Thesis (M.D.) -- University of Adelaide, Dept. of Medicine, 1974.

Chronic hepatitis has multiple aetiologies which include viral hepatitis (hepatitis B, B+D and C), autoimmune hepatitis and drugs. In sub-Saharan Africa the major aetiological factor is chronic hepatitis B virus infection. In this region 10-15% of the population is chronically infected with the virus and 76% have serological evidence of past exposure to the hepatitis B virus. HDV infection has not been documented in Southern Africa but studies from Northern Africa show antibody positivity for HDV of 21-31 % in patients with chronic HBV infection. Drug-induced hepatitis is also increasingly being recognised as a significant entity. This study arose from the observation that there are a significant number of patients with autoimmune hepatitis on follow-up at the Groote Schuur Hospital liver clinic. This group includes patients who test negative for the standard autoimmune markers done at Groote Schuur Hospital but have liver histology that is typical of classical autoimmune hepatitis. They also show a clinical and biochemical response to steroid and azathioprine therapy that is indistinguishable from that seen in classical autoimmune hepatitis cases on similar treatment. This study is retrospective and covers the period 1978 - 1996. The patients studied are those with chronic hepatitis as defined by the International Working Party in 1995, i.e. patients with necro-inflammatory disease of the liver lasting at least 6 months. This includes hepatitis B, B + D, C, autoimmune hepatitis and drug-induced liver disease. Several other liver diseases that may present with clinical and histological features of chronic hepatitis are specifically excluded. These include Wilson's disease, Primary biliary cirrhosis, Primary sclerosing cholangitis, alpha-1-antitrypsin deficiency, alcohol abuse and iron over load states.

No description available.

A human isolate of hepatitis A virus (HAV) strain HAS-15 was adapted to rapid growth FRhK-4 cells and a one-step growth curve was determined. Detectable virion production was absent for approximately 20 h post-infection (p.i.) and was followed by a 4-day logarithmic phase of virus production. A maximum intracellular virus titer of 10⁹ radioimmunofocus-forming units (RFU) per milliliter was achieved and remained essentially constant for a period of up to 14 days p.i. An adsorption study with HAV HAS-15 using FRhK-4 cells demonstrated greater than 99.9% of infectious virus adsorbed at 25 C in less than 20 min. Milligram amounts of purified HAV HAS-15 were obtained from persistently-infected RFhK-4 cells. The HAV polypeptides were separated by sodium dodecyl sulfate - polyacrylamide gel electrophoresis and transferred to nitocellulose for detection by an enzyme-linked immunotransfer blot (EITB) procedure. HAV nucleotide-derived amino acid sequence was subjected to computer analysis to identify potential immunogenic regions within the HAV capsid polypeptides. Synthetic peptides corresponding to selected regions of each of the larger putative capsid polypeptides were coupled to keyhole limpet hemocyanin and used to immunize rabbits. EITB reactivities of HAV specific anti-peptide sera have allowed the identification of the gene order for the larger HAV P1 gene products and the determination of the following molecular weights: HAV VP2 or 1B (MW 27,000), HAV VP3 or 1C (MW 29,000), and HAV VP1 or 1D (MW 33,000). The disposition of the HAV capsid polypeptides with respect to the virion external surface was evaluated by EITB reactivity of HAV polypeptides with specific antisera. Hyperimmune rabbit anti-157S HAV and human IgM reacted with VP1, VP2, and VP3, while IgG reacted predominantly with VP1 and VP2. Further evaluation of the HAV virion structure was attempted by examining the relative accessibility of the virion polypeptides to various labeling reagents. Reaction of intact virions with Iodogen resulted in the predominant labeling of VP1 while labeling of VP2 and 3 was barely detectable. Selective labeling of VP1 under controlled conditions, combined with the anti-HAV IgG immunologic reactivity against VP1 and VP2, suggests that these two capsid components are more exposed on the virion surface and may play an important role in the generation of neutralizing antibodies.

