Emergence of the long form of hepatitis delta virus antigen in transfected cells after intrahepatic transfection and during natural infection.
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Antigens, Viral↗
Biomedical subjects
J Monjardino
Publications and source records attributed to J Monjardino.
Characterization of stable hepatitis delta expressing hepatoma cell lines: effect of HDAg on cell growth.
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Antigens, Viral↗
Immunisation of woodchucks with hepatitis delta antigen expressed by recombinant vaccinia and baculoviruses, controls HDV superinfection.
We report the investigation of the role of humoral and cell mediated immune responses on hepatitis delta virus (HDV) superinfection of woodchucks chronically infected with woodchuck hepatitis virus (WHV). The animals were immunised with baculovirus or vaccinia virus recombinant hepatitis delta antigen (HDAg) but none showed detectable anti-HD titres prior to challenge with HDV. Following infection, both immunised and control animals developed HD-antigenaemia first detected after 2-3 weeks and lasting for up to 8 weeks. In spite of the presence of HDAg, in immunised animals HDV-RNA could only be detected by nested PCR in contrast with the controls, which were positive by dot blot hybridisation. No serum HDAg or HDV-RNA was detected after the acute episode over the six month follow-up period but intrahepatic HDAg was reported in post-mortem biopsies carried out at six months. Our results demonstrate that immunisation of woodchucks with HDAg expressed by vaccinia or baculovirus does not elicit a humoral immune response. The finding of a marked antigenaemia in the absence of serum HDV-RNA indicates a significant reduction in the number of circulating infectious virions possibly due to a cytotoxic T-cell response.
Animals↗
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.
Animals↗
Effect of cyclosporin-A in woodchucks with chronic hepatitis delta virus infection.
Four woodchucks chronically infected with hepatitis delta virus (HDV) were treated with cyclosporin-A (CyA) for 11 weeks. All animals had detectable HDAg in the liver and two of them were also positive for HDAg and HDV-RNA in serum. Reappearance of HDV in serum was noted in one of the two non-viraemic animals and increased viraemia in the two viraemic. HDV-RNA levels became elevated within a week of starting treatment and an inverse relationship between HDV-RNA and WHV-DNA became apparent during the treatment period. With discontinuation of treatment, HDV-RNA levels either returned to pretreatment levels or became negative. The remaining animal showed no return of viraemia during CyA treatment; HDV-RNA remained negative and WHV-DNA levels did not change. Liver biopsies revealed a slight increase in lobular activity during CyA treatment in the animals showing increased viraemia. These data are consistent with the hypothesis that the host immune response exerts a negative control on the level of HDV viraemia and that HDV influences HBV replication independently of the host immune response. In an animal that may have been clearing HDV, immunosuppression did not result in recurrence of viraemia.
Animals↗
Mapping of 5' ends of virion-derived HBV DNA.
Mapping of the 5' ends of virion-derived hepatitis B virus DNA molecules was carried out after cloning and sequencing linearized genomes made fully double stranded. Of five clones obtained, three of four minus strand termini mapped to the G in position 1826 and one to the T in position 1827. Plus strand 5' ends were more heterogeneous with evidence from one clone for the presence of an RNA-translocated primer from the 5' end of pregenomic RNA as well as two other termini, mapped to positions 1599 and 1601, the second 3' nucleotide of DR2 and the first nucleotide 3' to DR2, respectively. These molecules were also shown to be functional templates for expression of HBcAg after tranfection or microinjection of Huh-7 human hepatoma cells.
Base Sequence↗
Serological response and detection of viraemia in acute hepatitis C virus infection.
The serological response during acute hepatitis C virus (HCV) infection was examined by enzyme-linked immunosorbent assay (ELISA) in sequential serum samples from 13 haemophiliacs following their first exposure to factor VIII concentrates contaminated with HCV. The commercially available C100-3 peptide and a new 22 kDa recombinant protein (p22) encoded by the nucleocapsid region of the viral genome were used for antibody detection, whilst a nested polymerase chain reaction (PCR) method was used for the detection of viraemia. In addition, eight sporadic cases of acute HCV infection were studied. The results in haemophiliacs demonstrated that seroconversion to the C100-3 antigen occurred in only one-third of the patients within 12 weeks of disease onset, but all of the patients had a diagnostic serological response to p22 during this phase of the disease. The new test was positive in all the sporadic cases at a time when the commercially available test was negative. Although PCR offers a sensitive method for the detection of recent HCV infection, the complex methodology makes it unsuitable for diagnostic laboratories. The new ELISA test with p22 may therefore have a useful diagnostic role in acute disease.
