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Expression of anti-HBV primary micro-RNA shuttles using an inducible promoter system.Mlambo, Tafadzwa 28 March 2014 (has links)
Hepatitis B virus (HBV) infection is an important global health concern and chronic carriers of the virus are at high risk of developing hepatocellular carcinoma (HCC) and cirrhosis. Current therapies are only partially effective, which emphasises the need for improved treatment strategies. Harnessing the RNA interference (RNAi) pathway as a treatment strategy against HBV has shown great promise. However, there are obstacles that need to be overcome before RNAi-based treatment of HBV infection is realised. These include problems of liver tissue targeting and dose regulation. This study investigated the use of a liver specific and mifepristone-inducible RNA polymerase (Pol) II promoter system for the specific and precise regulation of anti-HBV sequence expression. The inducible system used consists of two expression cassettes; one containing the
regulator/transactivator protein and another containing the transgene. Natural primary microRNA (pri-miR) mimics, pri-miR-31/5 and pri-miR-31/5/8/9, were used as anti-HBV sequences. Firefly luciferase gene expression was used to test modulation by the inducible system and to determine optimal induction conditions. The pri-miR-31/5, pri-miR-31/5/8/9 and luciferase encoding fragments were incorporated into the plasmid vector pRS17 that bears the inducible promoter, creating pRS-31/5, pRS-31/5/8/9 and pRS-Luc respectively. Firefly luciferase expression with this system was shown to be inducible and mifepristone dose-dependent. Effective knockdown of HBV gene expression was achieved with both pRS-31/5 and pRS-31/5/8/9 in vitro and in vivo. However, with high vector amounts, similar efficiency in silencing of HBV gene expression was observed in the presence and absence of the inducer mifepristone suggesting leaky expression of the pri-miRs. To confirm this, knockdown studies were carried out with the pri-miR-31/5/8/9-expressing cassette separated from the transactivator cassette. HBV gene expression knockdown was observed with the pri-miR-31/5/8/9 cassette alone confirming leaky expression from the inducible system. Leakiness appears to be as a result of the E1B promoter driving the expression of the pri-miRs in the absence of mifepristone. However, reducing the vector amounts decreased basal expression and improved the inducibility of the system in cell culture studies. Successful propagation of an inducible and liver-specific RNAi-activating expression system will address the difficulty of achieving dose control of RNAi effectors and contribute to advancing the use of RNAi for HBV treatment.
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Hepatitis-B-associated glomerular disease : a clinicopathological study of Hepatitis B virus associated Membranous Glomerulonephritis in Namibian and South African children 1974 - 2005 and a comparison with Hepatitis B associated Membranous Glomerulonephritis as well as Idiopathic Membranous Glomerulonephritis in adultsBates, William D. 12 1900 (has links)
Thesis (DMed)--Stellenbosch University, 2011. / ENGLISH ABSTRACT: Background and Objective: The most common cause of severe
proteinuria/nephrotic syndrome (NS) in children worldwide is minimal change disease
(MCD). This is also the pattern observed in white and Indian children in South Africa
(SA). By contrast, black and mixed race/coloured children of Southern Africa in the
1960s to 1990s were shown to have a different pattern of NS. One of the main
differences was the frequency of hepatitis B virus (HBV) associated
glomerulonephritis, usually membranous glomerulonephritis (MGN). The objective of
this project was a clinicopathological study of this subgroup of nephrotic children to
document the disease further and in particular to seek correlations between
pathological and clinical features including prognosis. A central focus was to
document the detailed ultrastructural examination of the renal biopsies of these
children and to correlate the spectrum of pathological features with demographic,
clinical, laboratory and prognostic features.
The hypothesis was that the clinicopathological features of HBV MGN in
children differed substantially from idiopathic MGN in general (children and
adults) and also from HBV MGN in adults and that HBV MGN in children should
be viewed as a distinct disease.
