Thesis (M.Res.(Med.))--University of Hong Kong, 2003. / Includes bibliographical references (leaves 68-77) Also available in print.

Hepatitis E is an acute, enterically transmitted disease of public health importance. The mechanism of pathogenesis of HEV is poorly understood due to the lack of an in vitro cell culture system and an ideal animal model system. With the discovery of avian HEV and its association with a hepatic disease (Hepatitis-Splenomegaly syndrome), chickens provide an excellent small homologous animal model system to study this important virus. The objectives of this dissertation were to utilize chickens as a model system to study the pathogenesis and replication of avian HEV under the natural route of infection, to identify potential extrahepatic replication sites, to determine and analyze the complete genomic sequence of the avirulent strain of avian HEV, and to study the compartive pathogenesis of the two isolates of avian HEV, the prototype pathogenic and avirulent strains of avian HEV. We attempted to experimentally infect specific-pathogen-free (SPF) adult chickens by the natural fecal-oral route in order to systematically study HEV pathogenesis and replication and to characterize the clinical course and pathological lesions associated with avian HEV infection. Sixty-week-old, specific-pathogen-free (SPF) chickens were inoculated with 5 x104.5 50% chicken infectious dose of avian HEV by oronasal route and IV route. All oronasally- and IV- inoculated chickens had seroconverted to avian HEV antibodies and fecal virus shedding was detected variably from 1 to 20 DPI in the IV group, and from 10 to 56 DPI in the oronasal group. Avian HEV RNA was detected in serum, bile, and liver samples earlier during the course of infection in IV-inoculated chickens than in oronasally-inoculated ones. Gross liver lesions including subcapsular hemorrhages and enlargement of right intermediate lobe and microscopic hepatic lesions in the liver characterized by lymphocytic periphlebitis and phlebitis were observed in inoculated chickens. This is the first report of experimental HEV infection via its natural route in a homologous animal model system. Very little is known about HEV pathogenesis and it has been hypothesized that HEV replicates in tissues other than liver. The replicating negative-strand viral RNA was detected by negative-strand-specific RT-PCR in liver, serum, colon, cecum, jejunum, ileum, duodenum and cecal tonsils,but not in other non-GIT tissues. Immunohistochemistry using an avian HEV capsid protein-specific anti-peptide antibody revealed positive signal in liver and GIT tissues including colon, jejunum, ileum, cecum, cecal tonsils and pancreas. The detection of avian HEV capsid antigen and replicative negative-strand viral RNA in the GIT tissues indicates that HEV replicates in the GI tract following infection by fecal-oral route. The complete genomic sequence of an avirulent strain of avian HEV was determined using primer walking strategy. The full-length genome of the avirulent strain is 6649 nts in length and has a nucleotide sequence identity of 90.1% with the prototype pathogenic strain. Numerous non-silent mutations were observed in ORF1, the region coding for the nonstructural proteins. Six unique non-silent mutations were identified in the capsid-encoding ORF2 region and the ORF3 had four non-silent mutations. Phylogenetic analysis based on full-length genomic sequence revealed that the avirulent strain is clustered together with the pathogenic avian HEV and represents a branch distinct from mammalian HEVs. In order to study the comparative pathogenesis between the pathogenic and avirulent strains of avian HEV, an infectious stock of the avirulent avian HEV was generated and infectivity titer was determined to be 5 x 102.5 CID50 per ml by experimentally infecting young SPF chickens. Six-week-old SPF chickens were inoculated with one of two strains of avian hepatitis E viruses, pathogenic avian HEV recovered from a chicken with HS syndrome and avirulent avian HEV isolated from a healthy chicken to study comparative pathogenesis. Most of the chickens seroconverted by 3 wpi in both pathogenic avian HEV and avirulent avian HEV groups. Avian HEV RNA was detected in feces and serum of the chickens from both the inoculated group from 1 wpi. Microscopic liver lesions included lymphocytic periphlebitis and phlebitis the overall hepatic lesion mean score was higher for the pathogenic avian HEV group compared to the avirulent avian HEV and control groups, suggestive of attenuation In summary, SPF chickens were experimentally infected with avian HEV by natural route to study the systematic pathogenesis and replication. Non-liver replication sites of avian HEV were also identified in a chicken model. The complete genomic sequence of an apparently avirulent strain of avian hepatitis E virus was determined and the comparative pathogenesis of avian hepatitis E virus isolates from a chicken with HS syndrome and from a healthy chicken was also studied by experimental infections in young SPF chickens. The results from this dissertation research have important implications for the understanding of HEV pathogenesis. / Ph. D.

