Fibrosing cholestatic hepatitis in a transplant recipient with hepatitis B virus precore mutant

A patient with hepatitis B virus (HBV) precore mutant (seropositive for hepatitis B surface antigen [HBsAg], anti-hepatitis B e antigen [HBeAg], and HBV DNA) who underwent orthotopic liver transplantation for end-stage liver disease is described. Sequencing of the HBV precore region of the pretransplant serum sample confirmed the presence of the precore stop-codon mutant (G-->A mutation in codon 1896) only. The patient received HBV immunoglobulin prophylaxis for 6 months but HBV recurred thereafter with a mild hepatitic flare, and he remained seropositive for HBsAg, anti-HBe, and HBV DNA. The initial hepatitic illness resolved in 3 months. The patient remained well for another 16 months before presenting with fibrosing cholestatic hepatitis (FCH). During his entire initial hepatitic flare, quiescent period, and final FCH phase, he remained seropositive for HBsAg, anti-HBe, and HBV DNA. Moreover, sequencing of the serum HBV DNA in final FCH phase showed the presence of the identical HBV precore mutant. Immunohistochemical staining showed extensive expression of HBsAg/pre-S1, pre-S2, and hepatitis B core antigen, but HBeAg was scarcely detectable. This case illustrates that (1) recurrence of HBV precore mutant infection can occur in liver; (2) it can give rise to FCH; and (3) hepatic accumulation of HBeAg is not essential for the development of FCH.     

The intrahepatic expression of the hepatitis B virus in chronic delta hepatitis

In order to evaluate the interference of hepatitis delta virus (HDV) in hepatitis B viral particle (HBsAg, HBcAg) expression in the liver of chronic HDV patients, 39 and 81 liver biopsies of HBsAg carriers seropositive for anti-HDV and anti-HDV negative controls, respectively, were studied. HBcAg was positive in 16.7% of the HBeAg-positive patients with HDAg in the liver and in 91,4% of controls. In contrast, in HBeAg- and anti-HDV negative patients the intrahepatic expression of HBcAg was detected in 32.6%. In anti-HDV negative patients the HBcAg liver expression correlated significantly with the HBeAg in serum (p < 0.00001). The distribution of HBcAg was exclusively cytoplasmatic in 30% of HDV-infected patients but mixed nuclear and cytoplasmic in 38.3% of the controls. The nuclear expression of HBcAg was decreased in chronic HDV infection. HBsAg was positive in 70.3% of patients who were anti-HDV positive and in 82.3% of controls. The membranous expression of HBsAg was detected less frequently in HDV-infected patients (p < 0.05) than in controls, while associated with HBeAg in serum of HBV carriers without HDV superinfection (p < 0.00001). The prevalence and the HBsAg cytoplasmic expression was not different for the chronic HDV infection or controls. Our results show: 1) decreased intrahepatic expression of HBcAg and membranous HBsAg in HBV carriers superinfected with HDV, suggesting decreased HBV replication in the liver of these patients. 2) the changing of HBcAg and HBsAg expression in the liver of HDV-infected patients, suggest not so much a decrease but rather a modulation in HBV replication.     

Response of patients with dual hepatitis B virus and C virus infection to interferon therapy

Patients with dual infection with hepatitis B virus (HBV) and delta virus (HDV) responded poorly to interferon (IFN) therapy. Little is known about the effect of IFN therapy in patients with HBV and hepatitis C virus (HCV) dual infection. The patients in two randomized controlled trials with chronic HBV infection were retrospectively assayed for HCV markers. The HBV responses to IFN therapy in patients with and without HCV markers were compared. An open trial was conducted in 4 patients who had lost their serum HBV surface antigen (HBsAg) but had continuing HCV viremia and hepatitis. Of the 15 patients seropositive for HCV marker(s), only 1 (6.7%) responded with seroclearance of HBV DNA and HBV e antigen, as compared with 46 (28%) of 164 HCV-negative patients (p = 0.058). Icteric hepatitis developed in 1 patient on emergence of serum HCV RNA in association with seroclearance of HBV DNA. In contrast, good response was demonstrated in 3 of the 4 patients who had lost serum HBsAg before therapy. The results suggest that IFN therapy is not only of limited value in patients with dual infection with HBV and HCV but also has a potential risk of severe hepatitis if the clearance of one virus removes its suppressive effect on and facilitates the emergence of the other. However, patients with continuing HCV hepatitis after termination of the chronic HBsAg carrier state responded well to IFN therapy.     

