PubMed Health⌕ Search

Biomedical subjects

E J Gowans

Publications and source records attributed to E J Gowans.

At least 19 recordsLinked to original sources
PubMed2002

Expression of the hepatitis C virus structural proteins in mammalian cells induces morphology similar to that in natural infection.

Like many positive-strand RNA viruses, replication of the hepatitis C virus (HCV) is associated with cytoplasmic membrane rearrangements. However, it is unclear which HCV proteins induce these ultrastructural features. This work examined the morphological changes induced by expression of the HCV structural proteins, core, E1 and E2, expressed from a Semliki Forest Virus (SFV) recombinant RNA replicon. Electron microscopy of cells expressing these proteins showed cytoplasmic vacuoles containing membranous and electron-dense material that were distinct from the type I cytoplasmic vacuoles induced during SFV replicon replication. Immunogold labelling showed that the core and E2 proteins localized to the external and internal membranes of these vacuoles, but at times were also associated with some of the internal amorphous material. Dual immunogold labelling with antibodies raised against the core protein and against an endoplasmic reticulum (ER)-resident protein (protein disulphide isomerase) showed that the HCV-induced vacuoles were associated with ER-labelled membranes. This report has identified an association between the HCV core and E2 proteins with induced cytoplasmic vacuoles which are morphologically similar to those observed in HCV-infected liver tissue, suggesting that the HCV structural proteins may be responsible for the induction of these vacuoles during HCV replication in vivo.

Animals↗
PubMed2001

Vitamin B12 and hepatitis C: molecular biology and human pathology.

Cobalamins are stored in high concentrations in the human liver and thus are available to participate in the regulation of hepatotropic virus functions. We show that cyanocobalamin (vitamin B12) inhibited the HCV internal ribosome entry site (IRES)-dependent translation of a reporter gene in vitro in a dose-dependent manner without significantly affecting the cap-dependent mechanism. Vitamin B12 failed to inhibit translation by IRES elements from encephalomyocarditis virus (EMCV) or classical swine fever virus (CSFV). We also demonstrate a relationship between the total cobalamin concentration in human sera and HCV viral load (a measure of viral replication in the host). The mean viral load was two orders of magnitude greater when the serum cobalamin concentration was above 200 pM (P < 0.003), suggesting that the total cobalamin concentration in an HCV-infected liver is biologically significant in HCV replication.

Antiviral Agents↗
PubMed2001

Development and characterisation of recombinant hepatitis delta virus-like particles.

Injection of particulate hepatitis B virus surface antigen (HBsAg) in mice leads to the induction of a HBsAg-specific class-I-restricted cytotoxic T lymphocyte (CTL) response. It is proposed that any protein internal to HBsAg will also be able to elicit a specific CTL response. In this study, several carboxy-terminal truncations of hepatitis C virus (HCV) core protein were fused to varying lengths of amino-terminal truncated large hepatitis delta antigen (L-HDAg). These constructs were analysed for their ability to be expressed and the particles secreted in the presence of HBsAg after transfection into HuH-7 cells. The secretion efficiency of the various HCV core-HDAg chimeric proteins was generally poor. Constructs containing full length HDAg appeared to be more stable than truncated versions and the length of the inserted protein was restricted to around 40 amino acids. Thus, the use of L-HDAg as a chimera to package foreign proteins is limited. Consequently, a polyepitope (polytope) containing a B-cell epitope from human papillomavirus (HPV 16) and multiple T-cell epitopes from the HCV polyprotein was used to create the construct, L-HDAg-polyB. This chimeric protein was shown to be reliant on the co-expression of HBsAg for secretion into the cell culture fluid and was secreted more efficiently than the previous HCV core-HDAg constructs. These L-HDAg-polyB virus-like particles (VLPs) had a buoyant density of approximately 1.2 g/cm3 in caesium chloride and approximately 1.15 g/cm3 in sucrose. The VLPs were also immunoprecipitated using an anti-HBs but not an anti-HD antibody. Thus, these recombinant VLPs have similar biophysical properties to L-HDAg VLPs.

Amino Acid Sequence↗
PubMed2001

Antigenicity and immunogenicity of novel chimeric hepatitis B surface antigen particles with exposed hepatitis C virus epitopes.

