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.

Hepatitis B virus (HBV), the prototype member of the family Hepadnaviridae, is hepatotropic and replicates by reverse transcription. HBV is responsible for the chronic infection of more than 240 million people worldwide, of which 65 million reside in Africa. The nine HBV genotypes (A to I) identified to date, are geographically distributed and exhibit different clinical manifestations and treatment responses. The term occult HBV infection (OBI) refers to a HBV infection in which HBV surface antigen (HBsAg) cannot be detected by conventional serological assays as has been defined by the Taormina expert panel. . HBV and human immune deficiency virus (HIV) are both endemic in many parts of the world and share common transmission routes. Worldwide, 10% of those infected with HIV are also chronically infected with HBV. HIV co-infection has been shown to be a risk factor for the development of OBI in individuals infected with HBV. The aim of this study was to characterize, at the molecular level, HBV from mono-infected and HBV/HIV co-infected individuals in Sudan The objectives of this study were the systematic and comparative analysis of HBV genotype D sequences, available in the public databases; the molecular characterization of HBV from mono-infected Sudanese liver disease patients and from HBV/HIV co-infected Sudanese patients; and the development and testing of bioinformatics tools to explore HBV sequence data generated using ultradeep pyrosequencing (UDPS) and comparison of UDPS results with those obtained from cloning based sequencing (CBS). All available complete genomes of genotype D of HBV from the GenBank database were analyzed. The intra-group divergence of the subgenotypes ranged from 0.8% + 0.5 for subgenotype D6 to 3.0% + 0.3 for subgenotype D8. Phylogenetic analysis of genotype D showed separation into six distinct clusters (subgenotypes D1, D2, D3/D6, D4, D5 and D7/D8), with good bootstrap support. The mean intergroup divergence between subgenotype D3 and subgenotype D6 was 2.6%, falling below the accepted threshold of 4% required to define a subgenotype. This suggests that subgenotypes D3 and D6 are the same subgenotype because they also share signature amino acids. Furthermore, subgenotype D8 is a genotype D/E recombinant, which clusters with subgenotype D7. This analysis provided an update on the classification of the subgenotypes of genotype D of HBV. Although HBsAg seroprevalence in Sudan, a central-African country, is greater than 8%, the only sequencing data for HBV, available prior to our study, was from asymptomatic blood donors, where genotype E predominates, followed by genotype D and subgenotype A2. Ninety-nine HBV-positive liver disease patients were enrolled in our study, including: 15 with hepatocellular carcinoma (HCC), 42 with cirrhosis, 30 asymptomatic carriers, 7 with acute hepatitis and 5 with chronic hepatitis. The surface and basic core promoter/precore (BCP/PC) regions, and the complete genome of HBV were sequenced. Eighty-two percent of the samples from HBV mono-infected liver disease patients were genotyped. Fifty-nine percent were infected with genotype D (74% D1, 10% D2, 3% D3 and 13% D6), 30% with genotype E, 8.5% with genotype A and 2.5% with a genotype D/E recombinant. Patients infected with genotype E had a higher frequency of HBeAg-positivity (29.2%) and higher viral loads compared to patients infected with genotype D. BCP/PC region mutations, including the G1896A mutation, seen in 37% of the HBeAg-negative individuals, could account for the HBeAg-negativity. A total of 358 Sudanese HIV-positive patients were enrolled. HBsAg was detected in 11.7% of the participants, indicating chronic HBV infection. HBV DNA was detected in 26.8% of the participants: 11.7% were HBsAg positive (overt infection) and the remaining 15.1% were HBsAg-negative (OBI). Fifty serum samples from the HBV/HIV DNA-positive co-infected participants were selected for genomic analysis of HBV. Of these, the HBV genotype of 37 was determined. The genotype distribution of HBV isolates from the HBV/HIV co-infected participants did not differ significantly from those from the HBV mono-infected patients: genotype D (46%), E (21.6%), A (18.9%) and a D/E recombinant (13.5%). Compared to the HBV isolates from mono-infected liver disease patients, the frequency of the D/E recombinant and genotype A was higher in HBV/HIV co-infected patients, as was the intragroup divergence of genotype E. No difference in BCP/PC mutations affecting HBeAg expression at the transcriptional and translational levels between genotype D and E was observed. The following mutations could account for the HBsAg-negativity: sM133T, sE164G, sV168G and sS174N. No primary drug resistance mutations were found. Two online bioinformatics tools, the ―Deep Threshold Tool (DDT)‖ and the ―Rosetta Tool‖, were built to analyze data generated from UDPS and CBS of the BCP/PC region of four Sudanese serum samples, infected with either genotype D or E of HBV, from HBeAgpositive and HBeAg negative patients. A total of 10952 reads were generated by UDPS on the 454 GS Junior platform. The Threshold was calculated using DDT based on probability of error of 0.5%. In total, 39 unique mutations were identified by UDPS, of which 25 were nonsynonymous. The ratio of nucleotide substitutions between isolates from HBeAg-negative and HBeAg-positive patients was 3.5:1. From the sequences analyzed, compared to genotype E isolates, genotype D isolates showed greater variation in the X, BCP/PC/C regions. Only 18 of the 39 positions identified by UDPS were detected by CBS. Using the specific criteria, that have been suggested previously, to define genotypes/subgenotypes of HBV, we determined that genotype D has six and not eight subgenotypes. The importance of HBV genotypes in clinical consequences of infection and response to antiviral treatment has led us to characterize HBV genotypes circulating in Sudan. HBV mono-infected patients and HBV/HIV co-infected individuals, were mainly infected with genotype D or E. HBV mono-infected patients, infected with genotype E, had higher HBeAg-positivity and higher viral loads than those infected with genotype D. The ratio of genotype A to non- A, as well as the genotype E intra-group divergence were higher in HBV/HIV co-infected individuals compared to HBV mono-infected individuals. OBI was found in 15.1% HBV/HIV co-infected patients and its clinical relevance remains to be determined. In order to overcome the limitations of Sanger sequencing, which include its high cost and inability to detect minor populations in quasispecies, next generation sequencing techniques have been developed. It was demonstrated that correct analysis of UDPS data required appropriate curation of read data, in order to clean the data and eliminate artefacts and that the appropriate consensus (reference) sequence should be used in order to identify variants correctly. CBS detected fewer than 50% of the substitutions detected by UDPS. This new technology may allow the detection of minor variants between the different genotypes of HBV and provide biomarkers for the prediction of clinical manifestation of HBV and response to antiviral therapy.

