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H C Thomas

Publications and source records attributed to H C Thomas.

At least 73 records · Page 4Linked to original sources
PubMed1997

Expression, purification, and partial characterization of HCV RNA polymerase.

The product of the NS5B gene of Hepatitis C Virus (HCV) has been expressed in Escherichia Coli both as a fusion protein with glutathione-S-transferase (GST) of molecular weight 91 KDa and at high level as a single protein of molecular weight 65 KDa. The protein was sequestered within inclusion bodies and a variety of procedures designed to minimize inclusion body formation proved unsuccessful. The method finally adopted involved the purification of inclusion bodies followed by the solubilization, purification, and refolding of the expressed protein. A good recovery and protein purity of the order of 80-90% were achieved. The purified protein was shown to possess RNA polymerase activity in an assay using polyA/oligoU as template. The enzymatic activity is rifampicin resistant, poly A dependent, and requires Mg++. The availability of purified HCV RNA polymerase will allow the study of viral replication and constitute the basis for testing new anti-viral drugs.

Cloning, Molecular↗
PubMed1997

Mapping of the cellular immune responses to woodchuck hepatitis core antigen epitopes in chronically infected woodchucks.

T-cell responses to hepatitis B virus nucleocapsid antigens (HBcAg and HBeAg) play an important role in disease outcome in those infected with hepatitis B virus (HBV). The woodchuck is naturally infected in the wild with woodchuck hepatitis virus (WHV), which shows a high degree of genetic homology to HBV and produces a similar pattern of infection in its natural host. Twenty-three overlapping peptides were constructed to cover the entire WHV core region and used to identify immunodominant cellular epitopes in the nucleocapsid antigen using peripheral blood lymphocytes from 12 chronic WHV carrier and 4 uninfected control animals. A peripheral blood lymphocyte response was seen in all of the chronic WHV carrier animals to at least one peptide, and in 8 of the 12 chronic carrier animals a response was observed to 5 common peptides: peptide analogues of amino acids 16-30, 38-52, 50-69, 76-90 and 91-105. Peptide 91-105 produced maximal proliferation in 5 out of 12 infected animals. In addition, a difference in response was observed between wild and laboratory infected animals; the latter appeared to have a lower response to peptides than animals infected in the wild. This study provides evidence that the woodchuck has a population of peripheral blood cells which are sensitised to epitopes within the nucleocapsid protein and provides a basis on which to develop the use of the woodchuck as an immunological model of HBV infection for testing therapeutic means of enhancing this response.

Animals↗
PubMed1997

The precore sequence of hepatitis B virus is required for nuclear localization of the core protein.

The cellular localization of the precore/core and core proteins was studied by immunofluorescence following transfection of 143 thymidine kinase-negative (TK ) and Hep-G2 cells with expression constructs containing wild-type (hepatitis B e antigen [HBeAg]-positive) and precore mutant (HBeAg-negative) sequences. Precore/core constructs with the wild-type phenotype result in strong nuclear staining, while, in contrast, constructs expressing core antigen alone have strong cytoplasmic staining. These differences in the pattern of immunofluorescence staining may be caused by expression of the precore/core protein, some of which may be translocated into the nucleus, following removal of the signal peptide. In vitro translation experiments showed that the main protein products obtained in the presence of microsomal membranes were the precore/core protein and a truncated product representing the same protein without its signal peptide. Core protein expression from the precore mutant constructs was very much reduced, indicating that translational re-initiation was not very efficient. The significance of the precore/core protein being present in the nucleus is not clear, but suggests that it may be important in the replicative cycle of the virus. Finally, HBeAg produced by some of the constructs could not be detected because amino acid substitutions affected antibody-binding epitopes.

Amino Acid Sequence↗
PubMed1997

Emergence and selection of HBV variants in an anti-HBe positive patient persistently infected with quasi-species.

BACKGROUND/AIMS: Infection with the hepatitis B e antigen (HBeAg) negative variant of hepatitis B virus (HBV) causes chronic liver disease characterised by occasional acute exacerbations. This virus exhibits a high prevalence of mutations in the core region. Our aim was to study the changes in the pre-core/core region of the virus in relation to exacerbations of the disease. METHODS/RESULTS: We performed direct sequencing on DNA amplified from 7 sequential sera taken over a 5-year period from a hepatitis B surface antigen (HBsAg) and anti-HBe positive Greek patient infected with the HBeAg negative variant. The patient had chronic hepatitis with several acute exacerbation episodes and underwent interferon therapy twice. We found significant variability in the core region at different time points. To determine whether these variants were present in the initial serum sample and subsequently selected under immune pressure or whether they arose de novo during the course of the disease, we cloned the pre-core/core region from 4 sera before and after episodes of acute exacerbation. Fifteen clones from each time point were sequenced. Fourteen nucleotide mutations in the pre-core/core region were recorded, 7 (50%) of which led to amino-acid substitutions. All the amino-acid changes occurred at recognised B- and CD4+ epitopes. The cloning results indicate the presence of quasi-species in all the samples investigated. Some of the variants present as a minor population in the first sample appear to have been selected and become dominant in subsequent sera. However, the emergence of novel variants, not present at a detectable level in earlier samples, during the course of the disease, was also established. The quasi-species nature of HBV only became apparent after the cloning experiments and was not obvious from the direct sequencing results. CONCLUSIONS: New dominant variants occurring during the course of the disease arose either by the selection of pre-existing mutants that were not detected by direct sequencing or by mutation of existing strains. All changes were located within B- and CD4+ epitopes. The continuous production and selection of variants may enable virus to evade elimination by the immune system, resulting in persistent infection.

