Hepatitis C virus replication in hepatocellular carcinoma.

Hepatitis C virus (HCV) replication is reported in both tumour and non-tumour tissue in a case of hepatocellular carcinoma. Viral replication was established by showing the presence of minus strand HCV RNA by PCR amplification, after excluding residual reverse transcriptase activity of Taq polymerase. No minus strand was found in serum derived virion RNA. PCR amplified products from both tumour and non-tumour parenchyma were sequenced in the 5' non-coding region and shown to be identical. The genotype of this Indonesian patient was found to be 1b (or II), the most prevalent type in the Far East.

Serum HCV RNA levels in chronic HCV hepatitis measured by quantitative PCR assay; correlation with serum AST.

A quantitative PCR assay is described for serum HCV RNA which is based on a recombinant competitive titrating RNA template. This template is derived from the 5' non-coding region of the genome and generates a shorter PCR product (93 bp) than that from the serum-derived wild type genomes (279) bp from the same sets of primers. By using this assay we have found serum HCV RNA concentrations ranging between 3.3 x 10(2) and 6.6 x 10(8) viral genomes/ml of serum in 15 samples from 10 different patients, 9 cases of chronic hepatitis and one case of fulminant hepatitis. Comparison of viraemias with serum transaminase levels has shown a good correlation for individual patients between these two parameters during chronic HCV hepatitis.

Use of biotinylated probes in serum hepatitis B virus DNA detection.

A dot-blot hybridisation assay for serum HBV DNA is described using a non-radioactive (biotinylated) DNA probe. The assay is both sensitive (1 pg of HBV DNA) and reproducible, and shows several advantages over similar assays which use 32P-labelled probes for the routine detection of infectious virus particles both in blood and in other biological fluids.

Purification of woodchuck hepatitis surface antigen using a monoclonal antibody raised against the antigen.

Woodchuck hepatitis virus (WHV) is closely related to the human hepatitis B virus (HBV) and infection of woodchucks with WHV creates a useful model for studies of immunity, pathogenesis and therapy of HBV infection. To increase the usefulness of this model, monoclonal antibodies were raised to woodchuck hepatitis surface antigen (WHsAg) and one of these antibodies was used to purify the antigen by affinity chromatography from serum, a simpler and quicker method of purification than the current ultracentrifugation methods. The bands found by SDS-polyacrylamide gel electophoresis of WHsAg were the major 25 and 29 kilodalton (kDa) bands and a triplet of 45, 51 and 55 kDa which are thought to be the glycosylated and unglycosylated middle and large WHsAg. Both the antibody and the antigen are valuable reagents for the study of WHV infection.

Replication of hepatitis delta virus.

Hepatitis delta virus (HDV) is a unique viroid-like human pathogen that is always associated with hepatitis B infection. Replication of HDV involves the transcription of genomic RNA, probably by the host RNA polymerase II, by a rolling circle mechanism followed by self-cleavage and self-ligation. Editing of antigenomic RNA, possibly involving the enzyme adenosine deaminase, generates two functionally distinct forms of delta antigen. The molecular basis for HDV pathogenicity remains uncertain.

Delta hepatitis. The disease and the virus.

Delta hepatitis virus is a new human pathogen always found associated with hepatitis B virus (HBV) causing both fulminant hepatitis and the accelerated progression of pre-existing HBV hepatitis. The virus is coated in HBsAg and contains circular single stranded RNA genome with very high intramolecular base-pairing, similar to the genomes of plant viroids, and the HDV antigen, a specific marker of HDV infection also found in the nuclei of infected hepatocytes. The genome is about 1700 nucleotides long and of minus polarity with a major constant antigenomic Open Reading Frame (ORF) which codes for the antigen. Replication of viral RNA appears to proceed by a rolling circle mechanism and specific self-cleavage and self-ligation of both genomic and antigenomic HDV RNA strands has been demonstrated in vitro. HDV appears to require HBsAg for its propagation and hepatotropism but not to depend on HBV for replication.

An investigation of acid-soluble nuclear proteins of human leucocytes in relation to fraction RP2-L, a component of neoplastic cells.