Patients and methods: The childhood (12 years and younger) patient cohort was
309 children with severe proteinuria/nephrotic syndrome who presented at Tygerberg
Hospital (TBH) over a 21 year period from 1974-1995, including 67 children from
Namibia. The study group was 71 children with HBV MGN who were followed up to
2005. The comparative adult group was 45 adults with MGN of whom 12 had HBV MGN and 33 idiopathic MGN. (A comparison could not be made with idiopathic MGN
in childhood as this centre only had 2 such patients during the study period.)
Demographic, clinical, laboratory and renal pathology data were collected, compared
and correlated.
Results: HBV associated MGN was the most frequent cause of NS in the Namibian
subgroup, 25/67 (37%) and the third most frequent, 71/309 (23%) in the childhood
cohort as a whole. The MGN group was 86% (71/83) of the total HBV childhood
nephrotic cohort, by far the dominant subgroup.
The average age of the 71 children with HBV MGN was 6.0 years (range 2-12 years)
at presentation and boys comprised 80% of the group. Hepatitis B envelope antigen
(HBeAg) was identified in the serum of 87% of children tested. Laboratory features
different from idiopathic MGN included more prominent haematuria, mildly raised
serum transaminases and more frequently lowered serum C3 and C4 levels. Light
microscopic examination of renal biopsies showed mesangial proliferation in all
patients but with minimal glomerular sclerosis and interstitial disease. On
ultrastructural examination mesangial and subendothelial deposits were common and
prominent as was mesangial interposition. The MGN of HBV in children therefore
frequently showed mesangiocapillary glomerulonephritis (MCGN) features in addition
to the subepithelial deposits of MGN. The subgroup of 23 whose renal biopsies
displayed severe mesangial interposition in addition to the subepithelial deposits of
MGN were termed the mixed HBV MGN-mesangiocapillary GN group. Virus like
bodies and tubuloreticular inclusion bodies were both found in more than 80% of
biopsies of childhood HBV MGN. HBeAg was identified in the subepithelial deposits
in the glomeruli. This was the first time this feature was demonstrated in Africa. The 46 South African children with HBV MGN showed a cumulative remission rate of 25% at 2 years and 52% at 4 years. Seven of the children (10%) of the total cohort
developed chronic renal failure (CRF). Age of 6 years and above at presentation and
severe mesangial deposits on biopsy correlated with fewer remissions and poorer
outcome. In 3 patients the interval between the diagnosis of HBV MGN and the onset
of CRF was more than 19 years with the longest being 23 years. The 358 cases of
childhood HBV MGN from Southern Africa constitute 37% of the reported childhood
patients.
Comparative data
A comparison was made between the 71 children with HBV MGN, 12 adults with
HBV MGN and 33 adults with idiopathic MGN. The main differences were that both
HBV MGN groups included only coloured and black patients and were more
predominantly male while the idiopathic MGN group included all races. In the HBV
patients, haematuria was more frequent and severe, liver enzymes were frequently
raised and C3 more frequently reduced than in the idiopathic cohort. Both groups of
adult MGN patients had normal C4 levels while the childhood HBV MGN group had
reduced C4 levels.
The immune complex pattern in both of the HBV MGN adult and childhood groups on
biopsy was similar with more mesangial and subendothelial deposits as well as
mesangial interposition than the idiopathic group. Despite this similarity between the
two HBV groups, both adult groups showed more glomerular sclerosis and interstitial
disease than the childhood group. The clinical outcome of the children’s cohort was
better than the other 2 groups with remission (52%) more frequent at 4 years (p<0.01) and better renal and patient survival.
Including the 83 cases from this series, at least 1243 renal biopsy proven cases of
HBV MGN have been reported in the English literature; children (80%) and adults (20%). The male gender predominance in both age groups for HBV MGN is similar
(children 79%; adults 84%) and significantly greater than for idiopathic MGN.