Adult↗
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.
Base Sequence↗
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.
Amino Acid Sequence↗
Co-expression of markers for hepatitis delta and hepatitis B viruses in human liver.
Delta hepatitis (HDV) infection can only occur in the presence of hepatitis B (HBV) infection, as HDV requires a coat of HBV surface antigen (HBsAg) for assembly of complete virus. A number of studies have examined the variation of HBV markers in serum and liver during establishment of HDV infection, but none has systematically examined the relationship between the two viruses in individual hepatocytes. Liver biopsies from five patients with HDV/HBV infection were stained for HBsAg, HBV core antigen (HBcAg) and hepatitis D (delta) antigen (HDAg). Double immunostaining was performed with a combination of indirect immunoperoxidase and alkaline phosphatase/antialkaline phosphatase techniques. HDV and HBV antigens were expressed in all five liver biopsies. Co-localization of HBsAg was seen in up to 39% of HDAg positive cells, and HBcAg in up to 8% of HDAg positive cells. HBcAg was detectable in approximately 9% of HBsAg positive cells, and HBsAg in approximately 12% of HBcAg positive cells. HDV can replicate without HBV but ultimately requires HBV to produce complete virus and subsequently infect other cells. In this study the majority of HDV positive cells did not appear to contain HBV markers. This might suggest delta virus replication without assembly, or possibly sequential production/assembly of the virus.
Adult↗
Serum HBV DNA detected by PCR in dot blot negative HBV chronic carriers with active liver disease.
A group of forty-nine HBV chronic carriers with histologically confirmed active liver disease and undetected serum HBV DNA by dot-blot hybridisation were re-investigated using the polymerase chain reaction (PCR) for amplification of serum DNA. The group comprised 16 persistently serum HBeAg-negative and thirty-three anti-HBe-positive patients. The use of PCR followed by Southern blot analysis has increased the sensitivity of HBV DNA detection to about 10-50 virions per ml of serum. Our results showed 14/16 (87.5%) of the HBeAg-positive group and 27/33 (81.8%) of the anti-HBe group to be positive for HBV DNA using PCR. Of the nine cases where HBV DNA was undetected four were positive for markers of hepatitis delta virus (HDV) infection. Demonstration of low level HBV replication associated with active liver disease in chronic HBV carriers where it was previously undetected meets a basic requirement for the proposed role of cytotoxic T lymphocyte-mediated immunopathogenesis in chronic hepatitis B and suggests a combined antiviral and immunotherapeutic approach to achieve eradication of the infection.
Base Sequence↗
An in situ hybridization, molecular biological and immunohistochemical study of hepatitis delta virus in woodchucks.
The presence of hepatitis delta virus genomic RNA and hepatitis delta antigen was investigated in woodchuck liver and extrahepatic tissues by in situ hybridization using synthetic radiolabeled probes, Northern-blot analysis and immunohistochemical staining for hepatitis delta antigen. Hepatitis D virus RNA and hepatitis delta antigen were detected in the nuclei of infected hepatocytes but in none of the other tissues examined. Northern-blot analysis of total cell RNA confirmed these findings and revealed a series of hepatitis D virus transcripts, including full-length genomic RNA and dimers and trimers of hepatitis D virus RNA that may represent replicative intermediates. Use of single-stranded probes showed genome-size monomers and dimers to be both of genomic and antigenomic polarity, although dimers were found to be predominantly antigenomic. These findings document the strict hepatotropism of hepatitis D virus and support the rolling-circle model of genome replication for this unique, defective RNA virus.
Animals↗
Genetically engineered molecular probes in the study of the natural history of chronic delta hepatitis.
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Antigens, Viral↗
Prevention and treatment of hepatitis delta virus infection.