Despite evidence that viral hepatitis poses a significant risk to public health, universal vaccination has not yet been implemented. The risk for viral hepatitis infection is particularly high among injection drug users and other individuals who do not attend regular health care visits. Jails provide a structural opportunity to vaccinate these high risk individuals. HAV and HBV vaccines administered on an accelerated three week schedule could dramatically decrease the lifetime risk for contracting viral hepatitis among jail detainees. Assuming that 75% of detainees would accept vaccination, 33% have previous exposure to HAV, 25% have previous exposure to HBV, and independent future healthcare costs were US $317,000, the US health care system would save $12 per individual with a vaccinate upon entry program in comparison to no intervention. This savings translates into an economic benefit amounting to about US$ 5,000,000 saved if all new jail inmates in a given year were immunized. A vaccination upon entry program for HAV/HBV in jails should be widely implemented with coordination between the corrections system and public health agencies to reduce the growing cost of viral hepatitis infection.

This thesis is focused on occurrence of viral hepatitises by clients in Psychiatric Hospital Červený Dvůr between 2000 and 2009. In my beachelor thesis was covered years from 2000 to 2004. Now I continue with this diploma thesis where I want the 10 year results join together to show how the situation is developing. The intravenous drug users are the aimed group. These drug users belong to a high-risk group in which hepatitises types A, B or C are spread the most thanks to their high-risk behaviour. These days researches and epidemiologic researches show that the intravenous drug users belong among the main high-risk groups endangered by infectious disease. Viral hepatitises represent a serious problem, mainly hepatitis type C, because there is no way of protection. This hepatitis is occuring without any symptoms of this disease by 50 ? 70 % people. By 70 ? 80 % it passes into a chronic stadium. It is possible to protect yourself by hepatitis type A vaccination. This hepatitis doesn´t change into a chronic stadium. It is easily spread by infected water or food. In our republic there occured several epidemics. Hepatitises started to spread among drug users and then they spread also among the rest of population. The last epidemic occured in 2008. Hepatitis type B represents high risks for the perceptive population. There is a vaccine and population have a chance to protect against it. In the Czech Republic there is vaccination carried out within special vaccination of people who have high-risk job, for example in health service. Children of HBsAg positive mothers are vaccinated after they are born. From 2001 hepatitis vaccination was put into regular vaccination of children aged under 1 year in our republic. Also 12 year old children are vaccinated from 2001. Hepatitis type B often becomes chronic and the mortality is 1 ? 2 %. Intravenous using of drug is important high-risk factor. It is one of the ways of transfering hepatitises type B and C. Next monitored indicators are the age of the clients, achieved level of education, basic drug, the age of the first usage of any drug, the age of intravenous application of drug and occurrence of hepatitises type A, B, C by clients. According to my research I used quantitative research which allows me to analyse problem of viral hepatitises by clients from Psychiatric Hospital in Červený Dvůr. The gained information was worked out by secondary analysis of medical data. The monitored group has 2499 clients in years 2000 ? 2009. The aim of this thesis is to get general knowledge about prevalence of viral hepatitises by clients of Psychiatric Hospital in Červený Dvůr from 2000 to 2009 and to consider their progress. The methodical procedure: there will be used a content analysis of medical data given by Psychiatric Hospital in Červený Dvůr. The result of this thesis is that it indicates and covered the develop of drug scene. Concerning the age the main group consists of 20 ? 24 year old men and women, next large group consists of clients aged 25 ? 29. The monitored group is also changing according to achieved level of education. In the first five years of research there dominated vocational school, but in the last five years there it changed into basic school. Among women there happened no change. There basic school still dominates. Concerning the drugs there also changed something. At the beginning there dominated clients using heroin, in the middle there meth dominated. Hepatitis type A in acute stadium was revealed by only two clients. Hepatitis type B noticed a slight increase. Hepatitis type C also noticed a sligh increase, but it is probably a sign of increase of clients with chronic form of this hepatitis.