Conclusions: The findings confirm that HBV MGN in children is a distinct form of
GN which broadens the classical morphologic description of MGN by often including
a number of mesangiocapillary GN features. The subgroup of renal biopsies with the
most severe mesangiocapillary GN features was classified as the mixed HBV MGNmesangiocapillary
GN group. The MGN spectrum as a whole comprised 86% of the
HBV positive childhood group. HBV MGN was the most frequent association with
NS/severe proteinuria in the Namibian subgroup (37%) and the third largest group
(19%) in the SA children. It showed a relatively high spontaneous remission rate but
at least 10% of the children developed renal failure. Age of 6 years and above at
presentation and severe mesangial deposits on biopsy correlated with fewer
remissions and poorer outcome. Extended follow up (more than 15 years) was
required to demonstrate renal failure in some patients in the poor outcome group.
Urbanisation, associated with lower HBV carrier rates, and HBV vaccination (initiated
routinely in 1995 in SA), have already lead to a sharply decreasing incidence of this
disease in SA. HBV MGN has been a valuable and possibly unique model of human
GN and MGN in particular in that the HBeAg has been identified in both the serum
and glomeruli enabling confirmation of the aetiological role of HBeAg. het ’n kumulatiewe remissie koers van 25% teen 2 jaar en van 52% teen 4 jaar
getoon. Sewe van die kinders (10%) van die hele kohort het kroniese nierversaking
(KNV) ontwikkel. Ouderdom van 6 jaar en meer by presentasie en erge mesangiale
neerslae in ‘n biopsie het met minder remissies en ’n swakker uitkoms gekorreleer.
Drie pasiënte het meer as 19 jaar na aanvanklike voordoening ooglopende KNV
ontwikkel, waarvan 23 jaar die langste interval was. Die 358 gevalle van kinderjare
HBV MGN van Suidelike-Afrika maak 37% uit van die gerapporteerde kinder
pasiënte.
Vergelykende data
’n Vergelyking is getref tussen die 71 kinders met HBV MGN, 12 volwassenes met
HBV MGN en 33 volwassenes met idiopatiese MGN. Die hoof verskille was dat beide
HBV groepe net kleurling en swart pasiënte ingesluit het en meer oorwegend manlik
was, terwyl die idiopatiese groep alle rasse ingesluit het. In die HBV pasiënte was
hematurie meer algemeen en erg, lewer ensieme meer dikwels verhoog en C3 meer
dikwels verlaag as in die idiopatiese kohort. Beide groepe van volwasse MGN
pasiënte het normale C4 vlakke getoon terwyl die kindergroep met HBV MGN
verlaagde C4 vlakke bewys het. Die immuunkompleks patroon in biopsies van die
HBV MGN volwasse en kindergroepe was soortgelyk met meer mesangiale en
subendoteliële neerslae asook meer mesangiale interposisie as in die idiopatiese
groep. Ten spyte van hierdie ooreenkoms tussen die twee HBV groepe, het die twee
volwasse groepe meer glomerulêre sklerose en interstisiële siekte as die kindergroep
vertoon. Die kliniese uitkoms van die kinderkohort was beter as die ander twee
groepe met remissie (52%) wat meer algemeen was teen 4 jaar (p< 0.01) en met
beter nier- en pasïent oorlewing. Ingeslote die 83 gevalle van hierdie reeks, is ten minste 1243 nierbiopsie bewysde
gevalle van HBV MGN in kinders (80%) en volwassenes (20%) in die Engelse
literatuur gerapporteer. Die manlike oorheersing in beide ouderdomsgroepe van HBV
MGN is soortgelyk (kinders 79%; volwassenes 84%) en betekenisvol meer as vir
idiopatiese MGN.