Effects of mutations within and adjacent to the terminal repeats of hepatitis B virus pregenomic RNA on viral DNA synthesis

The viral polymerase and several cis-acting sequences are essential for hepadnaviral DNA replication, but additional host factors are likely to be involved in this process. We previously identified two sequences, UBS and DBS (upstream and downstream binding sites), present in multiple copies in and adjacent to the pregenomic RNA (pgRNA) terminal redundancy, that were specifically recognized by a 65-kDa host factor, p65. The possible roles of these two sequences in hepatitis B virus (HBV) replication were investigated in the context of the intact viral genome. UBS is contained within the terminal redundancy of pgRNA, and the 5' copy of this sequence is essential for viral replication. Mutations within the central core of UBS ablate p65 binding and selectively block synthesis of plus-strand DNA, without affecting RNA packaging or minus-strand synthesis. The DBS sequence, which is located downstream of the pgRNA polyadenylation site, overlaps the core (C) protein coding region. All mutations introduced into this site severely affected viral replication. However, these effects were shown to result from dominant negative effects of mutant core polypeptides rather than from cis-acting effects on RNA recognition. Thus, the 5' UBS but not DBS sites play important cis-acting roles in HBV DNA replication; however, the involvement of p65 in these roles remains a matter for investigation.     

Immunohistochemical study of hepatitis B and D virus antigens (HBsAg, HBcAg, HDAg) in chronic liver diseases

Authors have investigated the hepatitis B and D virus antigens in the liver tissue of 30 HBsAg and/or anti-HD seropositive patients (23 males, 7 females, age: 20-65, mean: 44 years) by immunohistochemical method. The immunohistochemical identification of HBsAg, HBcAg and HDAg in 42 liver sample (36 obtained by percutaneous biopsy, 6 from dissection) of 30 patients was performed with Dako and Sorin Biomedica kits. The detailed virus serologic examinations were carried out with Biomedica and Abbott kits by radioimmunoassay and ELISA methods. Examining 36 liver tissue samples of 27 HBsAg seropositive patients, HBsAg could be demonstrated in 31 cases. Each patient suffering of active HBV and/or HDV replication was HBsAg positive by immunohistochemistry, while the tissue samples of patients in integrational phase of HBV infection were positive in only 9 cases of 14. There was no HBsAg tissue positivity in HBsAg seronegative cases. 7 of 16 tissue samples of 12 patients classified to active HBV replication state were HBcAg positive by immunohistochemistry. HBcAg could be detected in the liver tissue of each HBe seropositive patient, while in only 3 of 8 cases with only IgM anti-HBc seropositivity (indicating low level of HBV replication). Tissue HBcAg positivity, indicating active virus replication, was verified in 2 of 11 patients classified to HBV integration state by serology. Authors detected HDAg tissue positivity only in cases with serologically active HDV replication (IgM anti-HD seropositive) and HDAg could also be identified from liver tissue in each IgM anti-HD seropositive case. No HBsAg, HBcAg and HDAg tissue positivity was observed in HBsAg negative cases. Authors emphasise mainly the importance of immunohistochemical detection of HBcAg and HDAg completing the serologic diagnosis of chronic HBV and HDV infections, helping the verification or exclusion of active virus replication being essential for selecting adequate therapy.     

Functional analysis of HBV genomes from patients with fulminant hepatitis

Two previous case reports suggest that hepatitis B virus (HBV) core promoter variants with a high replication competence contribute to the pathogenesis of fulminant hepatitis B (FHB). We recently found in HBV genomes from patients with FHB an accumulation of mutations within the core promoter region. Therefore, the aim of this study was to investigate the phenotype of these HBV variants. Replication competence and expression of hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) of viral genomes from seven patients with FHB and one patient with fulminant recurrent hepatitis after liver transplantation were analyzed by transfection experiments in human hepatoma cells. Compared with wild-type virus, the HBV variants from the seven patients with FHB produced similar or slightly lower levels of intracellular replicative intermediates and extracellular viral particles. In contrast, the HBV genomes from the patient with fulminant recurrent hepatitis synthesized and secreted significantly more HBV DNA. All genomes tested expressed similar or even higher levels of HBeAg compared with wild-type virus, except for those from four patients with a precore stop codon mutation in the respective dominant viral populations. The level of HBsAg produced by all variant genomes was similar or reduced compared with wild-type virus. These data indicate that in some cases HBV variants with enhanced replication competence and/or a defect in HBeAg expression may contribute to the development of FHB. However, neither phenotype is an essential prerequisite; thus, an additional role of other viral or host factors in the pathogenesis of FHB is suggested.     