1. The claim that tumour cells contain a specific nuclear protein was investigated. The presence of this component was confirmed in Walker tumour cells by the chromatography on CM-cellulose of nuclear proteins labelled with [(14)C]lysine. This protein was studied further in a number of human leucocyte cells. 2. The labelling of leucocyte nuclear proteins with [(14)C]lysine was attempted during incubation and culture in vitro. Incorporation of the label into acid-soluble nuclear proteins was highest in normal lymphocytes cultured with phytohaemagglutinin, followed by chronic-myeloid-leukaemic leucocytes and mixed samples of normal leucocytes incubated in plasma. Little incorporation was seen in similar extracts of chronic-lymphatic or normal leucocytes. 3. Lymphocytes were the only cells that gave nuclear extracts with amino acid analysis similar to that of unfractionated histones. 4. Little of the [(14)C]lysine in nuclear extracts of incubated leucocytes proved to be of chromosomal origin. No evidence was found of an RP2-L component in the highly labelled nuclear extracts of phytohaemagglutinin-treated lymphocytes until after 6 days of culture with [(14)C]lysine. This component was soluble in saline. 5. Evidence is presented that fraction RP2-L is a non-histone protein constituent of cell nuclei whose labelling with [(14)C]lysine may be dependent on the metabolic state of the cell. Thus this component is not specific to the neoplastic state.

Cloning and characterisation of a delta virus cDNA sequence derived from a human source.

We report the extraction of delta virus RNA from the serum of a delta-virus-infected patient and the subsequent cloning and analysis of a 380-nucleotide-long cDNA (D380). The nucleotide sequence of D380 shows overall differences of approximately 20% when compared with previously published sequences and does not include the viroid consensus sequence previously reported (Wang et al: Nature 323:508-514, 1986). A potentially coding open reading frame extending over the whole length of the D380 has been identified. Our results demonstrate the existence of genetic heterogeneity amongst different delta virus isolates.

Cloning and analysis of integrated hepatitis B virus DNA of the adr subtype derived from a human primary liver cell carcinoma.

A 10 kb genomic DNA fragment derived from a human primary liver cell carcinoma (PLC) and containing integrated hepatitis B virus (HBV) DNA was cloned and analysed. Physical mapping showed the viral DNA to comprise a linear sequence of at least 2.8 kb (87%) of the HBV genome and to be of the adr subtype. Integration appeared to have occurred in the region of the viral genome spanning the cohesive ends. The cellular flanking DNA sequences to one side of the integrated viral DNA contained repeats of the Alu family. The finding of no apparent rearrangements of the integrated HBV DNA sequences in this clone is in contrast to the situation in the huSP and PLC/PRF/5 PLC cell lines in which the integrated viral DNA sequences are greatly rearranged and suggests that such rearrangements may be atypical of solid PLCs.

Integration of hepatitis B virus DNA through a mutational hot spot within the cohesive region in a case of hepatocellular carcinoma.

Integrated hepatitis B virus (HBV) DNA previously cloned from a hepatocellular carcinoma genomic library derived from a Japanese patient was characterized further. Sequence analysis of restriction fragments bearing the virus-host junctions defined 3125 nucleotides of essentially un-rearranged HBV DNA of the adr subtype with the right junction mapping within the cohesive region at position 1970 and the left within the pre-core at position 1886. The right viral-host junction contains a 7 bp repeat (TGTAGGC) and a possible 2 bp inversion. The integrated HBV DNA includes the complete open reading frames for core, pre-S, S and polymerase and a 3' truncated X gene, and lacks most of the pre-core. Integration has occurred at a mutational hot spot of the viral genome and appears to be located in a region of semi-repetitive genomic DNA 3' to the beta-globin gene cluster.

Cloning and sequencing of the structural region and expression of putative core gene of hepatitis C virus from a British case of chronic sporadic hepatitis.

We report the cloning and sequencing of the putative structural region of the hepatitis C virus (HCV) genome (2229 nucleotides) from an isolate derived from a British case of chronic sporadic non-A, non-B hepatitis. The overall sequence shows a higher similarity with one type of HCV, HCV1 (92%), than with HCV2 (80%), is very highly conserved at the 5' end (99%) preceding the long open reading frame, is well conserved also in the putative core region (90 to 97%), but shows marked variation in the putative envelope region, particularly in the envelope 2/non-structural 1 region (70%). The putative core gene was cloned in pJ3 omega under the early simian virus 40 promoter and expressed in human hepatoma cells. A predominantly cytoplasmic 22K polypeptide was expressed which was antigenically reactive with serum from chronically infected HCV patients.

Editing efficiency of hepatitis delta virus RNA is related to the course of infection in woodchucks.