A hepatite A é uma zoonose causada pelo vírus da Hepatite A (HAV), um picornavirus que tem como hospedeiros naturais os primatas humanos e não humanos. Existe apenas um sorotipo, porém várias cepas, divididas em 7 génotipos, sendo que 3 destes são estritamente de cepas humanas e 3 contêm apenas cepas símias. O genótipo III possui tanto cepas humanas como de primatas não humanos. A variação genética entre as cepas de um mesmo genótipo é de no máximo 15%. Já a variação antigênica entre todas as cepas é praticamente inexistente; por isso pode-se utilizar os testes diagnósticos empregados para humanos em primatas não humanos. A infecção pelo vírus da hepatite A se dá pela via fecal-oral, ou seja, o animal ingere o vírus por meio de alimento ou objetos contaminado e, após replicação do vírus no fígado, ele é eliminado nas fezes. A manifestação clínica da doença em primatas e crianças é geralmente assintomática; mas quando presente, é inespecífica e varia de quadros brandos até a morte do animal. O diagnóstico é feito por métodos sorológicos, através da detecção de anticorpos específicos anti-HAV, ou através da detecção de antígeno viral nas fezes durante a fase aguda da doença. A presença de IgM anti-HAV indica infecção aguda ou recente, ao contrário dos anticorpos do grupo IgG, que são encontrados a partir da fase de convalecença e permanecem presentes por vários anos. O objetivo deste trabalho foi de pesquisar a soroprevalência de anticorpos anti-HAV em primatas neotropicais, e também a presença de antígeno viral nas fezes daqueles animais que estivessem apresentando infecção aguda. Para tanto foram testadas 421 amostras de soro de primatas neotropicais de 32 espécies, além de animais de 4 grupos de diferentes híbridos. Dentre os animais estudados, 13,5% (57/421) eram de vida livre, 29,7% (125/421) eram provenientes do Centro de Primatologia do Rio de Janeiro, 4% (17/421) de criadores, 3,8% (16/421) do DEPAVE e 48,9% (206/421) de zoológicos do Estado de São Paulo. As amostras foram testadas para IgM e anti-HAV total com teste imunoenzimático. As amostras positivas ou suspeitas foram reanalisadas. Todas as amostras foram negativas para IgM, ou seja, nenhum animal testado apresentava infecção aguda, portanto a pesquisa para o antígeno viral não pode ser realizada. Em relação ao anti-HAV total, todas os animais de vida livre foram negativos, assim como os animais mantidos no Departamento de Parques e Áreas Verdes do Município de São Paulo (DEPAVE). As porcentagens de animais positivos do CPRJ e de zoológicos/criadores foram respectivamente 4% (5/125) e 7,6% (17/223), demonstrando que uma parcela da população de primatas em cativeiro já esteve em contato com este vírus. A prevalência encontrada de anticorpos anti-HAV neste trabalho ficou abaixo do esperado, pois sabe-se que o índice de animais positivos em cativeiro é bastante alto. As razões para esta baixa prevalência são discutidas. Já a menor frequência de animais positivos observada na população do CPRJ era esperada, pois como se trata de um centro de pesquisas onde não há visitação pública, os animais têm menos contato com humanos, e estes são, sabidamente, a maior fonte de infecção para os primatas não humanos mantidos em cativeiro. Estes resultados nos levam a supor que a hepatite A não é uma doença de risco para a população de primatas neotropicais, tanto de cativeiro como de vida livre. / Hepatitis A virus (HAV) is a picornavirus that causes hepatitis A, a zoonotic disease. This virus has only humans and nonhumans primates as its natural hosts. Just one serotype is known, but several strains have been recognized. Those strains are divided into 7 genotypes, 3 of them being exclusively from human strains, other 3 exclusively from Old World primates strains and the genotype III that has human and primate strains. The maximum genetic difference between strains from the same genotype is 15% and the antigenic difference between all strains is almost none. This is why it is possible to use human diagnostic tests for nonhuman primates. The infection route of the HAV is fecal-oral. After the ingestion of the virus by infected food or contaminated objects, the virus replicates in the liver of the animal and reaches the intestines through the bile, being eliminated with the feces. The disease in primates and children is assymptomatic, but when present, it is unspecific and varies from mild signs to death. The diagnosis is made by serological tests or identification from the viral antigen in sera or feces at the acute phase from the disease. The presence of anti-HAV IgM shows acute or recent infection. On the other hand, anti-HAV IgG is found from the convalescent phase of the disease on, through several years. The aim of this project was to