Gevolgtrekkings: Die bevindinge bevestig dat HBV MGN in kinders ’n afsonderlike
vorm van GN is wat die klassieke beskrywing van MGN verbreed deur die algemene
insluiting van ’n aantal mesangiokapillêre GN kenmerke. Die ondergroep van nier
biopsies met erge mesangiokapillêre GN kenmerke is as die gemengde HBV MGNmesangiokapillêre
GN groep geklassifiseer. Die MGN spektrum in geheel het 86%
van die HBV positiewe kindergroep behels. HBV MGN was die mees algemene
assosiasie met NS/erge proteïenurie in die Namibiese subgroep (37%) en die derde
grootse groep (19%) onder die SA kinders. Die siekte het ’n relatiewe hoë spontane
remissiekoers getoon, maar ten minste 10% van die kinders het nierversaking
ontwikkel. Ouderdom van 6 jaar en meer by presentasie en erge mesangiale
neerslae in ‘n nierbiopsie het met minder remissies en ’n slegter uitkoms gekorreleer.
Uitgebreide opvolg (meer as 15 jaar) was nodig om nierversaking in sommige van
die swak uitkomsgroep aan te toon.
Verstedeliking is geassosieerd met laer HBV draersyfers en hierdie faktor saam met
algemene HBV inenting in die kinderjare (wat in 1995 in SA begin was), het ’n skerp
daling in die voorkoms van hierdie siekte in SA teweeg gebring. HBV MGN is ’n
waardevolle en moontlik unieke model van menslike GN en MGN, veral omdat die
HBeAg in beide die serum en glomeruli identifiseer kon word om die etiologiese rol
van HBeAg te bevestig. / AFRIKAANSE OPSOMMING: Agtergrond en Doelwit: Die algemeenste oorsaak van erge proteïenurie/nefrotiese
sindroom (NS) in kinders wêreldwyd is minimale veranderingsiekte. Hierdie patroon
kom ook voor in blanke- en Indiër kinders in Suid-Afrika. In teenstelling hiermee is
aangetoon dat swart en kleurling/gemengde ras kinders in Suider Afrika tussen die
jare 1960s tot 1990s ’n ander patroon van nefrotiese sindroom gehad het. Een van
die hoof verskille was die algemene voorkoms van hepatitis B virus (HBV)
geassosieerde glomerulonefritis, gewoonlik membraneuse glomerulonefritis (MGN).
Die doelwit van hierdie projek was ’n klinies-patologiese studie van hierdie subgroep
van nefrotiese kinders ten einde die siekte verder te beskryf en veral om korrelasies
te tref tussen patologiese en kliniese kenmerke insluitende prognose. Die
gedetaileerde ultrastrukturele ondersoek van die kinders se nierbiopsies en die
korrelasie van die spektrum patologiese kenmerke met demografiese, kliniese,
laboratorium en prognostiese kenmerke was ‘n sentrale fokusarea.
Die hipotese was dat die klinies-patologiese kenmerke van HBV MGN in
kinders wesenlik van idiopatiese MGN in die algemeen verskil (in kinders en
volwassenes) en ook van HBV MGN in volwassenes, en dat die beeld in kinders
as ’n afsonderlike siekte beskou behoort te word.
Pasiënte en metodes: Die kinder kohort (12 jaar en jonger) was 309 kinders met
erge proteïenurie/nefrotiese sindroom wie in Tygerberg Hospitaal (TBH) behandel
was oor ‘n 21 jarige periode vanaf 1974 tot 1995, insluitende 67 kinders van Namibië.
Die studiegroep was 71 kinders met HBV MGN wie waar moontlik tot 2005 opgevolg was. Die vergelykende volwasse groep was 45 volwassenes met MGN van wie 12
HBV MGN gehad het en 33 idiopatiese MGN. (’n Vergelyking met idiopatiese MGN
in kinders kon nie gedoen word nie omdat hierdie sentrum net twee sulke pasiënte
tydens die studietyd behandel het.) Demografiese, kliniese, laboratorium en
nierpatologie inligting is versamel, vergelyk en gekorreleer.