The small envelope protein of hepatitis B virus (HBsAg-S) can self-assemble into highly organized virus like particles (VLPs) and induce an effective immune response. In this study, a restriction enzyme site was engineered into the cDNA of HBsAg-S at a position corresponding to the exposed site within the hydrophilic a determinant region (amino acid [aa] 127-128) to create a novel HBsAg vaccine vector allowing surface orientation of the inserted sequence. We inserted sequences of various lengths from hypervariable region 1 (HVR1) of the hepatitis C virus (HCV) E2 protein containing immunodominant epitopes and demonstrated secretion of the recombinant HBsAg VLPs from transfected mammalian cells. A number of different recombinant proteins were synthesized, and HBsAg VLPs containing inserts up to 36 aa were secreted with an efficiency similar to that of wild-type HBsAg. The HVR1 region exposed on the particles retained an antigenic structure similar to that recognized immunologically during natural infection. VLPs containing epitopes from either HCV-1a or -1b strains were produced that induced strain-specific antibody responses in immunized mice. Injection of a combination of these VLPs induced antibodies against both HVR1 epitopes that resulted in higher titers than were achieved by vaccination with the individual VLPs, suggesting a synergistic effect. This may lead to the development of recombinant particles which are able to induce a broad anti-HCV immune response against the HCV quasispecies or other quasispecies-like infectious agents.

Animals↗
PubMed2000

Properties of the bovine viral diarrhoea virus replicase in extracts of infected MDBK cells.

An assay for the bovine viral diarrhoea virus (BVDV) replicase was developed using extracts from BVDV-infected cells. The replicase activity was maximal approximately 8 h post-infection as measured by the generation of a genomic length radiolabelled RNA. Using a semi-denaturing gel system, three virus-specific in vitro radiolabelled nascent RNA species were identified. A fast-migrating RNA was demonstrated to be the double-stranded replicative form (RF). A second form was shown to be a partially single-stranded/partially double-stranded RNA, characteristic of the replicative intermediate (RI). A third form, which was often undetectable, migrated between the RF and RI and was probably genomic viral RNA. The optimal replicase activity was dependent on 5-10 mM Mg2+ and although it was also active in 1-2 mM Mn2+ it was inhibited at higher concentrations. The optimum KCl concentration for labelling of the RI and RF were different, suggestive of at least two distinct replicase activities. These results are supportive of a semi-conservative model of BVDV RNA replication.

Animals↗
PubMed2000

Specific detection of minus-strand hepatitis C virus RNA by reverse-transcription polymerase chain reaction on PolyA(+)-purified RNA.

A full-length complementary DNA (cDNA) clone of the hepatitis C virus (HCV) genome was used to prepare full-length plus- and minus-strand RNA. The minus-strand RNA, which contains a polyA(+) tract complementary to the polyU tract found in the plus strand (genomic) RNA, but not the plus strand RNA, was captured with a commercial polyA(+)-tract isolation system. After elution, the minus strand was amplified by reverse-transcription polymerase chain reaction (RT-PCR). The combination of this procedure and RT-PCR using rTth resulted in an unprecedented level of discrimination of 10 logs(10). HCV minus-strand RNA isolation was unaffected by the addition of an excess of 10(4) of plus strands or by the addition of cellular RNA, and although the polyA(+) isolation step removed 99. 99% of plus strands, there was no loss of minus-strand signal. Minus-strand RNA was detected in RNA extracted from 4/4 liver samples and 4/8 peripheral blood mononuclear cells (PBMC) samples examined. Because the titer of plus-strand HCV RNA in any sample makes a significant contribution to false, random, and self-priming, removal of the plus strand in this manner results in the most accurate method yet devised to confirm the replication of HCV in a population of cells.

Hepatitis C↗
PubMed2000

Distribution of markers of hepatitis C virus infection throughout the body.