Adult↗
PubMed1997

Hepatitis G virus infection: clinical characteristics and response to interferon.

A new member of the Flaviviridae family has recently been cloned and completely sequenced. The new virus, tentatively named hepatitis G virus (HGV) and known to be closely related to GB virus C (GBV-C), is transmitted by blood and blood products, intravenous drug use and other behaviour associated with a high risk of parenteral exposure to blood. The association of the virus with hepatitis is demonstrated by the presence of raised liver transaminase (alanine aminotransferase, ALT) levels in patients infected with HGV in the absence of other identifiable causes of hepatitis. No patient sera from groups exposed to blood and blood products were found to be positive when tested for the presence of GBV-A or GBV-B sequences, two other recently described flaviviruses. Forty-five per cent of the HGV-infected patients investigated had normal ALT suggesting the existence of a normal carrier state. Persistent infection of up to 13 years duration was observed. Co-infection with hepatitis B or hepatitis C viruses (HBV and HCV) was commonly seen presumably because of shared risk factors. None of five patients with fulminant hepatic failure was positive for HGV infection. The virus is sensitive to interferon-alpha, but sustained responses were not seen with the treatment regimens used for HBV and HCV. Viral titres increased during immunosuppression following liver transplantation and the higher levels of viraemia were in one case accompanied by elavated ALT. Whether HGV (GBV-C) replicates in the liver in some or all cases remains to be established. Preliminary data suggest that it is present within peripheral blood lymphocytes.

Antiviral Agents↗
PubMed1997

Predicted secondary structure of the hepatitis G virus and GB virus-A 5'untranslated regions consistent with an internal ribosome entry site.

We describe comparative sequence analysis of 20 isolates of the recently discovered hepatitis G virus (HGV) and propose a model of the RNA secondary structure at the 3' end of the 5' untranslated region (UTR) of this virus. A single AUG starting at nucleotide position 552, which was in-frame and continuous with the putative polyprotein, was conserved in all 20 isolates and appeared to be the most likely site for the initiation of polyprotein synthesis. This consensus AUG was 14 amino acid residues upstream of a sequence with identity to the envelope protein E1 of hepatitis C virus (HCV), but the actual function of this N-terminal peptide of HGV is still not certain. None of the isolates encoded a sequence with similarity to the nucleocapsid protein of any known virus. The RNA secondary structure of the fragment under study was determined using thermodynamic modelling and validated using the results of comparative sequence analysis. Further support for the model was gained from the prediction of significant sequence and structural homology in the RNA secondary structure of the complete 5'-UTR of GB virus-A (GBV-A). A possible mechanism for translation initiation in HGV and GBV-A was suggested by the identification of features in common with the internal ribosome entry sites (IRESs) of HCV and picornaviruses.

Amino Acid Sequence↗
PubMed1997

Current status of hepatitis G virus (GBV-C) in transfusion: is it relevant?

A new flavivirus, provisionally designated hepatitis G virus or GBV-C has recently been described. The virus is parenterally transmitted by exposure to blood through transfusion, intravenous drug use and haemodialysis. Heat- or chemically-treated blood products are associated with reduced risk of infection. The virus may also be transmitted from mother to infant and by the sexual route. Although hepatitis G virus has been detected in patients with acute and chronic hepatitis, a causative role of the virus in such cases has not been established. The majority of long term carriers of the virus appear to have no liver, biochemical or histological abnormalities.

Flaviviridae↗
PubMed1997

Lamivudine therapy for chronic hepatitis B: a six-month randomized dose-ranging study.

BACKGROUND & AIMS: Lamivudine inhibits hepatitis B virus replication. This study investigated 6 months of lamivudine treatment at three doses. METHODS: Fifty-one patients (43% white, 49% Asian) with chronic hepatitis B were randomly assigned to receive 25, 100, or 300 mg of lamivudine orally once daily for 24 weeks with 24 weeks' follow-up. RESULTS: Serum hepatitis B DNA by liquid hybridization decreased in all patients and was undetectable at the end of the treatment in 7 of 12 (58%, 25 mg), 13 of 14 (93%, 100 mg), and 14 of 16 (88%, 300 mg) patients. Of the 36 patients with abnormal alanine aminotransferase (ALT) levels at baseline, 7 of 11 (64%, 25 mg), 5 of 11 (45%, 100 mg), and 5 of 14 (36%, 300 mg) normalized ALT at treatment completion. Quantitative decreases hepatitis Be antigen and hepatitis B surface antigen concentrations were observed at all doses. In most patients, markers of replication returned after treatment. Two patients (4%) were anti-HBe positive at the end of follow-up. Lamivudine was well tolerated. The incidence of adverse events was similar across all dose groups. However, 2 patients developed temporary hepatic decompensation after increase in transaminase levels after treatment. CONCLUSIONS: Lamivudine was well tolerated and induced sustained suppression of hepatitis B replication during treatment in all patients at all doses. These data support investigation of longer treatment durations of 100 mg once daily.