Resultate: HBV geassosieerde MGN was die algemeenste oorsaak van NS in die
Namibiese subgroep, 25/67 (37%) en die derde mees algemeen, 71/309 (23%) in die
kinder kohort as geheel. Die MGN groep was 86% (71/83) van die totale HBV kinder
nefrotiese kohort en verreweg die oorheersende subgroep.
Die gemiddelde ouderdom van die 71 kinders met HBV MGN by presentering was
6.0 jaar (reikwydte 2-12 jaar) en seuns het 80% van die groep behels. Hepatitis B
omhullingsantigeen (envelope antigen- HBeAg) is aangetoon in die serum van 87%
van die kinders wie daarvoor getoets is. Laboratoriumkenmerke wat van idiopatiese
MGN verskil het, het ingesluit meer prominente hematurie, gering verhoogde serum
transaminases en meer dikwels verlaagde serum C3 en C4 vlakke. Ligmikroskopiese
ondersoek van die nierbiopsies het mesangiale proliferasie in elke pasiënt getoon,
maar met minimale glomerulêre sklerose en interstisiële siekte. Met ultrastrukturele
ondersoek was mesangiale en subendoteliële neerslae asook mesangiale
interposisie algemeen. Die MGN van HBV in kinders het dus dikwels kenmerke van
mesangiokapillêre glomerulonefritis getoon bo en behalwe die subepiteliële neerslae
van MGN. Die ondergroep van 23 van wie die nierbiopsies erge mesangiale
interposisie aangetoon het asook die subepiteliale neerslae van MGN is die
gemengde HBV MGN-mesangiokapillêre GN groep genoem. Virustipe liggaampies
en tubuloretikulêre insluitingsliggaampies is in meer as 80% van die biopsies
bevestig. HBeAg was in die subepiteliële neerslae identifiseer. Dit was die eerste
keer dat hierdie kenmerk in Afrika identifiseer is. Die 46 Suid-Afrikaanse kinders
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Mucosal DNA vaccines for regionally unique pathogens: hepatitis B virus and penicillium marneffeiWong, Lei-po., 黃利寶. January 2002 (has links)
published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy
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Association of cytokine gene polymorphisms with susceptibility and disease progression in chronic hepatitis B virus (HBV) infectionLee, Wing-yan, 李穎欣 January 2007 (has links)
published_or_final_version / abstract / Paediatrics and Adolescent Medicine / Master / Master of Philosophy
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Relationship of serological markers, basic core promoter and precore mutations to genotypes of Hepatitis B virusLo, Kin-hang, Ken., 盧建恆. January 2009 (has links)
published_or_final_version / Medicine / Master / Master of Medical Sciences
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Recombinant expression and bioinformatic analysis of the Hepatitis B virus X proteinThompson, Liam Jed 18 September 2012 (has links)
There are an estimated 350 million people chronically infected with Hepatitis B Virus (HBV), of which
approximately 600 000 die each year from HBV complications including cirrhosis and liver cancer.
The X protein from HBV (HBx) has been implicated in the progression of chronic HBV to liver cancer
and has been reported to manipulate several critical cellular pathways. These include the cell cycle,
the tumour suppressor protein p53, protein degradation and signal transduction pathways. The role
of these interactions in HBV replication and the viral lifecycle is currently unknown. The lack of
animal models and infectable cell lines together with solubility and stability issues related to the
HBx protein have made progress difficult. The reliance on approximate cellular and animal models
has yielded many discordant studies that have confounded our interpretations of the role of HBx.
There have been no novel approaches attempting to express HBx at a quantity and quality sufficient
for high resolution X-ray and nuclear magnetic resonance structural determination. Additionally no
bioinformatic analyses have been applied to HBx, and thus distinctive features of HBx that may be
responsible for these challenges have not been reported.