Chronic hepatic porphyria and hepatitis B and C virus infections

BACKGROUND: It is known that in patients with porphyria cutanea tarda (PCT) there is an increased prevalence of the hepatitis B virus (HBV) and the hepatitis C virus (HCV). The incidence of anti-HCV in PCT in our country is 21.7% in estimations by the second generation method, however, the incidence of HBV in PCT was not assessed so far. METHODS AND RESULTS: In 60 patients with PCT antigens and antibodies against HBV and HCV were assessed (by the anti-HCV third generation ELISA method) and in subjects with signs of HBV or HCV. HBV DNA and HCV RNA were assessed by the method of the polymerase chain reaction. PCT without detectable HBV or HCV infection was found in 45 subjects (68%). HBV infection only was confirmed in seven subjects (10.6%), however none of the patients had positive HBsAg in serum. All had only antibodies against HBV. HCV infection only was detected in seven patients (10.6%) and HBV and HCV co-infection also in seven patients (10.6%). In the group of patients with HBV and HCV co-infection there was not a single HBsAg positive subject. The mean ALT serum activity was significantly higher as compared with subjects with HBV or HCV infection only (p < 0.05) and the histological finding on liver biopsy was more serious. CONCLUSION: HBV (21%) and HCV (21%) infection participates significantly in the clinical picture of PCT. A special subgroup is formed by patients with PCT and HBV and HCV co-infection who have as a rule a higher ALT activity and more severe histological changes in the liver. The incidence of HBV and HCV infection in PCT in the Czech Republic is double as compared with Germany or Great Britain.     

Analysis of hepatitis B virus populations in an interferon-alpha-treated patient reveals predominant mutations in the C-gene and changing e-antigenicity

It is largely unknown whether hepatitis B virus (HBV) sequence variation during chronic infection hampers HBV immune recognition or the antiviral effect of cytokines on HBV production. Here we have analyzed which region of the HBV genome changes most drastically during an interferon-alpha (IFNalpha)-stimulated immune response. In addition, we have investigated whether the mutations affect viral replication, gene expression, and immune recognition of the mutant viral proteins. The study was performed with full-length HBV genomes taken longitudinally from a patient who transiently cleared HBV and seroconverted to anti-HBe during a long-term IFNalpha treatment. We found a replacement of the predominant virus population during IFNalpha therapy The virus populations differed mainly by a cluster of nucleotide changes in the C-gene and a pre-S2 deletion. Most of the newly emerging mutations localized within core/HBe B-cell epitopes, changed HBe antigenicity toward mono- and polyclonal antibodies, and also influenced the reactivity of the anti-HBc/e antibodies of the patient. All genomes tested expressed less HBeAg than wild-type HBV, while replication and IFNalpha susceptibility were similar. These data indicate that IFNalpha therapy can lead to the emergence of HBV variants with mutations mainly affecting recognition of the core/HBe proteins by antibodies. Taken together, the type of core/HBe-specific B-cell immune response, the sequence of the corresponding epitopes, and the HBe expression level appear to contribute to the decision on viral clearance or persistence.     