research the seroprevalence of anti-HAV antibodies in New World primates and detect the viral antigen in feces from those animals that had acute infection. Sera from 421 animals of 32 different species were tested. From these animals, 13,5% (57/421) were wild animals, 29,7% (125/421) were from the Centro de Primatologia do Rio de Janeiro (CPRJ), 4% (17/421) from breeders, 3,8% (16/421) from Departamento de Parques e Áreas Verdes (DEPAVE) and 48,9% (206/421) were zoo animals. The sera were tested, by immune-enzymatic tests, for the presence of IgM and total anti-HAV antibodies. All the sera were negative for IgM, which means that no animal had acute infection when tested. All wild animals were negative for total anti-HAV, as were the animals from DEPAVE. Four percent (5/125) from the CPRJ animals and 7,6% (17/223) from the zoos’/breeders’ animals were positive for total anti-HAV, showing that a number of captive animals have already been in contact with the virus. The prevalence of anti-HAV antibodies found in this study was lower than expected, as it is known that the number of positive animals in captivity is high. The possible reasons for such low prevalence are discussed. Since humans are the major risk factor for primate infection with HAV, the lower prevalence found at the CPRJ was predictable, because this is a research center, where visitors are not allowed, so the animals have less contact with humans in there, than they do in zoos. Our results lead us to think that hepatitis A is not a disease of high risk for either wild or zoo New World primates.

by Lau Tze Chin, Gene. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1991. / Includes bibliographical references (Leaves 170-195). / Abstract --- p.1 / Acknowledgement --- p.3 / List of tables --- p.4 / List of figures --- p.6 / List of abbreviations --- p.7 / Chapter Chapter One - --- Introduction --- p.9 / Chapter 1.1. --- Historical Aspects --- p.9 / Chapter 1.2. --- Classification of hepatitis B virus --- p.12 / Chapter 1.2.1. --- Hepadnaviruses --- p.12 / Chapter 1.2.2. --- Comparative properties of hepadnaviruses --- p.13 / Chapter 1.2.2.1. --- Physical properties --- p.13 / Chapter 1.2.2.2. --- Genetic relatedness --- p.15 / Chapter 1.2.2.3. --- Pathogenesis --- p.16 / Chapter 1.3. --- Structural and morphological properties of HBV --- p.17 / Chapter 1.4. --- Molecular biology of HBV --- p.20 / Chapter 1.4.1. --- Molecular structure of HBV --- p.20 / Chapter 1.4.1.1. --- Biochemistry of the virion envelope --- p.20 / Chapter 1.4.1.2. --- The nucleocapsid --- p.21 / Chapter 1.4.1.3. --- Structural features of HBV genome --- p.23 / Chapter 1.4.2. --- Genetic organization of HBV --- p.24 / Chapter 1.4.3. --- Infection cycle of HBV --- p.29 / Chapter 1.4.3.1. --- Viral attachment and internalization --- p.29 / Chapter 1.4.3.2. --- Replication of HBV --- p.30 / Chapter 1.4.3.3. --- Gene expression and regulation --- p.31 / Chapter 1.4.3.4. --- Host-virus DNA interaction --- p.33 / Chapter 1.5. --- Epidemiology and transmission of HBV --- p.34 / Chapter 1.5.1. --- World wide prevalence --- p.35 / Chapter 1.5.1.1. --- HBsAg prevalence --- p.35 / Chapter 1.5.1.2. --- Cumulative rate of HBV infection --- p.35 / Chapter 1.5.1.3. --- Age specific pattern of HBV infection --- p.36 / Chapter 1.5.2. --- Epidemiological pattern of HBV in Hong Kong --- p.37 / Chapter 1.5.3. --- Mode of transmission --- p.38 / Chapter 1.6. --- Clinical outcomes of HBV infection --- p.38 / Chapter 1.6.1. --- Acute infection --- p.41 / Chapter 1.6.2. --- Chronic infection --- p.42 / Chapter 1.6.3. --- Primary hepatocellular carcinoma --- p.43 / Chapter 1.7. --- Laboratory diagnosis of hepatitis B --- p.44 / Chapter 1.7.1. --- The HBV markers --- p.47 / Chapter 1.7.1.1. --- HBsAg and anti-HBs --- p.47 / Chapter 1.7.1.2. --- HBcAg and Anti-HBc --- p.47 / Chapter 1.7.1.3. --- HBeAg and anti-HBe --- p.49 / Chapter 1.7.1.4. --- HBV-associated DM polymerase --- p.49 / Chapter 1.7.1.5. --- HBV-DNA --- p.49 / Chapter 1.7.2. --- Methodology in the detection of hepatitis B markers --- p.50 / Chapter 1.7.2.1. --- Direct detection of HBV and HBV antigens --- p.50 / Chapter 1.7.2.2. --- Serological detection of HBV markers --- p.51 / Chapter 1.7.2.3. --- HBV-associated DNA polymerase assay --- p.51 / Chapter 1.7.2.4. --- Molecular technique for the detection and quantitation of HBV-DNA --- p.52 / Chapter 1.8. --- Antiviral therapy in hepatitis B --- p.52 / Chapter 1.8.1. --- Therapeutic agents for treatment of HBV infection --- p.53 / Chapter 1.8.1.1. --- Steroids --- p.53 / Chapter 1.8.2.2. --- Nucleoside analogs --- p.54 / Chapter 1.8.1.3. --- Interferon --- p.55 / Chapter 1.8.2. --- Clinical trials of interferons --- p.55 / Chapter 1.9. --- Extrahepatic tissue tropism