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.

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.

Localization of hepatitis delta virus RNA in the nucleus of human cells.

Hepatitis delta virus (HDV) is a human pathogen that can greatly increase the severity of liver damage caused by an hepatitis B infection. HDV contains a circular, single-stranded RNA genome that encodes a unique protein, the delta antigen. Two forms of the delta antigen, deltaAg-S and deltaAg-L, are derived from a single open reading frame by RNA editing. Here we analyze the subcellular distribution of HDV RNA and its spatial relationship to known intranuclear structures. The human hepatoma cell line Huh7 was stably transfected with wild-type HDV cDNA and the viral RNAs were localized by in situ hybridization and fluorescence confocal microscopy. HDV RNA is detected throughout the nucleoplasm, with additional concentration in focal structures closely associated with nuclear speckles or clusters of interchromatin granules. Both the smaller form of the delta antigen (deltaAg-S), which is required for HDV genomic replication, and the larger form of the delta antigen (deltaAg-L), which represses replication, co-localize with delta RNA throughout the nucleoplasm and in the foci. However, the foci do not incorporate bromo-UTP and do not concentrate either RNA polymerase II or cleavage and polyadenylation factors required for viral RNA synthesis and 3' end processing, respectively. Thus, it is unlikely that the delta foci represent major sites of viral transcription or replication. In conclusion, the data show that viral RNA-protein complexes accumulate in structures closely associated with interchromatin granules, a subnuclear domain highly enriched in small nuclear ribonucleoproteins, poly(A+) RNA, and RNA splicing protein factors. This implies a specific compartmentalization of ribonucleoproteins in the nucleus.

Comparison of the rate of sequence variation in the hypervariable region of E2/NS1 region of hepatitis C virus in normal and hypogammaglobulinemic patients.

The hypervariable region (HVR) of the E2/NS1 region of hepatitis C virus (HCV) varies greatly between viral isolates with high rates of genomic change reported during the course of chronic infection. The HVR is thought to encode a structurally unconstrained envelope protein containing several linear B cell epitopes recognized by neutralizing antibody. It has been postulated that amino acid changes in the HVR could result from humoral immune pressure leading to the selection of escape mutants. The aim of this study was to compare the rates of nucleotide and amino acid variation in the HVR of control patients to patients with common variable immunodeficiency (CVID) where the effect of the humoral immune system is reduced. Five controls and four patients with CVID were studied. Serum samples were taken over periods of between 1 and 6 years. HCV was detected by polymerase chain reaction (PCR) with primers derived from conserved flanking regions of the HVR. PCR products were cloned into a plasmid vector and recombinant clones identified by restriction enzyme digestion. Purified DNA from at least three individual clones from each time point was sequenced by the dideoxynucleotide chain-termination method. Consensus sequences were extracted from the three clones, and the DNA and deduced protein sequences were compared. Control patients had a mean rate of nucleotide change of 6.954 nucleotide substitutions per year, compared with patients with CVID with a rate of 0.415 nucleotide substitutions per year (P < .02). The corresponding rates for amino acid variation were 3.868 amino acid substitutions per year for the control patients compared with 0.185 amino acid substitutions per year for the patients with CVID. These findings suggest that in the absence of humoral immune selective pressure, the frequency of occurrence of genetic variation in the major viral species is reduced. The mutations occur, but in the absence of immune selection remain as minor species. The evolution of viral mutants capable of evading the host's immune system may contribute to the ability of HCV to establish chronic infection.

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.

A comparison of the Invos 3100 and the Critikon 2020 near-infrared spectrophotometers as monitors of cerebral oxygenation.

Assessment of cerebral oxygenation using near-infrared spectroscopy in intensive care is increasing. We compared the ability of the Invos 3100 and the Critikon 2020 monitors to produce stable and consistent readings of regional cerebral oxygen saturation in resting volunteers. Failure to obtain any stable reading with the Critikon occurred in eight out of 18 subjects (44.4%) and with the Invos in three out of 15 subjects (20%). The Critikon showed a significantly higher failure rate in male subjects (p = 0.0011). Differences in recorded values of cerebral oxygen saturation (Critikon-Invos) ranged from -4.7% to 12.6% and were significantly related to the average saturation level (p < 0.0001). The within-monitor variability was significantly higher for the Invos (p = 0.0124). Neither monitor is able to give stable and consistent readings over time, particularly in male subjects. The unacceptably high failure rate of the recently introduced Critikon 2020 will limit or prevent its clinical use.