Variations of hepatitis B virus core gene sequence in Western patients with chronic hepatitis B virus infection

Heterogeneity of the hepatitis B virus (HBV) core gene has been reported to be associated with the presence of active liver disease in Japanese patients with chronic HBV infection. This study evaluated the significance of HBV core gene heterogeneity in Western patients with chronic HBV infection. The hepatitis B virus precore/core gene from 45 patients (inactive:active liver disease ratio 16:29) was amplified from serum by polymerase chain reaction (PCR). Gel electrophoresis was employed to detect large deletions. The PCR amplicons from 13 patients (all HBV serotype adw but with a different spectrum of liver disease) were cloned and sequenced. Hepatitis B surface antigen (HBsAg) serotypes were tested by enzyme immunoassay (EIA) and hepatic expression of HBV antigens was assessed by immunohistochemistry. The HBV core gene was amplified from the serum of all 45 patients. Three patients had mixed infection with both precore mutant and wild-type HBV and all three had active liver disease. No patient had a large deletion of the HBV core gene. Hepatitis B virus core gene sequence variations were more common in the midcore region and there was no difference in the number of silent and missense substitutions between those with inactive and active liver disease. There was no correlation between the nucleotide or encoded amino acid substitutions and the clinical and biochemical parameters, including the subsequent response to interferon-alpha therapy (n = 37) or hepatic HBV antigen expression. Variation of the HBV core gene was not found to be preferentially associated with active liver disease in Western patients with chronic HBV infection. The pattern of hepatitis B core gene variation is in accord with the genomic organization of HBV.     

Nested polymerase chain reaction (PCR) and multiple cloned antibody capture PCR for the examination of serum hepatitis B virus DNA in negative hepatitis B surface antigen patients suffering from liver diseases

In order to find out rapidly the causes of the liver diseases suffered by patients with negative hepatitis B surface antigen (HBsAg), nested polymerase chain reaction (PCR) and multiple cloned antibody capture PCR techniques were established to examine serum hepatitis B virus (HBV) DNA. By using both techniques along with the examination of hepatitis C virus (HCV) infection, the causes of chronic liver diseases with negative HBsAg were studied. It is found that nested-PCR can increase the sensitivity of single PCR more than 1,000 fold and multiple cloned antibody capture-PCR can detect concentration of HBV DNA as low as 0.1-0.01 pg/L. HBV DNA positive patients were found in 45.5%, 30.8%, 13.3% and 100% respectively of the patients suffering from liver cirhosis with negative HBsAg (group A, 22 cases), chronic hepatitis with negative HBsAg (group B, 13 cases), normal subjects with negative HBsAg and positive hepatitis B core antibody (HBcAb, group C, 30 cases) and liver cirhosis with positive HBsAg and negative HBeAg (group D, 12 cases). HBV DNA can be also found in the serum of HBsAb positive patients and subjects supposed to be healthy, 81.8% and 53.8% of the patients were infected with HBV and/or HCV in group A and group B respectively. All these results suggest that nested-PCR and multiple cloned antibody capture-PCR are rapid and highly sensitive methods for detection of serum HBV DNA. HBV infection is an important cause of chronic liver diseases in patients with negative HBsAg. The causes of most of the HBsAg-negative chronic liver diseases are related with infection of viruses. The clinical significance of serum HBsAb in naturally infected patients should be reconsidered.     

Changes in ceftazidime concentration in the CSF following overdose in acute kidney failure]

The diagnosis of liver diseases induced by hepatitis B virus (HBV) is supported by the detection of HBV surface antigen (HBsAg) in serum. The present study aimed to investigate the presence of HBV deoxyribonucleic acid (DNA) in patients with liver cirrhosis using a polymerase chain reaction (PCR) based on primers derived from the pre-S1 and pre-core regions. HBsAg was detected in 10 of 48 patients (21%), total anti-hepatitis B core antigen (HBc) antibodies in 54%, anti-hepatitis B e antigen (HBeAg) in 14.6%, anti-HBc immunoglobulin M in 8%, and anti-HBs in 26%; none had detectable HBeAg. HBV DNA was detected in 73% of the cirrhotic patients. All cirrhotic patients with HBsAg also had HBV DNA; HBV DNA was detected in 64.5% of those without HBsAg. We conclude that the clearance of HBsAg does not necessarily indicate termination of viraemia in patients with liver cirrhosis and the detection of HBV DNA using a PCR based on primers from the pre-S1 and pre-core regions should be included in the diagnosis of HBV infection.     