This thesis describes the detailed experimentation to express and purify HBx in a functional, soluble
and stable form. The study focussed on Saccharomyces cerevisiae and Semliki Forest Virus
(SFV) expression systems, together with the use of a solubility-enhancing Maltose Binding Protein
protein tag (MBP). The S. cerevisiae-based pYES2 and YEp and mammalian expression vectors
showed production of HBx protein. However HBx that had been expressed using S. cerevisiae and
human cells could not be reliably detected in Western blots using antibodies raised against E. coliexpressed
HBx. This result was despite the positive visualisation of HBx using the same antibodies
and immunofluorescence microscopy. This validated previous reports describing the variable antigenicity
of HBx. Furthermore these findings supported the decision to develop eukaryotic-based
HBx expression vectors as results suggested structural differences between eukaryote and prokaryote
expressed protein. HBx was subsequently detected and purified in a soluble and active form
using an MBP tag as well as a SFV expression vector. All of these options provide an excellent point
from which further work at optimising HBx expression and structural elucidation can occur.
Bioinformatic analysis of HBx suggested the presence of protein disorder and protease sensitive
sites within the negative regulatory domain of HBx. Literature descriptions of the molecular promiscuity that protein disorder allows, offers an explanation for the presence of the discordant findings on
HBx interactions and functions. It is generally accepted that proteins containing disorder are tightly
regulated and thus experimental systems employing overexpression methodologies may encourage
cellular toxicity and non-specific interactions through the use of short linear motifs. Evolutionary
analysis of HBx sequences revealed that the eight HBV genotypes (A-H) showed concordance regarding
synonymous and non-synonymous substitutions at the overlapping and non-overlapping
domains of hbx. Substitutions in hbx were most common at positions where a synonymous substitution
occurred in the overlapping partner gene. The presence of sites under positive, neutral and
negative selection were identified across the length of HBx. The different genotypes showed positive
selection indicating selective pressures unique to each, thus offering a contributing explanation for
the variable disease severity observed between the subtypes.
Overall, this thesis has provided novel methods to express and purify HBx in S. cerevisiae and
mammalian cells. These methods, together with an increased understanding of the nature of HBx
sequences through bioinformatic analysis, pave the way to conduct both structural studies and biological
assays to elucidate the genuine roles of HBx in the HBV lifecycle and its contribution to the
progression to liver cancer.
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Optimisation of expressed RNA interference effecters for the inhibition of hepatitis B virus ereplicationEly, Abdullah 23 February 2010 (has links)
PhD, Faculty of Health Sciences, University of the Witwatersrand, 2009 / Chronic infection with the hepatitis B virus (HBV) is a major risk factor for
cirrhosis and hepatocellular carcinoma, which is the sixth most common cancer worldwide.
Available treatment for chronic HBV infection has limited efficacy in preventing
associated complications. The compact and multifunctional nature of the viral genome
limits its mutability making HBV an ideal candidate for therapy based on nucleic acid
hybridisation. The potent and specific gene silencing that can be achieved with RNA
interference (RNAi) has fueled interest in exploiting this pathway as a therapeutic
modality. Synthetic and expressed RNA sequences have been used to activate RNAi.
These engineered sequences mimic natural substrates of the RNAi pathway, which allows
them to enter and reprogramme the pathway to effect silencing of intended targets.
Tradionally expressed RNAi activators have been transcribed as short hairpin RNA
(shRNA) sequences from RNA polymerase III (Pol III) promoters. These shRNA mimic
precursor microRNA (pre-miRNA) and consequently enter the RNAi pathway at a
relatively late stage. Overexpression of shRNA sequences from Pol III promoters,
specifically the U6 promoter, has been associated with toxic side effects and has raised
concerns about the use of expressed RNAi activators. Another concern of developing
therapeutic RNAi expression cassettes is the emergence of HBV mutants that are resistant
to silencing by a single expressed RNAi effecter. These points have highlighted the need
for the development expressed RNAi activators that are effective at low concentrations and
capable of combinatorial silencing. To address these issues the aim of this study was to
assess the feasibility of anti HBV effecter sequences that mimic an early substrate (viz.
primary miRNA or pri-miRNA) of the RNAi pathway. Pri-miRNA expression is typically
under the transcriptional control of Pol II promoters. Consequently RNAi activators that
Abstract - xi -
mimic pri-miRNA, so-called pri-miR shuttles, may be expressed from Pol II promoters.