Adult↗
PubMed1997

Host factors in chronic viral hepatitis.

The clinical outcomes of both hepatitis B and C virus infection are immensely variable, ranging from subclinical, self-limiting infection to end-stage liver disease with hepatocellular carcinoma. Knowledge of the host factors that determine these outcomes is important for the understanding and management of these diseases and may in the future guide rational drug development. Epidemiologic studies have elucidated the role of age (at the time of infection) and sex on disease outcome and the complex role of HIV coinfection has become clearer with time. More recently, investigation of genetic susceptibility to the most adverse outcomes of infection has identified the importance of polymorphisms in the MHC class I and II loci, mannose-binding protein, and the TNF alpha promoter. However, relative to malaria, the study of genetic susceptibility in viral hepatitis is still in its infancy.

Adolescent↗
PubMed1997

Genetic diversity between hepatitis G virus isolates: analysis of nucleotide variation in the NS-3 and putative 'core' peptide genes.

Significant variation was found, between 46 isolates of hepatitis G virus (HGV), following direct sequencing of subgenomic PCR fragments from either or both the NS-3 and putative 'core' peptide. Nucleotide sequences of most HGV NS-3 fragments varied by 10-30% and of most putative 'core' peptide fragments by 2-20%. HGV was therefore shown to be much less variable than hepatitis C virus (HCV) and pairwise comparisons of HGV sequences demonstrated a single distinct distribution of evolutionary distances. Construction of phylogenetic trees, bootstrap analysis and calculation of mean distances between possible subtypes also indicated one level of variation between HGV NS-3 and putative 'core' peptide sequences, and the suggested degree of variation between isolates was similar to that between HCV subtypes. No evidence for clustering of sequences into multiple subtypes or genotypes was found. Although very small subgenomic fragments of HCV are indicative of the viral genotype it seems that the assignment of genetic groups is not possible for HGV using such small subgenomic fragments. The relatively limited genetic variation observed in HGV may reflect a relatively low level of host selection pressure stemming from the low level of host immunity stimulated by this virus.

DNA Primers↗
PubMed1997

Identification, prevalence and aspects of molecular biology of hepatitis G virus.

Hepatitis G virus (HGV) is a newly identified member of the Flaviviridae family. The positive-sense RNA genome of the virus contains a single open reading frame that encodes the viral polyprotein. Its genomic organization is similar to that of the hepatitis C virus (HCV) with which it has only 25% homology at the nucleotide level. Nucleotide sequences from the NS-3 helicase region of HCV varied by 10-30%. HGV is therefore much less variable than HCV. Construction of phylogenetic trees, and calculation of mean distances between possible subtypes, indicated one level of variation in NS-3 sequences: the degree of variation between isolates was similar to that observed between HCV subtypes. Thus no evidence for clustering of sequences into multiple genotypes was found. The virus is transmissible through blood transfusion and by exposure to blood products or intravenous drug use, and may result in acute or chronic hepatitis. A causative role of HGV in acute and chronic hepatitis is not yet established. Concurrent infection with hepatitis B virus (HBV) and HCV is common and persistent viraemia has been documented for many years, in many cases, in the absence of transaminase elevations.

Antiviral Agents↗
PubMed1997

The relationship of histology to genotype in chronic HCV infection.

The histological description of chronic hepatitis is undergoing considerable change at present. It has become important to define chronic hepatitis aetiologically and then define levels of necro-inflammatory change (grade) and fibrosis (stage). The aim of this study was to compare the ability of different histological scoring systems to detect differences in the pathological changes associated with infection with the different HCV genotypes that are known to have different natural histories. The histological appearances of liver biopsies from 29 HCV infected patients were compared by the Knodell histological activity index (HAI), modified histological activity index and the Scheuer histological scoring system. HCV genotyping was performed for each patient by sequence analysis of the 5' non-coding region. The histological appearances from HCV 1 infected patients showed a tendency towards more active necro-inflammatory changes when compared with those from HCV 2 or 3 infected patients. The levels of fibrosis were similar for all genotypes. The modified HAI and Scheuer scoring systems detected differences, not revealed by the Knodell system, in the types of inflammatory pathology produced by the different genotypes of HCV. In particular these scoring systems noted significant differences in the component scores of inflammation, in addition to the total inflammatory scores. In conclusion, the recently introduced scoring systems were able to detect differences in liver pathology produced by infection of similar duration with different viral genotypes. As genotype is considered an important determinant of disease progression and response to anti-viral therapy, it is likely that those scoring systems correlating with genotype will yield more useful histological information than those that do not.

Adult↗