Use of pharmacological agents in elucidating the mechanism of insulin action

Hepatitis B virus (HBV) mutants have recently been identified in patients with acute or fulminant as well as chronic infections. Naturally occurring mutations have been identified in all viral genes and regulatory elements. Mutations in the gene coding for the hepatitis B surface antigen (HBsAg) may result in infection or viral persistence despite the presence of antibodies against HBsAg (anti-HBs) ("vaccine escape" or "immune escape"). Mutations in the gene encoding the pre-core/core protein (pre-core stop codon mutant) result in a loss of hepatitis B e antigen (HBeAg) and sero-conversion to antibodies to HBeAg (anti-HBe) with persistence of HBV replication (HBeAg minus mutant). Mutations in the core gene may lead among others to an immune escape due to a T cell receptor antagonism. Mutations in the polymerase gene can be associated with viral persistence or resistance to nucleoside analogues. Thus, HBV mutations may affect the natural course of infection, viral clearance and response to antiviral therapy. The exact contribution of specific mutations to diagnosis and therapy of HBV infection as well as patient management in clinical practice remain to be established.     

Pre-core mutants of hepatitis B virus in patients receiving immunosuppressive treatment after orthotopic liver transplantation

Orthotopic liver transplantation (OLT) is a possible treatment for acute or chronic liver failure due to hepatitis B virus (HBV) infection, but reinfection of the graft can be a serious complication. The aim of this study was to monitor HBV markers, to analyse pre-core-/core-mutations as well as to identify the viral population causing reinfection after OLT, and to investigate the emergence or disappearance of these mutants in patients receiving immunosuppressive treatment. Fifty-four pre-and posttransplant serum samples of 17 patients were analysed. All patients underwent OLT for HBV-related liver disease and had HBV-DNA before and after OLT. Total DNA was extracted from all sera and a 240 bp fragment comprising the pre-core region of HBV was amplified by polymerase chain reaction (PCR). Pre-core mutants of HBV were determined by direct sequencing of these PCR products and by sequencing of PCR clones. Eight of 17 patients were infected with pre-core wildtype HBV before OLT (group A). Seven of eight patients of group A were reinfected by pre-core wildtype HBV after OLT. In one of eight patients in addition to wildtype HBV a mutant strain (nt. 1899 G-->A) was detected. Nine of 17 patients were infected with pre-core mutant HBV before OLT (group B). Six of nine patients of group B were reinfected with the same mutant population; in one, an additional pre-core mutation emerged; two patients lost pre-core mutant HBV (nt. 1896 and 1899 G-->A). In one of the latter two, a pre-core start-codon mutant (nt. 1816 G-->T), not detectable before OLT, emerged, in the other a nt. 1897 G-->A stop-codon mutant persisted. Five patients of each group were followed-up for more than 24 (25 to 58) months on immunosuppressive therapy. In all five patients of group A, pre-core wildtype of HBV persisted during long-term follow up. Two of five patients of group B were infected stably with a stop-codon HBV-mutant nt. 1896. In three patients, the nt. 1896 stop-codon mutant disappeared during immunosuppressive therapy. However, in one of the latter three, an HBV stop-codon mutant nt. 1897 persisted. In conclusion, most patients who underwent OLT for HBV-related disease were reinfected with the same virus population that existed before OLT. In rare cases, new mutants emerged after OLT or preexisting mutants were lost. During long-term follow-up on immunosuppressive therapy, in the majority of patients pre-core mutants disappeared and wildtype HBV became the predominant virus strain.     

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.     

Transmission of a homogenous hepatitis B virus population of A1896-containing strains leading to mild resolving acute hepatitis and seroconversion to hepatitis B e antigen antibodies in an adult

The pre-core variant, A1896, which switches off hepatitis B e antigen (HBeAg) production, is common in hepatitis B e antigen antibody (anti-HBe)-positive chronic hepatitis patients. It has been observed in occasional case reports of acute hepatitis. However, transmission in the absence of HBeAg-producing strains, leading to acute nonfulminant hepatitis and clearance in adults, has not been reported. Here, we show that this event can occur, further confirming that A1896 strains are "wild-type" and can lead to all the same outcomes as G1896 strains. This is in keeping with phylogenetic evidence that A1896 is transmitted independently on a large scale in the population and explains anti-HBe- positive persons who have not had an HBeAg-positive phase documented.     

