Thesis ( M Med (Virological Pathology))--University of Limpopo, 2010. / Introduction: Hepatitis B virus (HBV) is a serious problem worldwide causing
various liver diseases such as chronic hepatitis and hepatocellular carcinoma (HCC).
The pathogenesis of HBV related HCC is not well established. Hepatitis B X protein
(HBx) plays an important role in the pathogenesis of HCC. HBx coded by HBV X
gene enhances several cellular pathways in hepatocytes which may lead to HCC.
The genetic variability of other HBV genomic regions plays a significant role in
diagnosis, vaccine development and drug resistance. However, the genetic
variability of HBV X gene is not well understood. In addition the dual basal core
promoter mutations found within the X gene have been implicated in the inhibition of
hepatitis B e antigen (HBeAg) expression. Studies focusing on HBV X gene are
scarce in South Africa. Consequently HBV X gene variability may reveal interesting
mutations and substitutions that are important in chronic liver diseases or HCC. This
study aimed at characterising HBV X gene at a molecular level isolated from patients
with different serological profiles.
Methods: This was an exploratory study which used 20 stored sera (-70°C)
collected from adult patients at Dr George Mukhari hospital, Pretoria. The samples
were already tested for HBsAg, anti-HBs, anti-HBc and HBeAg serological markers
(Elecsys, Roche Diagnostics, Penzburg, Germany). HBV DNA extraction was
performed from serum using High Pure Viral Nucleic Acid Assay (Roche Diagnostics,
Penzburg, Germany). Nested PCR assay was used for the amplification of 465
nucleotide HBV X gene. Sequencing of PCR positive samples was done using
spectruMedix SCE2410 genetic analysis system. Six samples selected, were cloned
into the pGEM®-T Easy vector system (Promega, Madison, USA). Three clones of
each sample were selected and their plasmids purified using Pure Yield™ Plasmid
Miniprep System (Promega, Madison, USA). The plasmid DNA was recovered using
optimised nested PCR assay and sequenced. A total of 38 sequences were
generated from the study and compared with reference strains retrieved from
GenBank. Phylogenetic analysis based on HBV X gene sequences was done using
MEGA 4 software to determine different genotype clusters.
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Results: HBV X gene was successfully detected and amplified in 20 study samples.
The sequenced HBV X gene products revealed mutations and insertions. Particularly
a six nucleotide insertion, GCATGG between nucleotides 1611 and 1618 which was
detected in five samples. In addition, the six cloned samples confirmed the six
nucleotide insertion and other mutations associated with inhibition of hepatitis B e
antigen (HBeAg) detected in the study. The substitutions within HBx were detected
in the N (1-50 amino acids) and C (51-154) terminals by comparing our sequences
with archival sequences from GenBank. Important substitutions found within the N
and C terminals were S31A, P38S, A42P, F73L, H94Y, P101S, K118T, D119N,
I127T/N, K130M and V131I. These substitutions are associated with various
biological functions and pathogenesis. Other substitutions with unknown functions
detected in the study include A2G, A3G, A4G, C6W, P42S and V116L. Further
mutations of T1753M, A1762T and G1764A associated with inhibition of HBeAg
expression were detected in most samples and only one sample had C1766T
mutation. Phylogenetic analysis resulted in A, C and D HBV genotypes. Five
samples and 11 clones clustered with genotype D, two samples and four clones
clustered with genotype C and finally 13 samples and 3 clones clustered with
genotype A.
Conclusion: HBV X gene was successfully characterised using various molecular
methods. HBx substitutions detected are involved in various pathogenic effects and
may present a risk of HCC for patients infected with HBV. Genotype D samples
displayed most mutations/substitutions and this can be regarded as an important
genotype with high risk of HCC. The detection of a six nucleotide insertion
(GCATGG) in 5 samples may emerge as a new variant of genotype D. Furthermore
triple mutations of T1753M/A1762T/G1764A within basal core promoter region were
detected mostly in HBeAg negative samples. However further analysis of HBV X
gene variability is needed.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:ul/oai:ulspace.ul.ac.za:10386/417 |
| Date | January 2010 |
| Creators | Malinga, Lesibana Anthony |
| Contributors | Selabe, S.G., Mphahlele, M.J. |
| Publisher | University of Limpopo ( Medunsa Campus ) |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | xvii, 91 leaves. |
| Relation | Adobe Acrobat Reader, version 6.0 |
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