Complex patterns of HCV infection are increasingly reported, particularly in highly exposed individuals, with multiple and variable subtype profiles seen in many chronic patients. This study aims to address some of the questions arising from this increasingly diverse and dynamic picture, both within hosts and at a population level. In Chapter 2 I find evidence for a highly dynamic infection profile in acute HCV, both in terms of viral load and the dominant subtype. I extrapolate these observations from individual patients to formulate a model of HCV transmission across a high-risk population in order to predict the impact of current and anticipated interventions in Chapters 3 and 4. I show that antiviral therapy and a putative vaccination can still have a significant impact on HCV prevalence at the population level, even when the latter offers only partial protection and in the epidemiological background of ongoing exposure. Thus, in an epidemic with more than one circulating strain it will be crucial for any individual or combination of interventions to target all variants present. In Chapter 5 I demonstrate that early viral load kinetics of patients initiating treatment are indicative of treatment outcome. Strain differences are also evident in the virologic response to treatment with hard-to-treat genotype 1 exhibiting a slower rate of viral load decline than genotypes 2 and 3.

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

Improved methods for the in situ hybridisation detection of messenger RNA ( mRNA ) in sections of liver tissue, were derived by use of an experimental system. This involved the use of tritiated-poly ( dT ) probes to detect poly ( A ) sequences attached to the 3 ' end of mRNA in sections of mouse liver that had been processed in various ways. The improved - methods were applied to the detection of hepatitis B virus ( HBV ) - and hepatitis delta virus ( HDV ) - RNA. In situ hybridisation and immunostaining techniques were then applied to studies of the pathogenesis of HBV and duck hepatitis B virus ( DHBV ) infection. In situ hybridisation studies of liver biopsy tissue from HBV - infected immunosuppressed renal transplant patients demonstrated an anatomical association between piecemeal necrosis and HBV replication at the cellular level in some patients. However, widespread replicative infection of hepatocytes also occurred in some patients in the presence of normal hepatocyte morphology and mild inflammatory changes indicating that at the cellular level virus replication was not necessarily a direct cytopathic process. These findings supported the view that hepatocyte Injury may : ( i ) result from immune - mediated damage directed against cells undergoing replicative, but not restricted infection ; ( ii ) eliminate cells undergoing replicative infection and favour clonal regeneration of cells undergoing restricted infection. Localisation of interferon - alpha ( IFN - alpha ) expression in liver tissue chronically infected with HBV and HDV, identified mononuclear cells and fibroblasts ( but not hepatocytes ) as the main producers of IFN - alpha. IFN - alpha - positive cells were associated with areas of liver tissue containing cells supporting virus replication and exhibiting the greatest degree of liver damage, suggesting that locally produced IFN - alpha may be a natural regulator of virus replication in chronic liver disease. Experimental DHBV infection of Pekin - Aylesbury ducks showed that virus inoculated either intravenously or intraperitoneally, gained access to randomly distributed hepatocytes without first replicating in other cell types in the liver. Virus was seen to disseminate to contiguous cells following anatomical boundaries by the third day post - inoculation. Markers of DHBV infection in liver and serum showed reproducible kinetics, and duck hepatocytes in this system appeared to be highly permissive as large amounts of DHBV DNA and DHBsAg were produced intracellularly without the development of ongoing cytopathology. Hepatocytes were the major cell type responsible for early significant DHBV replication, in contrast to pancreas, kidney, spleen and circulating mononuclear cells where significant levels of infection were detected only after the first week of infection and the onset of viraemia. / Thesis (Ph.D.)--Department of Microbiology and Immunology, 1989.