The detection of markers of hepatitis C virus (HCV) infection is complicated by the presence of virus in circulating blood. Consequently, it is necessary to target the negative-strand virus RNA or the nonstructural proteins. This has proved challenging, due to unforeseen difficulties in the specific detection of negative-strand viral RNA. However, recent data suggest that although the liver is the major target organ, the virus may be able to replicate in peripheral blood mononuclear cells, lymph nodes, and pancreas and to a more limited degree in bone marrow cells, thyroid, adrenal glands, and spleen. An analysis of the distribution of virus-infected cells in the liver has proved equally challenging. It is clear from the paucity of data generated by immunoblot and Northern blot hybridization that the level of HCV replication in the liver is very low, and this has necessitated studies to detect the viral RNA and antigens by in situ hybridization and immunohistochemistry, respectively. The results of these studies are quite disparate. Those related to in situ hybridization are nonreproducible and reflect wide disparities in the methodology used by different research workers. The data are often internally inconsistent or are inconsistent with data generated by nucleic acid amplification methods to detect the viral RNA in purified RNA extracts or inconsistent with our current understanding of the virus replication cycle. In contrast, the detection of the viral proteins is more reproducible, and some consensus can be reached. It is clear that frozen sections rather than formalin-fixed paraffin-embedded tissue are preferable. Indeed, it is likely that the use of the latter samples can generate nonspecific positive reactions. Surprisingly, many of the most useful data were derived by the use of polyclonal human antibodies from chronic carriers. HCV proteins were detected in the cytoplasm of infected hepatocytes, often in a punctate granular pattern in some hepatocytes and occasionally in monocytes and other cell types. There is often no correlation between HCV antigen expression and the degree of liver cell injury, although patients with lower levels of antigen expression are more likely to respond to interferon therapy.

Antigen-Antibody Complex↗
PubMed1999

A cellular protein which binds hepatitis B virus but not hepatitis B surface antigen.

The envelope of hepatitis B virus (HBV) consists of three related proteins known as the large (L), middle (M) and small (S) hepatitis B surface antigens (HBsAg). L-HBsAg has a 108-119 amino acid extension at the N terminus compared with M-HBsAg and contains the preS1 sequence of the HBV envelope. Previous research has identified this region as the likely virus attachment protein which is thought to interact with the cellular receptor for the virus. However, as the receptor has still not been identified unequivocally, we used the preS1 region of L-HBsAg to screen a human liver cDNA library by the yeast two-hybrid system. Several positive clones were isolated which encoded cellular proteins that interacted with the HBV preS1 protein. The specificity was examined in an independent manner in experiments in which baculovirus-derived glutathione S-transferase (GST)-preS1 was incubated with 35S-labelled protein expressed by in vitro translation from the positive clones. The intensity of the interactions using this alternative approach mirrored those observed in the yeast two-hybrid system and two proteins (an unidentified protein and a mitochondrial protein) were selected for further study. The specificity of the binding reaction between the preS1 protein and these two proteins was further confirmed in a competition assay; HBV purified from serum, but not purified HBsAg, was able to compete with preS1 and thus block GST-preS1 binding to the unidentified protein but not to the mitochondrial protein. The unidentified protein was then expressed as a fusion protein with GST and this was able to bind HBV virions in a direct manner.

Baculoviridae↗
PubMed1998

The replicative intermediate molecule of bovine viral diarrhoea virus contains multiple nascent strands.

The replication of bovine viral diarrhoea virus (BVDV) RNA is considered to involve replicative intermediates (RI) and replicative forms (RF) of virus RNA. These RNA species were radiolabelled metabolically by 3H-uridine in BVDV-infected cells and separated by sucrose gradient centrifugation. RNA in the fractions was then digested with RNase A in high salt (2 x SSC) and the RNase-resistance was determined. Using the Baltimore formula, this led to the calculation that the RI of BVDV contained 6-7 nascent strands on the template. This suggests that the structure of the BVDV RI is similar to that of poliovirus and dengue 2 virus.

Animals↗
PubMed1998

Molecular cloning of an Australian isolate of hepatitis C virus.

The genomic sequence of an Australian isolate of hepatitis C virus (HCV) was determined from overlapping cDNA clones obtained from a small amount (1.2 ml) of serum from a single individual with hepatitis C. The isolate (HCV-A) comprises 9379 nucleotides (nt) including 324 nt of a 5' untranslated region (5'UTR), a single long open reading frame of 9033 nt encoding a polyprotein of 3010 amino acids (aa), and 22 nt of a 3' untranslated region (3'UTR). Sequence analysis of a 251 nt region within the 5'UTR and a 222 nt region within NS5B showed the genotype of HCV-A to be subtype 1b. A striking difference in the amino acid sequence of the hypervariable region 1 (HVR-1), and not in the surrounding sequence, was seen in cDNA clones synthesised from serum taken 52 weeks after the initial sample, indicating a significant population diversity of HCV genomes.

Amino Acid Sequence↗
PubMed1998

A stable cell line with a proportion of cells persistently infected with bovine viral diarrhoea virus.