of HBV --- p.62 / Chapter 1.10. --- Objective and design of study --- p.65 / Chapter 1.10.1. --- Objectives of study --- p.65 / Chapter 1.10.2. --- Study design --- p.66 / Chapter 1.10.2.1. --- Cross-sectional study --- p.67 / Chapter 1.10.2.2. --- Longitudinal study --- p.67 / Chapter 2.1. --- Materials --- p.71 / Chapter 2.1.1. --- Patients recruitment and clinical materials --- p.71 / Chapter 2.1.1.1. --- Cross-sectional study --- p.71 / Chapter 2.1.1.2. --- Longitudinal study --- p.71 / Chapter 2.1.2. --- Bacteria] stock --- p.71 / Chapter 2.1.3. --- "Chemicals, equipments and consumables" --- p.72 / Chapter 2.1.4. --- Buffers and solutions --- p.72 / Chapter 2.1.4.1. --- Phosphate buffer saline (PBS) --- p.72 / Chapter 2.1.4.2. --- Leucocyte lysis buffer (X 5)(LLB) --- p.72 / Chapter 2.1.4.3. --- Buffer equilibrated phenol (BEP) --- p.76 / Chapter 2.1.4.4. --- Phenol-Chloroform mixture --- p.76 / Chapter 2.1.4.5. --- 3.0M sodium acetate (pH 5.2) --- p.76 / Chapter 2.1.4.6. --- Tris-EDTA buffer (pH 8.0) (TE) --- p.76 / Chapter 2.1.4.7. --- Stock salmom sperm DNA solution --- p.77 / Chapter 2.1.4.8. --- Tracking dye --- p.77 / Chapter 2.1.4.9. --- Tris-borate electrophoresis buffer (TBE) --- p.77 / Chapter 2.1.4.10. --- Luria-Bertani Broth (LB) --- p.77 / Chapter 2.1.4.11. --- Solution ] --- p.78 / Chapter 2.1.4.12. --- Solution ]] --- p.78 / Chapter 2.1.4.13. --- Potassium acetate buffer (pH 5.4) --- p.78 / Chapter 2.1.4.14. --- Column elution buffer (CEB) --- p.78 / Chapter 2.1.4.15. --- NPMEB solution --- p.79 / Chapter 2.1.4.16. --- Neutralizing solution --- p.79 / Chapter 2.1.4.17. --- Standard saline citrate (SSC) --- p.79 / Chapter 2.1.4.18. --- Denhardt solution --- p.79 / Chapter 2.1.4.19. --- Prehybridization solution (PS) --- p.80 / Chapter 2.1.4.20. --- NETFAP Solution --- p.80 / Chapter 2.1.4.21. --- Heparin solution --- p.81 / Chapter 2.1.4.22. --- Hybridization mix for oligo-nucleotide probe --- p.81 / Chapter 2.1.4.23. --- NEPS solution (pH 7.0) --- p.81 / Chapter 2.1.4.24. --- Restriction endonuclease and buffer --- p.82 / Chapter 2.2. --- Methods --- p.82 / Chapter 2.2.1. --- Sample preparations --- p.82 / Chapter 2.2.1.1. --- Isolation of plasma and peripheral blood leucocytes (PBL) --- p.82 / Chapter 2.2.1.2. --- Extraction of DNA from Peripheral blood leucocytes --- p.83 / Chapter 2.2.1.3. --- Quantitation of Peripheral blood leucocyte DNA --- p.83 / Chapter 2.2.2. --- Preparation of radio-labelled HBV-DNA probe --- p.84 / Chapter 2.2.2.1. --- Plating and selection of bacterial stock --- p.84 / Chapter 2.2.2.2. --- Growth of E. coli HB101 and amplification of pAM6 --- p.84 / Chapter 2.2.2.3. --- Harvesting of E. coli and extraction of plasmid pAM6 --- p.84 / Chapter 2.2.2.4. --- Purification of plasmid pAM6 --- p.86 / Chapter 2.2.2.5. --- Large scale isolation and purification of HBV genome from plasmid pAM6 --- p.86 / Chapter 2.2.2.6. --- Radio-labelling of HBV-DNA --- p.88 / Chapter 2.2.2.6.1. --- Nick-translation of total HBV-DNA genome --- p.88 / Chapter 2.2.2.6.2. --- Multi-primer labelling of total HBV- DNA genome --- p.88 / Chapter 2.2.2.6.3. --- End-labeling of 21-base HBV oligo- nucleotide --- p.88 / Chapter 2.2.2.6.4. --- Determination of labelling efficiency --- p.89 / Chapter 2.2.2.7. --- Purification of labelled HBV-DNA probe --- p.90 / Chapter 2.2.2.7.1. --- Total genomic HBV-DNA probe (pAM6 probe) --- p.90 / Chapter 2.2.2.7.2. --- Oligo-nucleotide HBV-DNA probe (oligo probe) --- p.90 / Chapter 2.2.3. --- Hybridization study of clinical samples --- p.91 / Chapter 2.2.3.1. --- Solution hybridization of sera samples --- p.91 / Chapter 2.2.3.2. --- Spot hybridization of sera samples --- p.91 / Chapter 2.2.3.2.1. --- "Pre-hybridization treatment of sera samples (adapted from Lin et al.,1987)" --- p.91 / Chapter 2.2.3.2.2. --- Pre-hybridization and hybridization of the membrane --- p.92 / Chapter 2.2.3.2.3. --- Washing of membrane --- p.92 / Chapter 2.2.3.2.4. --- Final treatment and autoradiography: --- p.92 / Chapter 2.2.3.3. --- Quantitation of HBV-DNA in the sera samples: --- p.93 / Chapter 2.2.4. --- Assay for serological Hepatitis B marker --- p.93 / Chapter Chapter Three - --- Results --- p.93 / Chapter 3.1. --- Preparation of HBV-DNA probes --- p.95 / Chapter 3.2. --- Radiolabelling of HBV-DNA --- p.95 / Chapter 3.3. --- Hybridization methodology --- p.98 / Chapter 3.4. --- Comparison of the performance of HBV-DNA probes --- p.100 / Chapter 3.4.1. --- Quantitation of serum HBV-DNA --- p.100 / Chapter 3.4.2. --- Comparative hybridization performance of different HBV-DNA probes --- p.105 / Chapter 3.5. --- Clinical application of HBV-DNA probe:Detection of HBV-DNAin serum and peripheral blood leucocytes (PBL) --- p.109 / Chapter 3.5.1. --- Cross-sectional