Hepatitis B virus (HBV) is hyperendemic to southern Africa, the Asia-Pacific region, and the Amazon Basin. HBV causes both acute, and chronic infection of the liver that result in a wide spectrum of liver diseases ranging from acute mild subclinical infection, and an asymptomatic carrier state (ASC) to severe clinical manifestations, including, severe acute and, chronic hepatitis, which can progress to cirrhosis, and the development of hepatocellular carcinoma (HCC). Several viral factors have been implicated in the activation, and inhibition of apoptosis. The development, and progression of this wide spectrum of liver diseases are associated with an unregulated increase or decrease in hepatocyte apoptosis as well as a loss of balance between cell proliferation, and apoptosis. In southern Africa, genotype A of HBV is the predominant genotype, with subgenotype A1 prevailing. Individuals infected with subgenotype A1 have several unique characteristics, including relatively lower levels of HBV DNA, and early seroconversion of hepatitis B e antigen (HBeAg) to antibodies against HBeAg during the course of HBV infection. Infection with this subgenotype is associated with rapid disease progression, and high frequency of HCC development. Moreover, patients infected with subgenotype A1 had increased levels of apoptosis when compared to the other subgenotypes. The G1862T mutation occurs most frequently in subgenotype A1. In the clinical setting, South African HCC patients infected with G1862T subgenotype A1 strains had higher levels of apoptosis while ASCs patients infected with G1862T subgenotype A1 strains had lower levels of apoptosis, when compared to those infected with wild-type. To date, G1862T has been functionally characterized in subgenotype A2, genotype B and in a genotype D HBV genome containing a subgenotype A1 precore (PreC) region. The objectives of our study were to construct 1.28 mer replication competent clones containing an endogenous HBV promoter for wild-type subgenotypes A1, A2, and D3 as well as mutant G1862T subgenotype A1, and to functionally characterize these strains in tissue culture. Transfection of Huh7 cells was used to follow the viral replication, expression of HBeAg, activation of the unfolded protein response (UPR), and subsequent apoptosis. The strategy used for the generation of these replication competent clones had several advantages. Very few PCR errors were introduced, and carry-over of enzymes, nonspecific products, and reaction reagents downstream was prevented. The clones contained endogenous HBV promoters, and enhancers, and were generated from a single complete genome of HBV. These replication competent clones may be used in future studies for the establishment of stable cell lines that constitutively express HBV proteins without the need for further manipulation. This strategy can be used for the generation of replication competent clones belonging to genotypes A to D, and G, and with a few minor modifications, for genotypes E, F, and H. Using the newly generated clones, their replication competence was demonstrated using transfection of Huh7 cells. Even in the absence of an exogenous promoter, these clones were able to support the expression of intracellular, and extracellular HBV DNA at levels of 108 to 109 genome copies/ml. HBcAg, HBeAg, and HBsAg were expressed for a period of five days, and the order of expression was similar to that seen during acute HBV infection. Comparison of transfection with a replication competent clone containing an exogenous HBV promoter demonstrated higher expression of HBV DNA, and proteins, as well as an earlier expression, and accumulation of HBeAg in the endoplasmic reticulum (ER) relative to the clone containing an endogenous HBV promoter. This initial increased accumulation of HBeAg in the ER did not affect the level of activation of the UPR, but