Bovine turbinate (BTu) and lamb testis (LT) cell lines persistently infected with bovine viral diarrhoea virus (BVDV) arose as a result of a single change of medium containing commercial foetal calf serum. Infected cells comprise 30% and 50% respectively, of the total cell population, determined by immunohistochemical staining. The ratio of positive cells has remained unchanged during successive passages. Characterization of the persistently-infected BTu cells (BTuI) showed that only full length viral RNA was detected by northern blot hybridisation, indicating that DI particles were not involved. Secreted and intracellular virus from these cells was fully infectious for fresh BTu and LT cells. The BTuI cell line was fully permissive for a cytopathic BVDV isolate and a bovine herpesvirus, but non-permissive for two non-cytopathic BVDV isolates. Attempts to induce the permissive state in the BVDV-negative cells of the BTuI culture by treatment with actinomycin D and 5'-aza-cytidine failed. These cells provide a convenient model to study aspects of BVDV pathogenesis and replication.

Animals↗
PubMed1998

Identification of novel sequences at the 5' terminus of the hepatitis C virus genome.

To permit accurate identification of the 5' end of the HCV genome, we used RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE) where an RNA molecule of known sequence (transcribed in vitro from a cDNA template) was ligated to RNA purified from a hepatitis C virus (HCV)-infected liver sample. After ligation, the product was amplified by nested polymerase chain reaction (PCR) and amplicons that were equal to or greater in size than those predicted from the recognized 5' terminus of the HCV genome were cloned into pBluescriptKS. Twelve clones were sequenced, including three that were identical and contained an additional eight nucleotides, namely CCCCCCCA. Thus, the HCV 5' untranslated region (UTR) is now recognized as comprising 349 nucleotides, although it is possible to speculate that these additional nucleotides are part of a second, as yet undetected, stem-loop at the extreme 5' terminus of the genome.

Base Sequence↗
PubMed1998

Exposure to GB virus type C or hepatitis G virus in selected Australian adult and children populations.

BACKGROUND: The epidemiology and disease association for the GB virus type C (GBV-C) or hepatitis G virus (HGV) are poorly understood. STUDY DESIGN AND METHODS: This study describes the exposure rates to GBV-C/HGV in diverse Australian population groups by testing for current infection and evidence of past infection with a reverse transcriptase polymerase chain reaction and an anti-E2 enzyme-linked immunosorbent assay, respectively. Subjects included volunteer blood donors, hepatitis C antibody (anti-HCV)-positive donors, children, hemodialysis patients, pregnant women attending a prenatal clinic, injecting drug users (IVDUs), and adult hemophiliacs. RESULTS: Combined GBV-C RNA and E2 antibody prevalence was 6.5 percent (6/93) in children, 13.3 percent (75/565) in blood donors, 14 percent (14/99) in pregnant women, 22.5 percent (18/80) in hemodialysis patients, 80 percent (56/70) in anti-HCV-positive donors, 88.6 percent (31/35) in IVDUs, and 85.7 percent (54/63) in adult hemophiliacs. Children had the lowest antibody rate, 1.1 percent, whereas the rate was 10.8 percent for blood donors and rose to 45.7 percent for IVDUs, 57.1 percent for anti-HCV-positive donors, and 74.6 percent for hemophiliacs. In contrast, current infection rates were comparable for children, blood donors, and pregnant women (5.4, 2.6, and 6%, respectively), rising to 11.1 percent for hemophiliacs, 24.3 percent for anti-HCV-positive donors, and 48.6 percent for IVDUs. Ten of 12 blood donors had persistent viremia, while 2 had recent infections, 1 with apparent resolution. CONCLUSION: Exposure to GBV-C can commence at an early age, although ongoing exposure may also occur among adults with no apparent risk factors. GBV-C RNA positivity was not associated with abnormal plasma alanine aminotransferase levels among blood donors.

Adult↗
PubMed1997

Expression and interaction of the hepatitis C virus structural proteins and the 5' untranslated region in baculovirus infected cells.

The structural proteins, core, E1 and a C-terminal truncated E2 (C-E1-E2p) as well as the 5'UTR linked to the core gene (5'UTR-C) of the hepatitis C virus were expressed in Sf9 cells. Expression of the C-E1-E2p polyprotein from a single vector resulted in expression of multiple forms suggesting that the cleavage of the polyprotein in the insect cells is incomplete. The structural proteins were expressed as insoluble forms and were solubilized by freeze/thaw cycles or detergent treatment. Analysis of the co-expressed (5'UTR-C and C-E1-E2p) proteins through a 20-60% sucrose gradient revealed that the core protein migrated rapidly into the gradient, in common with the sedimentation profile for core protein expressed from the core gene only. Neither E1 nor E2 proteins were involved in the core-core protein interaction. Expression of the 5'UTR-C in insect cells resulted in a high level of mRNA transcription but no translation of core protein, presumably due to a failure to initiate HCV translation by an internal ribosome entry mechanism, since the mRNA transcript was translated in vitro, suggesting that the 5'UTR can influence HCV tropism.