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.

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.

Base changes at positions 1014 and 578 of delta virus RNA in Greek isolates maintain base pair in rod conformation with efficient RNA editing.

Analysis of delta hepatitis virus (HDV) genomic RNA, derived from Greek patients from an area where HDV infection is associated with low pathogenicity, is described. In all isolates sequenced, which included 18/18 HDV cDNA clones derived from 6 different patients, irrespective of pathogenicity, a base change (T-->C) was found in position 1014. No significant differences in editing efficiency were found between isolates from inactive and active forms of the disease, although L-antigen was present in low to undetectable levels in the serum of 5/6 patients. An additional mutation was identified at position 578 (A-->G), which reestablishes the canonical base pair G/C with the mutated 1014 when the genome adopts the "rod-like" conformation. This finding supports the presence of this genome conformation in vivo and the requirement for the Watson-Crick base pair 1014/578. A mutation, found at amino acid position 170 (serine-->asparagine), appears to segregate with patients with inactive disease.

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.

Hepatitis C virus type I(1a) in northern China.

Twenty-four patients with hepatitis C virus (HCV) antibody from the Chinese North Western province of Jilin were further analysed by the immunoblot assay-2 (RIBA-2), reverse transcription-polymerase chain reaction (RT-PCR) for serum HCV RNA detection, and direct sequence-genotyping. Good concordance was found between the original second generation HCV antibody ELISA, RIBA-2, and serum HCV RNA. The occurrence of genotype I (1a), a genotype not previously reported in China, is described in 5(20.8%) of 24 cases, in association with genotypes II(1b) and III(2a) which were found in 16(66.7%) and 3(12.5%) of 24 cases, respectively. Imported blood products were unlikely to be the source of infection with genotype I (1a) but could not be definitively ruled out.

Hepatitis delta virus replication in vitro is not affected by interferon-alpha or -gamma despite intact cellular responses to interferon and dsRNA.

The hepatitis delta virus (HDV) genome consists of circular ssRNA which has extensive intramolecular complementarity and can form a dsRNA rod-like structure. If such RNA species were to exist in an unmasked form in cells, they would be expected to induce interferon (IFN) expression and activate two IFN-inducible dsRNA-dependent enzymes with anti-viral activity, namely the dsRNA-dependent protein kinase (PKR) and 2',5' oligoadenylate (2',5' A) synthetase. Since the virus replicates to high copy number for prolonged periods in infected cells it is apparently able to evade these antiviral mechanisms. The RNA genome may be masked and fail to induce or activate the antiviral response, or the virus may inhibit such a response. Treatment of a hepatoma cell line, Huh7, and a fibrosarcoma cell line, HT1080, stably transfected with a trimeric HDV cDNA construct, with IFN-alpha or IFN-gamma for up to seven days failed to influence the level of expression of genomic or antigenomic HDV RNA, or delta antigen (Ag). This is consistent with either failure of activation or inhibition of the IFN response. However the induction of several IFN-responsive genes, including PKR, 2',5' A synthetase and class I MHC is normal and cotransfection of a construct expressing delta Ag did not affect expression from an IFN-inducible chloramphenicol acetyltransferase construct. In addition, the activation of PKR is not inhibited in HDV-expressing cells and antiviral assays suggest that the ability of these cells to mount an antiviral response to at least two cytopathic viruses is unaffected. IFN-beta is inducible normally by dsRNA in cells transfected with the delta cDNA trimer. We conclude that HDV replication is not inhibited by IFN-alpha or IFN-gamma, even though the responses of cells expressing HDV RNA and antigen to IFN and dsRNA are intact.

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.

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.

Partial control of hepatitis delta virus superinfection by immunisation of woodchucks (Marmota monax) with hepatitis delta antigen expressed by a recombinant vaccinia or baculovirus.

We have successfully limited the level of hepatitis delta viraemia occurring after superinfection of hepadna-virus infected woodchucks by prior immunisation with the short form of the hepatitis delta virus antigen expressed by a recombinant baculovirus or vaccinia virus. This phenomenon occurred in the absence of detectable circulating antibody to hepatitis delta virus antigen and in the absence of evidence of priming of the humoral immune response and may reflect the induction of a cytotoxic T-cell response. The latter would control viraemia by rapid lysis of delta antigen expressing hepatocytes. It is suggested that the T-cell epitopes involved may be located on the carboxyl end of the delta protein (amino acids 77-195).

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.