature virions, nor an accumulation of viral DNA
within the cell.
Negligible HBsAg expression was observed on days 1, 3 and 5 following transfection
with the overlength construct that contained the 784 bp fragment derived from a patient
with occult HBV infection. The VL following transfection with this construct was higher
than both positive controls at all three time points, in both the supernatant and lysate
compartment. This is again consistent with the clinical characteristics of the patient,
which also had high viral loads. This finding is novel, since the in vitro experiments
mimicked the phenotype of occult infection seen in the patient in vivo.
As far as we are aware, we are the first group to have constructed plasmids with deletion
mutants and an occult mutant in a subgenotype A1 backbone and to show that they
express viral proteins and viral DNA following transfection into HuH7 cells. These
constructs will be important resource for further research into the high
hepatocarcinogenic potential of subgenotype A1.
Description
Degree of Master in Science of Medicine
A dissertation submitted to the Faculty of Health Sciences at the University
of the Witwatersrand, Johannesburg, South Africa in fulfillment of the
requirements for the degree of Master in Science of Medicine
Johannesburg, 2016