Initially a panel of shRNA expression cassettes driven by a Pol III promoter was
constructed and silencing of HBV replication assessed. Pri-miR shuttles were then
designed by incorporating guide sequences of the most effective anti HBV U6 shRNA into
naturally occurring pri-miR-122 and pri-miR-31. Potent inhibition of viral replication was
observed with both Pol III and Pol II-driven pri-miR shuttle expression cassettes in vitro
and in vivo. Subsequently liver-specific pri-miR-122 and multimeric pri-miR-31 shuttle
expression cassettes were created. Pri-miR-122 shuttle sequences expressed from the
alpha-1 antitrypsin promoter and HBV basic core promoter exhibited the best liver-specific
silencing. Polycistronic pri-miR-31 shuttle sequences were shown to produce multiple
RNAi activators capable of silencing multiple target sequences. Silencing by the pri-miR
shuttle sequences was independent of toxic effects that arise from induction of the
interferon response or saturation of the endogenous miRNA pathway. Pri-miR shuttles
clearly represent an improved option for the use of expressed shRNA and brings
therapeutic RNAi technology a step closer to clinical application.
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Development of a diagnostic ELISA for the hepatitis B x-protein using monoclonal antibodiesMashinini, Bongiwe 27 September 2010 (has links)
MSc (Med), Faculty of Health Sciences, University of the Witwatersrand / The hepatitis B virus remains a major public health problem even after decades of its discovery. Horizontal transmission during early childhood is the predominant mode of transmission in highly endemic regions such as sub-Saharan Africa. Infection exhibits a wide spectrum of clinical manifestations, from an asymptomatic stage to severe liver disease which may result in hepatocellular carcinoma (HCC). The HBV X protein (HBx) has been implicated in carcinogenesis, which often has a poor prognosis, consequently the use of highly specific monoclonal antibodies (mAbs) directed against HBx in an enzyme-linked immunosorbent assay (ELISA) could lead to early identification of HBV carriers at risk of developing liver cancer. A variety of mixed hybridoma cell cultures secreting anti-HBx antibodies were cloned and sub-cloned by “limiting dilution”. Clonal supernatants were assessed for anti-HBx antibody production by Indirect ELISA and Western/Immunoblotting. Monoclonal antibodies were then characterized according to their relative binding affinity (Indirect ELISA) and relative epitope specificity (Competitive ELISA). One of our monoclonal antibodies was found to bind to the same epitope on HBx as the commercial anti-HBx antibody and with the same high affinity.
In the developed Sandwich ELISA, our monoclonal antibody proved effective as the „detecting‟ antibody when the commercial anti-HBx antibody was deployed as the
„capture‟ antibody. This Sandwich ELISA will be further developed in our laboratory with the object of applying it to patient sera.
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Construction and screening of a DNA library to detect integrated hepatitis B virus DNABondonno, Catherine Patricia 16 August 2016 (has links)
Degree awarded with distinction on 6 December l995.
A dissertation submitted to the Faculty of Science, University the Witwatersrand,
in fulfilment of the requirements for the degree of Master of Science.