study --- p.112 / Chapter 3.5.1.1. --- Frequency of HBV-DNA detection in relation to different clinical manifestations --- p.112 / Chapter 3.5.1.2. --- Frequency of HBV-DNA detection in relation to the serological status --- p.114 / Chapter 3.5.1.3. --- Distribution of serum and PBL HBV-DNA level in chronic hepatitis B patients in relation to the different HBV-related manifestations --- p.119 / Chapter 3.5.2. --- Longitudinal study of patients with chronic hepatitis B under interferon therapy with prednisolone pretreatment --- p.123 / Chapter 3.5.2.1. --- Features of patients under study --- p.123 / Chapter 3.5.2.2. --- Correlation between the occurrence of HBV- DNA and HBeAg in serum --- p.123 / Chapter 3.5.2.3. --- Outcome of clinical trial: --- p.126 / Chapter 3.5.2.3.1. --- Number of patients responding to therapy: --- p.126 / Chapter 3.5.2.3.2. --- Variation in serum HBV markers during the course of study --- p.128 / Chapter 3.5.2.3.3. --- Change of HBV-DNA statusin peripheral blood leucocytes --- p.134 / Chapter Chapter Four - --- Dicussion --- p.140 / Chapter 4.1. --- Preparation of HBV-DNA hybridization probes --- p.140 / Chapter 4.1.1. --- Source of HBV-DNA --- p.140 / Chapter 4.1.2. --- Raidolabelling of HBV-DNA --- p.141 / Chapter 4.2. --- Hybridization methodology --- p.141 / Chapter 4.2.1. --- Optimization of hybridization conditions --- p.141 / Chapter 4.2.2. --- Comparison of the performance among different HBV- DNA probes --- p.144 / Chapter 4.3. --- Detection of HBV-DNA in clinical serum samples --- p.148 / Chapter 4.3.1. --- Crossectional study of patients with various categories of HBV related diseases --- p.148 / Chapter 4.3.1.1. --- HBV-DNA detection in serum --- p.148 / Chapter 4.3.1.2. --- Detection of HBV-DNA in peripheral blood mononuclear cells --- p.153 / Chapter 4.3.2. --- Longitudinal studies of patients undergoing antiviral therapy --- p.159 / Chapter 4.3.2.1. --- Serum HBV-DNA and HBeAg --- p.159 / Chapter 4.3.2.2. --- HBV-DNA in peripheral blood leucocytes --- p.163 / Conclusion --- p.166 / Future perspectives --- p.168 / References --- p.170

Introdução: Pacientes com infecção crônica pelo VHC e superinfecção pelo vírus da hepatite A (VHA) ou o vírus da hepatite B (VHB), têm maior morbi-mortalidade quando comparados com pacientes que apresentam infecção aguda somente pelo VHA ou VHB. A mortalidade associada à hepatite A aguda pode estar particularmente elevada em pacientes com pré-existência de hepatite crônica causada pelo VHC. Por esta razão, a imunização ativa com vacinas contra o VHA e o VHB vem a ser obrigatória nesta população, e consequentemente esta sorologia deve ser determinada. Objetivos: O objetivo deste trabalho foi avaliar a prevalência de marcadores sorológicos da hepatite A e hepatite B em 1.000 pacientes com infecção crônica pelo VHC atendidos no Ambulatório de Hepatites da Divisão de Gastroenterologia e Hepatologia Clínica do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo. Resultados: O anti-VHA IgG foi positivo em 923 de 1000 pacientes (92,3%). Quando estratificados por idade, o anti-VHA IgG foi encontrado em 61% dos pacientes entre 20 e 29 anos, 70% entre 30 e 39 anos, 85% entre 40 e 49 anos, 94% entre 50 e 59 anos e 99% nos pacientes com mais de 60 anos . O anti-HBc total foi positivo em 244 pacientes (24%). Estratificados por idade, em 4,3% dos pacientes entre 20 e 29 anos, 17% entre 30e 39 anos, 21% entre 40 e 49 anos, 24% entre 50 e 59 anos, e 28% dos pacientes com mais de 60 anos. Dos 244 pacientes anti-HBc IgG positivos, 0,8% são HBsAg positivo, 8,5% anti-HBc IgG isolado e 16% anti-HBs positivo. Conclusões: A prevalência de anti-VHA IgG nod nossos pacientes com hepatite C crônica foi semelhante à da população geral no município de São Paulo. No entanto, o anti-HBc totaI foi maior em nossos pacientes, quando comparada historicamente à população geral dos países ocidentais, sugerindo fatores de risco semelhantes para as hepatites B e C, o que enfatiza a importância dos programas de imunização nesta população / Background and Aims: Patients with chronic HCV and superinfection by hepatitis A virus (HAV) or hepatitis B virus (HBV) have higher morbidity and mortality when compared with those without HCV. For this reason, HAV and HBV active immunization has become mandatory in this population and hence their serological markers must be determined. The aim of this study was to evaluate the prevalence of serological markers of HAV and HBV infection in patients with chronic HCV. Methods: 1.000 chronic HCV infected patients at the University of Sao Paulo School of Medicine outpatient Liver Clinic were evaluated for the prevalence of serological markers of HAV and HBV infection. Results: Anti-HAV IgG was positive in 923 of 1000 