led to an increased level of total cell death as a consequence of necrosis. When comparing the different subgenotypes following transfection into Huh7 cells, subgenotype D3 replicated at a lower level, as measured by HBsAg, and HBV DNA levels, with HBeAg passing through the secretory pathway earlier, when compared to cells transfected with genotype A. There was no difference in the intracellular, and extracellular HBsAg between cells transfected with either subgenotype A1 or A2. However, cells transfected with subgenotype A1 had higher levels of intracellular replicative intermediates, HBeAg, and HBcAg, and lower extracellular expression of HBeAg from days 1 to 3, when compared to cells transfected with subgenotype A2. The intracellular retention of the PreC/ core (C) precursor protein in cells transfected with subgenotype A1 was clearly demonstrated by its lower expression in the secretory pathway, and its higher co-localization in the nucleus, using indirect immunofluorescence. This intracellular retention led to greater ER stress, and an earlier, and prolonged activation of the UPR. This correlated well with the higher PERK, ATF6, and IRE1/XBP1 activity seen on days 3 than on day 5. These findings suggest that the prolonged activation of the UPR in cells transfected with subgenotype A1 led to increased apoptosis, and subsequent induction of liver damage, and may therefore, be a contributing factor to the higher hepatocarcinogenic potential of subgenotype A1. Our study demonstrated that G1862T reduced replication, and led to the initial temporal retardation of intracellular core-particle-associated HBV DNA. Although, G1862T did not affect HBsAg expression, it led to a decreased expression of HBcAg, and HBeAg. The decreased expression of extracellular HBeAg was probably as a result of decreased cleavage efficiency by the signal peptide, which consequently led to the retardation of the PreC/C precursor protein in the ER, and ER-Golgi intermediate compartment (ERGIC), and its decreased expression in the nucleus. This retardation, and accumulation led to the earlier activation of all three UPR pathways, but not to increased apoptosis. Therefore, it is evident that G1862T does not completely abolish HBeAg expression, but affects the rate of HBeAg maturation, and its expression through the secretory pathway. These findings suggest that in response to the accumulation of HBeAg in the ER, the UPR was activated resulting in the alteration of the capacity to overcome this stress, consequently leading to a new homeostasis of the ER being reached. The capacity of the ER is increased, with no further activation of the UPR and apoptosis, which facilitates maturation of HBeAg. In conclusion, our study for the first time demonstrated that there are a number of factors that influence the expression of proteins in HBV transfection studies including the type of transcriptional promoter, the different genotypes/subgenotypes of HBV, the use of protein expressing as opposed to replication competent clones, and the presence, and absence of mutations, such as the G1862T. Therefore, when comparing the outcomes of various experiments these factors should be taken into consideration, and the results interpreted with caution, because experiments may not be strictly speaking comparable. Importantly, replication competent clones were generated from strains circulating in southern Africa. The generation of these clones is an important step in further functional characterization of African strains of HBV, and their comparison to strains circulating other geographical regions of the world. These strains, in particular, subgenotype A1 can develop unique mutations, such as the G1862T, which we demonstrated can influence the expression of HBeAg, in a way that it can possibly account for the higher hepatocarcinogenic potential of subgenotype A1.