Relativistic density-dependent Hartree-Fock approach for finite nuclei

Two precore predominant mutations of human hepatitis B virus (HBV) at either nucleotide (nt) 1896 or nt 1899 often occur in combination. At nt 1896, a G to A mutation creates a TAG stop codon at codon 28 of precore protein. At nt 1899, a G to A mutation changes glycine at codon 29 to aspartic acid. To assess the effect of each individual mutation as well as any interaction between these two mutations, HBV derivatives bearing one or both precore predominant mutations have been constructed. HBV e-Ag-negative mutants bearing a TAG stop codon mutation at codon 28 uniformly replicate at least 20-fold better than mutants bearing a TGA stop codon at the same amino acid position, irrespective of the sequence context at nt 1899. A single mutation at nt 1899, changing the wild-type G to a pyrimidine (T or C) is deleterious to viral RNA encapsidation and DNA replication. Our results explain in part why only a purine (G or A) at nt 1899, never a pyrimidine, is observed in natural HBV genomes. The effects caused by these two closely linked mutations on viral replication are not independent of each other. The stringent selection for a highly efficient RNA encapsidation element may play a crucial role in the natural occurrence of these two closely linked precore mutations. The putative 27-amino-acid peptide resulting from the truncation of precore by the nt 1896 mutation has no apparent effect on viral replication. The preferential occurrence of the G to A mutation at nt 1896 and 1899, instead of at other nonpredominant positions, is likely to be a combined consequence of both selection and higher intrinsic mutation frequency at these positions.     

A randomized, controlled trial of a 24-month course of interferon alfa 2b in patients with chronic hepatitis B who had hepatitis B virus DNA without hepatitis B e antigen in serum

Short-term interferon treatment of serum hepatitis B e antigen (HBeAg)-negative carriers with serum hepatitis B virus (HBV) DNA and histological features of chronic hepatitis B has been largely unsuccessful. In a pilot study of long-term treatment, 42 such patients were randomly assigned to 6 million units of interferon alfa 2b (IFN-alpha2b) three times per week for 24 consecutive months (n = 21, 4 with cirrhosis) or to no therapy (n = 21, 3 with cirrhosis). Five patients (24%) discontinued therapy because of treatment-related adverse reactions. Serum levels of alanine transaminase (ALT) became persistently normal and HBV DNA undetectable by dot-blot assay in 8 patients receiving interferon and in 2 untreated controls (38% vs. 10%; P = .03). Hepatitis flare-ups disappeared in 17 patients during therapy compared with 6 controls (81% vs. 29%; P < .001). During a median period of 22 months after interferon was stopped, 2 treated patients (10%) lost serum hepatitis B surface antigen (HBsAg) and seroconverted to antibodies to hepatitis B surface antigen (anti-HBs). Serum ALT remained persistently normal and HBV DNA undetectable by dot-blot assay in 6 initial responders and 1 initial nonresponder, compared with none of the 21 untreated controls (sustained response: 33% vs. 0; P < .001). Comparative analysis of pre- and posttreatment liver biopsies showed that mean Knodell scores dropped in the treated group (10.3 to 5.3; P = .01), but not in the untreated group (9.3 to 9.8; not significant). In conclusion, a 24-month course of treatment with 6 MU IFN-alpha2b was well tolerated by most patients, led to sustained suppression of HBV in one third, and attenuated hepatitis in 81% of patients.     

Treatment of chronic viral hepatitis

Recent advances have been made in the treatment of chronic viral hepatitis, mainly with recombinant interferon (IFN) alpha. However, the present treatment of chronic viral hepatitis is not entirely satisfactory because the efficacy is inconstant and/or incomplete. In chronic hepatitis B IFN-alpha induces a sustained interruption of hepatitis B virus (HBV) replication, with a HBeAg to anti-HBe seroconversion in about 30% of patients. Patients most likely to respond are those with no immunosuppression, HBV infection acquired during adulthood or active liver disease with low HBV replication. Responders usually show a significant decrease in serum HBV DNA levels during the first 2 months of therapy, followed by a significant increase in the level of aminotransferases. New nucleoside analogues might be useful in combination with IFN-alpha in the treatment of those who do not respond to IFN therapy. In chronic hepatitis B-D, the rate of sustained response to IFN-alpha therapy is low. To be effective, IFN-alpha must be used at a high dosage (9-10 mega units) with a long duration (1 year). In chronic hepatitis C, IFN-alpha at a dosage of 3 mega units over 6 months, induces a sustained response in about 20% of patients. A higher dosage of IFN (5-10 mega units) and a longer duration of treatment increases the rate of sustained response but is associated with poor tolerance. Non-responders to a first course of IFN do not respond to a second course of treatment. In patients who respond but relapse after treatment, the rate of sustained response after a second course of IFN needs to be assessed. Ribavirin, which has a significant antiviral effect on hepatitis C virus, might be useful in combination with IFN-alpha. At the dosage (3-6 mega units) usually used, IFN-alpha is relatively well tolerated. In about 10% of the patients therapy is interrupted, mainly because of severe fatigue, thyroid dysfunction or depression.     