patients (92.3%). When stratified by age, the anti-HAV IgG was found in 61% of patients between 20-29 years, 70% between 30-39 years, 85% between 40-49 years, 94% between 50-59 years, and 99% over 60 years of age. Anti-HBc IgG was positive in 244 patients (24%). Stratified by age, anti-HBc IgG was found in 4.3% of patients between 20-29 years, 17% between 30-39 years, 21% between 40 -49 years, 24% between 50-59 years, and 28% of patients over 60 years of age. Of the 244 anti-HBc IgG positive patients, 0.8% were also HBsAg positive, 8.5% were anti-HBc IgG isolated and 16% were also anti-HBs positive. Conclusions: The prevalence of anti-HAV IgG was similar to the general population in the city of São Paulo. However, anti-HBc IgG was higher in our chronic HCV patients, when compared historically to the general population of western countries, suggesting similar risk factors for HBV and HCV acquisition, so emphasizing the importance of immunization programs in this population. Keywords: Hepatitis C, Chronic; Hepatitis C; Hepacivirus, Prevalence; Hepatitis A; Hepatitis B Título: Prevalência de Marcadores Sorológicos das Hepatites A e B em Pacientes com Hepatite C Crônica atendidos no Ambulatório de Hepatites do Serviço de Gastroenterologia Clínica do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo - HCFMUSP Background and Aims: Patients with chronic HCV and superinfection by hepatitis A virus (HAV) or hepatitis B virus (HBV) have higher morbidity and mortality when compared with those without HCV. For this reason, HAV and HBV active immunization has become mandatory in this population and hence their serological markers must be determined. The aim of this study was to evaluate the prevalence of serological markers of HAV and HBV infection in patients with chronic HCV. Methods: 1.000 chronic HCV infected patients at the University of Sao Paulo School of Medicine outpatient Liver Clinic were evaluated for the prevalence of serological markers of HAV and HBV infection. Results: Anti-HAV IgG was positive in 923 of 1000 patients (92.3%). When stratified by age, the anti-HAV IgG was found in 61% of patients between 20-29 years, 70% between 30-39 years, 85% between 40-49 years, 94% between 50-59 years, and 99% over 60 years of age. Anti-HBc IgG was positive in 244 patients (24%). Stratified by age, anti-HBc IgG was found in 4.3% of patients between 20-29 years, 17% between 30-39 years, 21% between 40 -49 years, 24% between 50-59 years, and 28% of patients over 60 years of age. Of the 244 anti-HBc IgG positive patients, 0.8% were also HBsAg positive, 8.5% were anti-HBc IgG isolated and 16% were also anti-HBs positive. Conclusions: The prevalence of anti-HAV IgG was similar to the general population in the city of São Paulo. However, anti-HBc IgG was higher in our chronic HCV patients, when compared historically to the general population of western countries, suggesting similar risk factors for HBV and HCV acquisition, so emphasizing the importance of immunization programs in this population

Deng Xue-Long. / "January 2003." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (p. 196-230). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

A hepatite A é uma zoonose causada pelo vírus da Hepatite A (HAV), um picornavirus que tem como hospedeiros naturais os primatas humanos e não humanos. Existe apenas um sorotipo, porém várias cepas, divididas em 7 génotipos, sendo que 3 destes são estritamente de cepas humanas e 3 contêm apenas cepas símias. O genótipo III possui tanto cepas humanas como de primatas não humanos. A variação genética entre as cepas de um mesmo genótipo é de no máximo 15%. Já a variação antigênica entre todas as cepas é praticamente inexistente; por isso pode-se utilizar os testes diagnósticos empregados para humanos em primatas não humanos. A infecção pelo vírus da hepatite A se dá pela via fecal-oral, ou seja, o animal ingere o vírus por meio de alimento ou objetos contaminado e, após replicação do vírus no fígado, ele é eliminado nas fezes. A manifestação clínica da doença em primatas e crianças é geralmente assintomática; mas quando presente, é inespecífica e varia de quadros brandos até a morte do animal. O diagnóstico é feito por métodos sorológicos, através da detecção de anticorpos específicos anti-HAV, ou através da detecção de antígeno viral nas fezes durante a fase aguda da doença. A presença de IgM anti-HAV indica infecção aguda ou recente, ao contrário dos anticorpos do grupo IgG, que são encontrados a partir da fase de convalecença e permanecem presentes por vários anos. O objetivo deste trabalho foi de pesquisar a soroprevalência de anticorpos anti-HAV em primatas neotropicais, e também a presença de antígeno viral nas fezes daqueles animais que estivessem