Animals↗
PubMed1997

The development and characterisation of a SV40 T-antigen positive cell line of human hepatic origin.

COS7 cells and other cell lines expressing the SV40 large T-antigen, have been of great benefit in transfection studies using transient expression vectors which contain the SV40 origin of replication, as these cells allow plasmid replication to a high copy number. As no T-antigen expressing cell line derived from a well characterised hepatocyte-like continuous cell line currently exists, the establishment of such a cell line for studies which require expression of hepatocyte-specific factors would be extremely useful. A HepG2-derived stable cell line (THT1) was therefore developed which demonstrates a high level of transfection efficiency whilst retaining hepatocyte-like features, such as the production of hepatitis B virus. The THT1 cell line displayed chromosomal integration of the SV40 T-antigen gene and nuclear expression of the antigen. The cell line also maintained the general morphological features of its parent cell line, and showed an increased rate of cell growth.

Animals↗
PubMed1997

Hepatitis B virus binding to leucocyte plasma membranes utilizes a different region of the preS1 domain to the hepatocyte receptor binding site and does not require receptors for opsonins.

A quantitative assay of hepatitis B virus (HBV) binding to hepatocyte plasma membranes was adapted to show that leucocyte plasma membranes bind serum-derived HBV saturably, and that this binding is inhibited using synthetic peptides representative of the large envelope protein of HBV. Using a panel of ligand-blocking monoclonal antibodies (mAb) to opsonin receptors, it was shown that the three classes of Fc gamma R and CR3 are not major receptors for HBV on leucocytes or hepatocytes. It was also shown that HBV does not utilize the receptor for IgA, Fc alpha R, for attachment to leucocytes, despite reported sequence homology between the large envelope protein of HBV and the Fc portion of human IgA. Evidence is presented that the receptor for HBV on leucocytes may differ from the hepatocyte receptor(s), based on synthetic peptide inhibition assays of HBV binding. Furthermore, it was observed that glycosaminoglycans influence the HBV-liver and leucocyte interactions, providing evidence that HBV attachment may be a multi-stage process.

Adult↗
PubMed1996

An ELISA for the detection of antibody to the core antigen of hepatitis C virus: use in diagnosis and analysis of indeterminate samples.

AIMS: In order to examine more carefully the natural history of hepatitis C virus infection and to determine a role for anti-core in the discrimination of indeterminate samples, a solid phase ELISA to detect antibody of the immunoglobulin G class to the hepatitis C virus core antigen was developed using purified protein expressed in Escherichia coli from a major portion of the core antigen coding region. METHODS/RESULTS: In a study which examined 651 samples submitted for routine testing by a commercial ELISA (Ortho), only 11 samples showed discrepant results; of these, 10 were Ortho ELISA positive, anti-core negative and one was Ortho ELISA negative anticore positive. Supplemental tests showed that 5/10 of these samples were anti-HCV negative by RIBA and the reciprocal 5 were negative for anti-C22 but positive for anti-C100 and anti-C33. The Ortho ELISA negative, anticore positive sample was weakly positive for anti-C22. The anti-core ELISA was then used to examine 67 indeterminate samples from the blood bank; 11/11 samples which were HCV-RNA positive were anti-core positive and 7/56 samples which were HCV-RNA negative were anti-core positive. The anti-core titre was then examined in two groups of indeterminate samples; group 1, polymerase chain reaction-positive, anti-core positive and group 2, polymerase chain reaction-negative, anti-core positive. The geometric mean anti-core titres in these groups were 1 x 10(-3.6) and 1 x 10(-2.3), respectively. Thus in this group of indeterminate samples, all samples (except one) with an anti-core titre > or = 1/200 were polymerase chain reaction-positive, confirming a close correlation between anti-core levels and hepatitis C viraemia. Anti-core was detected with equal efficiency in patients infected with genotypes which differed to that used to express the recombinant core antigen.

Antibody Specificity↗