The prevention of HDV infection can readily be achieved by immunisation with the envelope proteins (HBsAg/pre-S) of the helper virus (HBV) on which HDV is dependent. In patients with established chronic HBV infection, superinfection with HDV may theoretically be controlled, but not prevented, by immunisation to the internal components of HDV which may be the target of a cytotoxic T-cell response to HDV infected hepatocytes. Such a response, by analogy to the effects of immunisation to HBcAg, may result in rapid lysis of infected hepatocytes thereby limiting the spread of HDV through the liver. In our preliminary experiments using recombinant HDAg (amino-acids 13-76) we have shown that a humoral immune response to HDV prior to HDV superinfection, does not control HDV infection but may facilitate it. We do not know whether this immunisation protocol successfully induced a cytotoxic T-cell response or whether the region of HDAg included is recognised by cytotoxic T-cells and involved in the immune destruction of HDV infected cells. Further studies of the mechanisms of lysis of HDV infected hepatocytes to determine the role, if any, of cytotoxic T-cells or humoral lytic mechanisms, is needed. Although short courses of alpha interferon inhibit HDV replication, relapses are common and amelioration of the inflammatory liver disease is not always seen. Longer periods of therapy may be effective if they can be continued until clearance of the helper virus (HBV), as well as HDV, has occurred. In three patients treated for greater than one year, clearance of HBsAg has occurred. If these patients remain in this state after stopping therapy, further studies of long term alpha interferon therapy may be fruitful.
Animals↗
Immunization of woodchucks with recombinant hepatitis delta antigen does not protect against hepatitis delta virus infection.
To assess the role of immunization against hepatitis delta antigen in the prevention of hepatitis delta virus infection, woodchuck carriers of woodchuck hepatitis virus were immunized with a 64 amino acid portion of hepatitis delta antigen from its N-terminal region. The protein was expressed in Escherichia coli and contained a major immunogenic epitope. A significant anti-hepatitis delta response was observed that did not, however, protect the animals from hepatitis delta virus superinfection. Unexpectedly, the period of detectable viremia was longer in the immunized than in the control animals. We conclude that immunization with this recombinant hepatitis delta antigen does not afford protection against subsequent hepatitis delta virus exposure.
Animals↗
Cloning and expression of an immunodominant region of the hepatitis delta antigen.
A cDNA clone prepared from hepatitis delta virus (HDV) RNA extracted from human serum was subcloned in the bacterial expression vector pPL31 to produce a fusion protein consisting of the first 98 amino acids of MS2 polymerase and of 64 amino acids from near the N-terminal region of hepatitis delta antigen (HDAg). The fusion protein was shown to be related to HDAg by a commercial sandwich immunoassay (Abbott) and immunoblotting with human anti-HDAg serum. Antiserum against the fusion protein was raised in rabbits and used to identify HDAg extracted from the serum and liver of an HDV-infected woodchuck and chimpanzee and from the serum of an HDV-infected human, by immunoblotting and immunohistology. A single, major polypeptide of 24K was detected in both serum and liver extracts, with a minor polypeptide of 26K sometimes present. Liver extracts also contained lower Mr polypeptides thought to be degradation products, the major species being 22.5K. The same pattern of staining was obtained with human anti-HDAg serum. Absorption experiments with the expressed protein and cross-competition experiments with the rabbit antiserum suggest that a major immunodominant region of HDAg is present near the N-terminal end of the antigen, between positions 1561 and 1368 on the genome. Both the expressed protein and rabbit antiserum were shown to be good diagnostic reagents.
Amino Acid Sequence↗
Treatment of chronic hepatitis delta virus (HDV) infection with human lymphoblastoid alpha interferon.
Ten patients with evidence of continuing HDV replication were treated with lymphoblastoid alpha-interferon and eight more were followed as a non-randomized control group. Four out of eight patients who completed one year of follow-up had cleared HDV-RNA from the serum whilst none of the control group did so. In these four responding cases there was a transient increase in transaminase levels during treatment and in two, this was followed by improvement. One patient also cleared HBV and seroconverted to anti-HBs (antibody to hepatitis B surface antigen--HBsAg). In one patient with sustained loss of HDV, recurrence of HDV infection was detected 18 months after completion of treatment. These data suggest that alpha-interferon can inhibit HDV replication in the short term but relapse after one to two years may occur. Inhibition of HDV-RNA is associated with improvement in the inflammatory liver disease and now larger studies are required to determine whether it influences survival.
Adult↗
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.
Base Sequence↗