apresentando infecção aguda. Para tanto foram testadas 421 amostras de soro de primatas neotropicais de 32 espécies, além de animais de 4 grupos de diferentes híbridos. Dentre os animais estudados, 13,5% (57/421) eram de vida livre, 29,7% (125/421) eram provenientes do Centro de Primatologia do Rio de Janeiro, 4% (17/421) de criadores, 3,8% (16/421) do DEPAVE e 48,9% (206/421) de zoológicos do Estado de São Paulo. As amostras foram testadas para IgM e anti-HAV total com teste imunoenzimático. As amostras positivas ou suspeitas foram reanalisadas. Todas as amostras foram negativas para IgM, ou seja, nenhum animal testado apresentava infecção aguda, portanto a pesquisa para o antígeno viral não pode ser realizada. Em relação ao anti-HAV total, todas os animais de vida livre foram negativos, assim como os animais mantidos no Departamento de Parques e Áreas Verdes do Município de São Paulo (DEPAVE). As porcentagens de animais positivos do CPRJ e de zoológicos/criadores foram respectivamente 4% (5/125) e 7,6% (17/223), demonstrando que uma parcela da população de primatas em cativeiro já esteve em contato com este vírus. A prevalência encontrada de anticorpos anti-HAV neste trabalho ficou abaixo do esperado, pois sabe-se que o índice de animais positivos em cativeiro é bastante alto. As razões para esta baixa prevalência são discutidas. Já a menor frequência de animais positivos observada na população do CPRJ era esperada, pois como se trata de um centro de pesquisas onde não há visitação pública, os animais têm menos contato com humanos, e estes são, sabidamente, a maior fonte de infecção para os primatas não humanos mantidos em cativeiro. Estes resultados nos levam a supor que a hepatite A não é uma doença de risco para a população de primatas neotropicais, tanto de cativeiro como de vida livre. / Hepatitis A virus (HAV) is a picornavirus that causes hepatitis A, a zoonotic disease. This virus has only humans and nonhumans primates as its natural hosts. Just one serotype is known, but several strains have been recognized. Those strains are divided into 7 genotypes, 3 of them being exclusively from human strains, other 3 exclusively from Old World primates strains and the genotype III that has human and primate strains. The maximum genetic difference between strains from the same genotype is 15% and the antigenic difference between all strains is almost none. This is why it is possible to use human diagnostic tests for nonhuman primates. The infection route of the HAV is fecal-oral. After the ingestion of the virus by infected food or contaminated objects, the virus replicates in the liver of the animal and reaches the intestines through the bile, being eliminated with the feces. The disease in primates and children is assymptomatic, but when present, it is unspecific and varies from mild signs to death. The diagnosis is made by serological tests or identification from the viral antigen in sera or feces at the acute phase from the disease. The presence of anti-HAV IgM shows acute or recent infection. On the other hand, anti-HAV IgG is found from the convalescent phase of the disease on, through several years. The aim of this project was to research the seroprevalence of anti-HAV antibodies in New World primates and detect the viral antigen in feces from those animals that had acute infection. Sera from 421 animals of 32 different species were tested. From these animals, 13,5% (57/421) were wild animals, 29,7% (125/421) were from the Centro de Primatologia do Rio de Janeiro (CPRJ), 4% (17/421) from breeders, 3,8% (16/421) from Departamento de Parques e Áreas Verdes (DEPAVE) and 48,9% (206/421) were zoo animals. The sera were tested, by immune-enzymatic tests, for the presence of IgM and total anti-HAV antibodies. All the sera were negative for IgM, which means that no animal had acute infection when tested. All wild animals were negative for total anti-HAV, as were the animals from DEPAVE. Four percent (5/125) from the CPRJ animals and 7,6% (17/223) from the zoos’/breeders’ animals were positive for total anti-HAV, showing that a number of captive animals have already been in contact with the virus. The prevalence of anti-HAV antibodies found in this study was lower than expected, as it is known that the number of positive animals in captivity is high. The possible reasons for such low prevalence are discussed. Since humans are the major risk factor for primate infection with HAV, the lower prevalence found at the CPRJ was predictable, because this is a research center, where visitors are not allowed, so the animals have less contact with humans in there, than they do in zoos. Our results lead us to think that hepatitis A is not a disease of high risk for either wild or zoo New World primates.