Improved methods for the in situ hybridisation detection of messenger RNA ( mRNA ) in sections of liver tissue, were derived by use of an experimental system. This involved the use of tritiated-poly ( dT ) probes to detect poly ( A ) sequences attached to the 3 ' end of mRNA in sections of mouse liver that had been processed in various ways. The improved - methods were applied to the detection of hepatitis B virus ( HBV ) - and hepatitis delta virus ( HDV ) - RNA. In situ hybridisation and immunostaining techniques were then applied to studies of the pathogenesis of HBV and duck hepatitis B virus ( DHBV ) infection. In situ hybridisation studies of liver biopsy tissue from HBV - infected immunosuppressed renal transplant patients demonstrated an anatomical association between piecemeal necrosis and HBV replication at the cellular level in some patients. However, widespread replicative infection of hepatocytes also occurred in some patients in the presence of normal hepatocyte morphology and mild inflammatory changes indicating that at the cellular level virus replication was not necessarily a direct cytopathic process. These findings supported the view that hepatocyte Injury may : ( i ) result from immune - mediated damage directed against cells undergoing replicative, but not restricted infection ; ( ii ) eliminate cells undergoing replicative infection and favour clonal regeneration of cells undergoing restricted infection. Localisation of interferon - alpha ( IFN - alpha ) expression in liver tissue chronically infected with HBV and HDV, identified mononuclear cells and fibroblasts ( but not hepatocytes ) as the main producers of IFN - alpha. IFN - alpha - positive cells were associated with areas of liver tissue containing cells supporting virus replication and exhibiting the greatest degree of liver damage, suggesting that locally produced IFN - alpha may be a natural regulator of virus replication in chronic liver disease. Experimental DHBV infection of Pekin - Aylesbury ducks showed that virus inoculated either intravenously or intraperitoneally, gained access to randomly distributed hepatocytes without first replicating in other cell types in the liver. Virus was seen to disseminate to contiguous cells following anatomical boundaries by the third day post - inoculation. Markers of DHBV infection in liver and serum showed reproducible kinetics, and duck hepatocytes in this system appeared to be highly permissive as large amounts of DHBV DNA and DHBsAg were produced intracellularly without the development of ongoing cytopathology. Hepatocytes were the major cell type responsible for early significant DHBV replication, in contrast to pancreas, kidney, spleen and circulating mononuclear cells where significant levels of infection were detected only after the first week of infection and the onset of viraemia. / Thesis (Ph.D.)--Department of Microbiology and Immunology, 1989.

Thesis (Ph. D.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves 157-186). Also available in print.

Hepatitis C virus (HCV) infection is a major global health burden affecting 1-2% of the world’s population. The majority of infected individuals will develop chronic infection and are at risk of cirrhosis and hepatocellular carcinoma. There is currently no preventative vaccine available for HCV. In the developed world, the highest HCV incidence and prevalence rate is amongst intravenous drug users (IDU). The duration, frequency of IDU, and sharing of drug injecting equipment contribute to particularly high rates of HCV infection in this population. Individuals at high risk of recurrent exposure to HCV infection from long term IDU have been recruited in Plymouth, UK, from 2003 onwards and if they remain negative for HCV infection are termed exposed uninfected (EU). Understanding the factors that prevent HCV infection in this cohort could give valuable insight into the mechanisms of natural resistance to HCV infection. The EU cohort was previously shown to have characteristics attributable to the activation of both the adaptive and the innate arms of the immune system with no known dominant, immune or non- immune, mechanism of HCV protection. The aim of this thesis was to attempt and identify this mechanism and for that purpose a comparative transcriptional profile study was initially performed between 3 groups: EU, individuals who spontaneously cleared HCV infection (SR) and patients with chronic HCV infection (CHCV). Of the differentially regulated genes, the association with resistance to HCV was strongest for Interleukin-27 (IL-27) which was significantly upregulated in EU compared to the 2 other groups and C X C motif chemokine 7 (CXCL7) which was significantly upregulated in EU relative to the CHCV group. The CD28 mediated T-helper cell signalling pathway was significantly upregulated in SR relative to the 2 other groups. We attempted to corroborate the above findings and we demonstrated that IL-27 is overexpressed in EU, compared to SR and CHCV. The possible role of IL-27 in natural protection from HCV infection remains to be elucidated and requires further study.