Thesis ( M Med ( Virology ) )-- University of Limpopo (Medunsa Campus), 2013. / Introduction: The hepatitis B virus (HBV) causes a serious type of liver disease referred to as hepatitis B, which is associated with various fatal sequelae following the onset of chronic infection. As a result of the global burden of chronic HBV infection, HBV-related mortality is currently estimated at 620 000 annual deaths worldwide (Hwang and Cheung, 2011). The World Health Assembly (WHA) recommended in 1992 that the hepatitis B vaccine be incorporated into national immunization programmes universally, especially in the hyperendemic regions of the world, in order to curb the global burden of hepatitis B (WHO, 1992). Accordingly, South Africa introduced the hepatitis B vaccine into the national Expanded Programme on Immunization (EPI-SA) in April 1995. Almost 17 years later, South Africa has not conducted any nationwide serosurveys to monitor the population impact of the hepatitis B vaccine. Instead, a number of field and laboratory studies have been conducted only in vaccinated children within the first 5 years of life and as such reports on the short¬term impact made by the hepatitis B vaccine in the country have largely relied on these studies (Tsebe et al., 2001; Schoub et al., 2002; Simani et al., 2008). The aim of the current study therefore was to assess the population impact made by the hepatitis B vaccine post its introduction into EPI-SA using an age stratified, cross-sectional study. The objectives were to compare the prevalence of HBV exposure between the post- and pre-vaccination populations of South Africa, determine the influence of HIV infection on the prevalence of HBV exposure between the post- and pre-vaccination populations of South Africa by performing a subset analyses, and lastly, to perform molecular characterization of hepatitis B surface and polymerase genes in HBV DNA positive individuals. Materials and Methods: This was an explorative and descriptive retrospective, cross¬ sectional study based on recently tested and stored blood samples from the NHLS fi Diagnostic Laboratory in the Department of Virology. For the purpose of this study, these samples were obtained after Ethics approval, and two target populations identified based on the year (Le. 1995) the hepatitis B vaccine was introduced into EPI-SA; a post-vaccination population consisting of 605 blood samples from individuals aged 1-15 years and a pre¬vaccination population consisting of 601 blood samples from individuals aged 16-25 years. The post-vaccination population was further stratified by age as follows; 1-5,6-10 and 11-15 years, in order to assess immunity and chronic carriage of HBV across the different age groups. All samples were tested for the following primary serological markers; HBsAg, anti¬HBc and anti-HBs, to determine the prevalence of HBV chronic carriage, past HBV exposure and immunity to HBV infection respectively. Samples were further assessed for the vi incidence of acute HBV infection by testing for IgM anti-HBc. All serological testing was performed using the Elecsys@ 2010 Immunoassay System. Samples with serological evidence of infection or exposure to HBV were selected and screened for HBV DNA using a real time PCR assay to determine the prevalence of active HBV infection within this group. Study subjects with records of their HIV status, either positive or negative, were also pooled for subset analyses in order to determine the influence of HIV infection on immunity and chronic carriage of HBV. Finally, samples positive for HBV DNA were subjected to molecular characterization of the hepatitis B surface (S) and polymerase (pol) genes. Results: Following serological screening, immunity to HBV infection was found to be significantly (p<0.001) higher (56.7%) in the post-vaccination population than in the pre-vaccination population (15.5%). Within the post-vaccination population alone, immunity was found to wane with increasing age from 76.1 % in those 1-5 years of age to 50.0% in those 6-10 years and 44.2% in those 11-15 years of age. Chronic carriage on the other hand was significantly (p=0.008) reduced in the post-vaccination population with 1.5% HBsAg prevalence as compared to 4.0% in the pre-vaccination population. Within the different age strata, chronic carriage increased with increasing age (0.5% in 1-5 years; 1.3% in 6-10 years; 2.5% in 11-15 years). Overall, no acute HBV infection was detected within the post-vaccination population, while a 14.6% prevalence rate of acute HBV infection was found for the pre-vaccination population. From the subset analyses, immunity was found to be significantly (p<0.001) higher in the HIV uninfected population as compared to the HIV infected population; 82.5% versus 22.0% in the post-vaccination population and 26.7% versus 0% in the pre-vaccination population, while chronic carriage was found to be higher in the HIV infected population than in the HIV uninfected population. Following molecular characterization of the HBV S gene, it was revealed that the majority of the viral isolates were genotype A, with only 1 genotype D isolate found. A number of notable amino acid i$ variations were also detected within the antigenic region of the HBsAg of viral isolates, inCluding the K122R, N131T, T143S, and E164D mutations. Conclusion: Introduction of the hepatitis B vaccine into EPI-SA has shown remarkable success in children under the age of 5 years. Overall, immunity and chronic carriage of HBV within the post-vaccination population has been greatly impacted by hepatitis B immunization. Within the HIV infected population, susceptibility to HBV infection remains a cause for concern. Finally, although amino acid variations within the viral HBsAg are present, vaccine escape-related mutants appear to be rare or even absent within the South African population. vii