March. 1995 / Hepatitis B virus (HBV) infection resulting in integration of the viral DNA into host
liver cell DNA is associated with the development of hepatocellular carcinoma
(HCC). This is indicated by epidemiological trends, molecular studies and studies of
animal models infected with viruses closely related to HBv. However, little is
known about the mechanism by which the integrated HBV DNA includes HCC
despite continuing analysis of the integrated HBV DNA and its surrounding cellular
sequences. [Abbreviated Abstract. Open document to view full version]
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Characterization of mutants and splice variants of hepatitis B virus isolated from South African black hepatocellular carcinoma patientsSkelton, Michelle 15 February 2010 (has links)
Ph.D. thesis, Faculty of Health Sciences,University of the Witwatersrand, 2009 / Hepatitis B virus (HBV) infection is endemic in Africa. As many as 98% of black
Africans are infected during their lives and about 10% (65 million) have chronic
HBV infection, which is the cause of 70-80% of all hepatocellular carcinoma (HCC)
cases. Despite this high prevalence of HBV and the high incidence of HCC in
Africa, relatively few complete HBV genomes from African HCC cases have been
deposited in international data bases. In order to gain a clearer understanding of
the role of genetic variants and mutants in the development of HCC, the complete
genomes of HBV isolated from southern African HCC patients were amplified and
molecularly characterized. HBV DNA was extracted from the serum forty HBsAgpositive
HCC patients. Twenty six complete genomes were successfully amplified,
cloned and sequenced from nine HCC patients.
Phylogenetic analyses of the complete genomes and the individual open reading
frames of HBV isolates from the HCC patients, led to the classification of all the
isolates within subgenotype A1. No isolates belonging to subgenotype A2 and
genotype D were identified, even though these genotypes/subgenotypes have
been shown to circulate in South Africa. Three patients contained the uncommon
combination of serological subtype ayw1 in the subgenotype A1 strain. This
combination has been found previously in South Africa and the Phillipines.
Seventy-eight percent of the patients carried HBV strains with the double basic
core promoter (BCP) mutation (1762T/1764A), previously shown to reduce HBeAg
expression. Furthermore, complete genome sequence analysis has revealed a
complex combination of mutations, which include at least three or five of these
residues 1753C1762T1764A1766T1768A1809T1812T occurring as the dominant
HBV strains isolated from 5/9 HCC patients. These mutations have previously
been shown to regulate gene expression at various levels, to enhance viral
replication and simultaneously decrease HBeAg expression.
All five HBV genomes isolated from one patient contained novel complex BCP
rearrangements, which introduced 2 HNF1 and 1 putative HNF3 transcription
factor binding sites. These mutations can enhance viral replication and
simultaneously abolish HBeAg expression at a transcriptional level. Furthermore,
truncated core proteins would be expressed from 4/5 isolates and none would
express wild-type HBx. Several mutations were identified in the pre-S/S genes of
2/5 isolates, which would result in the expression of novel 3’ truncated medium
surface proteins (MHBst) and large surface proteins (LHBst). The majority of the
mutations would contribute to hepatocyte pathogenesis and transformation by
activating cell proliferating pathways.
Two patients also contained rare HBV variants not previously identified in HBV
strains from southern Africa. These included an HBV splice variant and a poly (dA)
variant from patient 10 and patient 6, respectively. These variants occurred in
combination with other isolates within the respective patients.
The envelope genes were characterised in a total of 18 HCC patients, the pre-S
gene of HBV contained deletions in 72% of the patients. Deletions across pre-
S1/pre-S2, pre-S2 initiation codon mutations with internal deletions, and S gene
nonsense mutations were prevalent. Mutated envelope proteins have been shown
to accumulate within the hepatocyte endoplasmic reticulum (ER) and are a
characteristic histopathological hallmark of HCC known as ground glass
hepatocytes. HBV induced ER stress has been shown to dysregulate several cell
cycle regulatory pathways, which contribute to HCC.
In addition several novel LHBst and MHBst have been described. These potential
transactivators require further investigation. The HBV mutations described in this
study have been associated with increased risk for HCC.
Despite the obvious heterogeneity HBV displays within and between patients,
there are common characteristics shared between the HBV variants which emerge
during the development of HCC. These include the BCP and pre-C
(1753C1762T1764A1766T1768A1809T1812T) mutations and the pre-S/S
mutations. These mutations are able to affect HBV replication and gene
expression, and may work synergistically to promote liver dysfunction and HCC.
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