Lamivudine treatment for chronic replicative hepatitis B virus infection after allogeneic bone marrow transplantation

The risk of severe hepatic damage in patients with chronic hepatitis B virus (HBV) infection is well known; more effective treatments for this infection are needed. Lamivudine is being studied in immunocompetent and immunosuppressed HBV infected patients. We report a patient suffering from chronic replicative HBV infection after allogeneic BMT, who responded to lamivudine therapy. A 24-year-old woman with CML received an allogeneic BMT from her HLA-identical sister in June 1992. Before transplant, her HBV status demonstrated viral contact without active infection (HBsAb+, HBcAb+ IgG, HBeAb+). Four months after BMT mild chronic liver GVHD appeared, requiring immunosuppressive treatment. Antibodies to HBV completely disappeared post-transplant. Acute icteric hepatitis occurred 2 years later, with HBsAg+, high level of HBV-DNA, HBeAg+ and HBcAb IgM+. Lamivudine 100 mg/day rapidly reduced transaminase levels and effected HBV-DNA disappearance within 2 months. The treatment was well tolerated; no hematological side-effects occurred. This preliminary observation warrants further investigation of lamivudine treatment in bone marrow transplanted patients with active HBV infection.     

Hepatitis B virus genomic heterogeneity: variation between quasispecies may confound molecular epidemiological analyses of transmission incidents

Nucleotide sequence variability studies were conducted on a 263-base pair fragment of the core-coding genomic region of hepatitis B virus (HBV), amplified by the polymerase chain reaction (PCR) from three surgeons with varying circulating levels of HBV, all of whom were thought to have transmitted HBV to their patients post-surgically. DNA sequencing was applied to amplicons obtained directly from serum and those cloned into plasmid vectors, and from single HBV molecules in serum separated by a limiting dilution procedure. In one surgeon, who had a titre of approximately 3 x 10(5) genome equivalents ml-1, the direct sequence was identical to none of 29 other sequences and differed by one base substitution from the sequence amplified from the single patient he infected. In another surgeon, who had a titre of approximately 2 x 10(6) genome equivalents ml-1, the direct sequence was identical to 17 of 36 (47%) sequences; however, the sequence common to all three infected patients was identical to a unique sequence in the surgeon that differed by three base substitutions from the direct sequence. By contrast, the direct sequence in the third surgeon, who had a titre of approximately 4 x 10(7) genome equivalents ml-1, was identical to 25 of 38 (66%) sequences, and to the sequence common to all 11 infected patients. Assessment of HBV DNA sequences directly amplified from clinical specimens may not be appropriate to studies of transmission in which the source of infection harbours a relatively dilute, heterogeneous mix of viral variants.     

Biodistribution, stability, and antiviral efficacy of liposome-entrapped phosphorothioate antisense oligodeoxynucleotides in ducks for the treatment of chronic duck hepatitis B virus infection

This study investigated the feasibility of using liposomes to increase the hepatic delivery and antiviral efficacy of phosphorothioate antisense oligodeoxynucleotides (PS-ODN) for the in vivo treatment of hepatitis B virus (HBV) infection. Ducks infected with duck hepatitis B virus (DHBV) were used as the model. We studied the stability of an antisense PS-ODN in duck plasma, its integrity during the process of liposome entrapment, its in vivo biodistribution, plasma clearance, and excretion. In addition, the intrahepatic distribution of a labeled free and liposome-entrapped ODN was also investigated. The results of our studies show that: 1) phosphorothioate ODN remain stable during the process of liposome entrapment; 2) are stable in duck plasma for many hours; 3) are rapidly cleared from the plasma when injected intravenously; 4) intravenous injection of antisense ODNs entrapped within liposomes enhances delivery of the ODN to the liver; and 5) inhibit DHBV replication. Serum DHBV DNA levels fell rapidly, with a corresponding decrease in intrahepatic viral replicative intermediates at the end of the 5-day study period. Although inhibition of viral replication and a fall in the target protein was observed, a marked inhibition of viral replication was also observed with high doses of a random-sequence ODN. Thus, it is not certain that inhibition of viral replication was entirely through an antisense mechanism. Therefore, liposomes may be effective vehicles to improve the delivery of antisense oligonucleotides to the liver for the therapy of hepatotropic viruses.     

Detection and sequence analysis of hepatitis B virus integration in peripheral blood mononuclear cells

A PCR-based technique was used to detect hepatitis B virus (HBV) integration in peripheral blood mononuclear cells from patients with chronic hepatitis B. Integrated HBV DNA sequences, with virus-cell junctions located in the cohesive region between direct repeat 1 (DR1) and DR2, were found in 2 of 10 studied patients.