Thesis (M Med (Virology))-- University of Limpopo (Medunsa Campus), 2013 / Introduction: The hepatitis B virus (HBV) causes a serious type of liver disease referred to as hepatitis B, which is associated with various fatal sequelae following the onset of chronic infection. As a result of the global burden of chronic HBV infection, HBV-related mortality is currently estimated at 620 000 annual deaths worldwide (Hwang and Cheung, 2011). The World Health Assembly (WHA) recommended in 1992 that the hepatitis B vaccine be incorporated into national immunization programmes universally, especially in the hyperendemic regions of the world, in order to curb the global burden of hepatitis B (WHO, 1992). Accordingly, South Africa introduced the hepatitis B vaccine into the national Expanded Programme on Immunization (EPI-SA) in April 1995. Almost 17 years later, South Africa has not conducted any nationwide serosurveys to monitor the population impact of the hepatitis B vaccine. Instead, a number of field and laboratory studies have been conducted only in vaccinated children within the first 5 years of life and as such reports on the short¬term impact made by the hepatitis B vaccine in the country have largely relied on these studies (Tsebe et al., 2001; Schoub et al., 2002; Simani et al., 2008). The aim of the current study therefore was to assess the population impact made by the hepatitis B vaccine post its introduction into EPI-SA using an age stratified, cross-sectional study. The objectives were to compare the prevalence of HBV exposure between the post- and pre-vaccination populations of South Africa, determine the influence of HIV infection on the prevalence of HBV exposure between the post- and pre-vaccination populations of South Africa by performing a subset analyses, and lastly, to perform molecular characterization of hepatitis B surface and polymerase genes in HBV DNA positive individuals. Materials and Methods: This was an explorative and descriptive retrospective, cross¬ sectional study based on recently tested and stored blood samples from the NHLS fi Diagnostic Laboratory in the Department of Virology. For the purpose of this study, these samples were obtained after Ethics approval, and two target populations identified based on the year (Le. 1995) the hepatitis B vaccine was introduced into EPI-SA; a post-vaccination population consisting of 605 blood samples from individuals aged 1-15 years and a pre¬vaccination population consisting of 601 blood samples from individuals aged 16-25 years. The post-vaccination population was further stratified by age as follows; 1-5,6-10 and 11-15 years, in order to assess immunity and chronic carriage of HBV across the different age groups. All samples were tested for the following primary serological markers; HBsAg, anti¬HBc and anti-HBs, to determine the prevalence of HBV chronic carriage, past HBV exposure and immunity to HBV infection respectively. Samples were further assessed for the vi incidence of acute HBV infection by testing for IgM anti-HBc. All serological testing was performed using the Elecsys@ 2010 Immunoassay System. Samples with serological evidence of infection or exposure to HBV were selected and screened for HBV DNA using a real time PCR assay to determine the prevalence of active HBV infection within this group. Study subjects with records of their HIV status, either positive or negative, were also pooled for subset analyses in order to determine the influence of HIV infection on immunity and chronic carriage of HBV. Finally, samples positive for HBV DNA were subjected to molecular characterization of the hepatitis B surface (S) and polymerase (pol) genes. Results: Following serological screening, immunity to HBV infection was found to be significantly (p<0.001) higher (56.7%) in the post-vaccination population than in the pre-vaccination population (15.5%). Within the post-vaccination population alone, immunity was found to wane with increasing age from 76.1 % in those 1-5 years of age to 50.0% in those 6-10 years and 44.2% in those 11-15 years of age. Chronic carriage on the other hand was significantly (p=0.008) reduced in the post-vaccination population with 1.5% HBsAg prevalence as compared to 4.0% in the pre-vaccination population. Within the different age strata, chronic carriage increased with increasing age (0.5% in 1-5 years; 1.3% in 6-10 years; 2.5% in 11-15 years). Overall, no acute HBV infection was detected within the post-vaccination population, while a 14.6% prevalence rate of acute HBV infection was found for the pre-vaccination population. From the subset analyses, immunity was found to be significantly (p<0.001) higher in the HIV uninfected population as compared to the HIV infected population; 82.5% versus 22.0% in the post-vaccination population and 26.7% versus 0% in the pre-vaccination population, while chronic carriage was found to be higher in the HIV infected population than in the HIV uninfected population. Following molecular characterization of the HBV S gene, it was revealed that the majority of the viral isolates were genotype A, with only 1 genotype D isolate found. A number of notable amino acid i$ variations were also detected within the antigenic region of the HBsAg of viral isolates, inCluding the K122R, N131T, T143S, and E164D mutations. Conclusion: Introduction of the hepatitis B vaccine into EPI-SA has shown remarkable success in children under the age of 5 years. Overall, immunity and chronic carriage of HBV within the post-vaccination population has been greatly impacted by hepatitis B immunization. Within the HIV infected population, susceptibility to HBV infection remains a cause for concern. Finally, although amino acid variations within the viral HBsAg are present, vaccine escape-related mutants appear to be rare or even absent within the South African population. vii

Thesis equivalent (M.P.H.)--University of Michigan, 1947.

Thesis (doctoral)--Lund University, 1995. / Added t.p. with thesis statement inserted.

Thesis (doctoral)--Lund University, 1995. / Added t.p. with thesis statement inserted.