Based on evidence in vitro which shows that the small form of hepatitis delta virus (HDV) antigen (S-HDAg) initiates virus replication, whereas the long form (L-HDAg) encoded by the edited L-genome inhibits replication, we first put forward the hypothesis that HDV RNA editing efficiency, i.e. the intracellular L/S-genome ratio, could be a determining factor on the course of the infection. In order to analyse the precise sequence of events after infection, woodchuck carriers of woodchuck hepatitis virus (WHV) were superinfected with HDV and sequential changes in HDV RNA editing efficiency were analysed in relation to the duration of viraemia. Our findings show that: (1) in both transiently and persistently viraemic woodchucks, the percentage of L-genome is higher at the early stage of onset of the disease than at the late stage; (2) at the early stage of onset the percentage of L-genome is higher in cases with transient viraemia than in those with persistent viraemia; (3) a relatively greater decrease in L-genome is seen later in transiently viraemic animals than in those that remain persistently viraemic. In view of the above findings in vivo and other supporting evidence in vitro, we propose a hypothesis for the pathogenesis of HDV. This hypothesis predicts the outcome of acute infection and we suggest a gene therapeutic approach for this disease based on the intracellular accumulation (or increase) of the L-genome.

Sequence, expression and reconstitution of an HCV genome from a British isolate derived from a single blood donation.

Morphological analysis of hepatitis C virus and development of antiviral drugs to eradicate this agent have been seriously hampered by the low viraemias observed during natural infection and the unavailability of a cell culture system for virus propagation. Recently a low-grade hepatitis has been reported in chimpanzees after intrahepatic transfection of full-length synthetic HCV RNA and successful infections shown to be critically dependent on the integrity and genetic homogeneity of the reconstituted clone. In this study we describe and characterize a full HCV RNA sequence derived from a case of chronic sporadic hepatitis. The genotype was shown to be 1a with a low level of intraclonal sequence heterogeneity, and processing of both structural and nonstructural proteins has been documented. The assembly of the full genome has also been achieved. The low level of intraclonal variation observed may reflect infection with a single isolate and the fact that cloning was performed on virus obtained from a single blood donation makes this clone a good candidate for future in vivo and in vitro transfection studies.

Sequence variation in the large envelope glycoprotein (E2/NS1) of hepatitis C virus during chronic infection.

Sequence variation in the putative large surface glycoprotein (E2/NS1) of hepatitis C virus (HCV) was analyzed over 18 months in 4 patients with chronic relapsing non-A, non-B liver disease, of whom 2 were treated with lymphoblastoid interferon-alpha. Sequence analysis showed marked heterogeneity between isolates from different patients at the hypervariable region mapping to the amino-terminus of E2/NS1 (amino acid position, 386-411) and the extended sequence analyzed again confirmed the existence of two types, HCV1 and HCV2. Sequential nucleotide analysis of the hypervariable region over 1 year for each patient showed 0-9 amino acid changes, more common in the untreated than in the interferon-alpha-treated patients (3 and 9 vs. 1 and 0). Peaks of raised serum transaminases in individual patients showed no consistent association with mutations within this region. However, results do not exclude the possibility that viral persistence may be due to the emergence of "escape mutants" in the hypervariable region.

Hypervariable region of hepatitis C virus envelope glycoprotein (E2/NS1) in an agammaglobulinemic patient.

In an agammaglobulinemic patient with chronic hepatitis C, a previously identified hypervariable region of the major envelope glycoprotein remained unchanged for 2.5 years. Serum-derived RNA amplified by reverse transcription-polymerase chain reaction was cloned in a bacterial vector, and a minimum of three independent clones were sequenced by dideoxy chain termination reaction. Comparison of consensus sequences from three different time points during the chronic phase of infection showed absolute homology at both amino acid and nucleotide levels. This finding provides support for the role of antibody selection in generating genetic variation and viral persistence; also, it is consistent with the hypothesis that an epitope within this region is the site of virus neutralization. The observations show that the hepatitis seen in hepatitis C virus infection is not dependent on the humoral immune response.

Cloning and sequencing of RNA of hepatitis delta virus isolated from human serum.

The RNA of hepatitis delta virus (HDV) 1682 nucleotides long, has been cloned from a human serum isolate. Comparison with the three complete published sequences shows that a region of the HDV genome, between positions 620 and 1350, which contains sequences involved in replication and possibly pathogenicity, is highly conserved.

Hepatitis delta virus and the host response: current status and future perspectives.

The demonstration that recombinant vaccinia can control the level of viraemia after HDV superinfection raises the possibility that established HBV carriers can be immunized against HDV. Furthermore the likelihood that this protective effect is dependent on the induction of a CTL response to HDV raises the possibility that the therapeutic effect of interferon operates by the induction of this cellular response. The failure of interferon to exert a direct inhibitory effect on HDV replication needs further investigation as does the nature of the immune responses involved in the protective effect induced by immunization with recombinant vaccinia.