ABSTRACT In sub-Saharan Africa a causal relationship has been established between hepatitis B virus (HBV) infection and membranous nephropathy (MN), especially in Black children. The most common method of treatment is interferon therapy, which is however, only effective in 30-40% of patients. The reason for this is unclear. The objective of this pilot study was to determine whether mutations in the complete surface gene of HBV isolated from Black children with HBV-associated MN before, during and after treatment with interferon, had any effect on treatment response and vice versa. HBV DNA was extracted from the serum of a responder, reverter and non-responder patient before, during (4 and 16 weeks) and after (40 weeks) IFN treatment. The preS1/preS2/S region was amplified and cloned, and the clones sequenced. Sequence analyses revealed the preS2 region to be the most variable in the reverter and non-responder and HBsAg was the most variable in the non-responder. Phylogenetic analysis showed that the viral population dynamics between the responder strains and the reverter/non-responder strains differed as a result of various mutations found within the surface gene. Thus the presence of mutations in preS2 and HBsAg of the non-responding patients may carry predictive markers for nonresponse but further investigation would be needed to conclusively prove this.

En las dos últimas décadas del siglo pasado, nuestro país atravesó una de las mayores crisis político-social, con una enorme cuota de violencia y muerte y cuya consecuencia socioeconómica de más impacto, fue un acelerado despoblamiento de las áreas rurales donde el fenómeno violentista era mayor, con su consiguiente masiva migración hacia los centros urbanos más importantes del país, principalmente a la capital de la república. Estos inmigrantes en su gran mayoría provenían de los valles interandinos y de la ceja de selva, precisamente las áreas hiperendémicas de hepatitis B. Este fenómeno, asumimos que podría tener un impacto importante sobre la prevalencia de la hepatitis B en la capital, especialmente en los estratos socioeconómicos bajos, como ya se insinuaba en dos reportes de finales de la década de los ’80. La influencia de estos dos fenómenos en todos los cambios sociales en el mundo, y por ello, la migración de decenas de miles de personas del interior del país hacia la capital que hacían prever un cambio en el patrón epidemiológico de la hepatitis B en Lima pasando de endemicidad baja a intermedia, nos ha llevado a pronosticar , un incremento de la prevalencia de la infección por el virus B en adolescentes, y el consiguiente riesgo de transmisión al neonato; además la población adolescente en el año 2003, es en su inmensa mayoría vulnerable a la infección por el VHB, pues las campañas de vacunación contra esta infección que inició el Ministerio de Salud en el año 1996, en los infantes de cero a un año, no alcanzó a los que ahora son adolescentes. Todos estos hechos han motivado nuestro interés en demostrar el incremento de la prevalencia de hepatitis B en este grupo poblacional, así como investigar todos los otros factores de riesgo que puedan contribuir a la transmisión de esta enfermedad. Este es el propósito de esta investigación. / Tesis

Thesis (MPH)--University of Limpopo, 2010. / Introduction: Hepatitis B infection is a serious blood-borne disease caused by the hepatitis B virus (HBV) which attacks the liver, and is the leading cause of liver cancer and cirrhosis of the liver. HBV can be transmitted through exposure to infected blood and human secretions through needle stick / sharps injuries and splashes. Thus nurses are at high risk for HBV infection. The aim of the study: To investigate the knowledge, attitudes and practices (KAP) regarding the prevention of hepatitis B virus infections, in final year college student nurses in Gauteng province. Methods: A cross-sectional quantitative survey on 350 final year nursing students was conducted in three Gauteng province nursing colleges, using an anonymous self administered questionnaire with questions on knowledge, attitudes, and practices regarding HBV. The data were analysed using SPSS (statistical package for social science studies). Results: Of 350 questionnaires distributed, 312 student nurses returned completed forms (response rate: 89.14% [312/350]). The majority were females (86.8% [270/331]) and were below 31 years of age (30.1% [93/309]). The majority (87.6% [271/310]) had good knowledge of the causes and prevention of HBV. The unvaccinated respondents had fairly low positive attitudes, with a mean, mode and median score of 1 (possible score from -4 to +4). The majority (79% [244/310]) practiced good compliance with universal precautions of, and the majority (64.9% [202/311]) were vaccinated. College A displayed significantly higher knowledge (p<0.001), positive attitudes (p=0.001) and safer practices (p<0.001) than college B and C.