Psychological adjustment of children with mild and moderately severe asthma

In chronic hepatitis B virus (HBV) infection seroconversion from hepatitis B e antigen (HBeAg) to hepatitis B e antibody (HBeAb) may be followed either by remission of the disease with low-level viraemia, or by continuing inflammation with high-level viraemia. In both situations the virus may acquire a mutation in the precore sequence which prevents it from encoding HBeAg. We now show that the number of amino acid substitutions in the HBV core is low in viral sequences from patients with HBeAg positive chronic liver disease and HBeAg negative HBeAb positive patients in remission, but the frequency of substitutions is high in HBeAg, negative HBeAb positive patients with active liver disease. Furthermore we show that these substitutions cluster in the promiscuous CD4+ T-helper-cell epitope and in HBV core/e antibody binding determinants, but are not found in regions recognized by major histocompatability complex (MHC) restricted cytotoxic T lymphocytes. Sequential viral sequences from patients before and after HBeAg/HbeAb seroconversion shows that core mutations arise either at the same time or after the precore stop mutation which prevents the virus from encoding HBeAg. These results are consistent with the hypothesis that after clearance of HBeAg, mutations in regions of the virus recognized by CD4+ helper T cells and B cells allow persistence of the HBe negative virus in HBeAb positive patients with viraemia and active hepatitis.     

Self-expanding metal oesophageal endoprostheses: which is best?

The main problem of children with HBeAg positive hepatitis B and associated hepatitis D is progression to liver cirrhosis with decompensation of liver function and need for liver replacement therapy within 15-20 years after infection. To determine whether interferon-alpha (IFN-alpha) therapy has a positive effect on HBV replication and inflammatory activity, we evaluated clinical and serological data of 8 children treated with IFN-alpha and 6 historic control patients without treatment. 4 of the nontreated patients seroconverted from HBeAg to anti-HBe between 7 to 17 years after initial diagnosis and showed decreased inflammatory activity in the liver. In the treatment group, the rate of seroconversion to anti-HBe (3 early, 2 late seroconverters) corresponded well to former trial results obtained in patients exclusively infected by HBV. Serum aminotransferase levels decreased or normalized in seroconverted children. In chronic HBV infection with associated hepatitis D (HDV) infection--compared to the spontaneous course of the disease--IFN-alpha therapy reduced inflammatory activity by earlier seroconversion to anti-HBe in responding patients. Moreover, viral replication and infectivity of hepatitis B was markedly reduced, but no effect on replication of HDV could be documented. Although long-term effects cannot be exactly estimated, at present IFN-alpha remains the only available treatment for HBeAg and anti-HDV positive children and seems to be of benefit for responding patients.     

Prevalence of mutations in core promoter/precore region in Japanese patients with chronic hepatitis B virus infection

We examined the frequency and significance of mutations in the core promoter and precore region in 103 Japanese patients with chronic hepatitis B virus (HBV) infection. HBV DNAs from the patients' sera were amplified by polymerase chain reaction and were directly sequenced. A double mutation (T1762 A1764) in the core promoter was frequently observed in the patients regardless of HBeAg status except for asymptomatic carriers with HBeAg. Furthermore, a mutation at nucleotide 1753 from T to C or G was frequently found in anti-HBe positive patients and was often accompanied by the double mutation. The A1896 mutation was found in only about one fourth of the patients with anti-HBe. These data suggest that the patients with chronic liver diseases frequently had a double mutation regardless of HBeAg status and a mutation at nucleotide 1753 might be associated with HBeAg-negative chronic hepatitis B virus infection.     

Estimation of the future numbers of patients with circulatory diseases in Japan based on the results of national patient surveys]

Virological response to treatment of chronic hepatitis B is defined as the loss of serum hepatitis B virus DNA (HBV DNA) and hepatitis B e antigen (HBeAg). The quantitative measurement of HBV DNA is useful for monitoring and predicting the response to therapy with interferon-alpha (IFN-alpha). In this study, we evaluated whether quantitative measurement of serum HBeAg and IgM antibody to hepatitis B core antigen (HBcAb) could also be used in this manner. Using a microparticle-capture enzyme immunoassay (IMx), a standard curve of fluorescence rate vs HBeAg concentration was constructed to provide quantitative results. The IgM HBcAb index was also measured using a microparticle enzyme immunoassay and serum HBV DNA was measured by a solution hybridization assay. We studied 48 patients who were initially positive for HBeAg and HBV DNA and who were treated with IFN-alpha2b. Their sera were serially evaluated for HBeAg concentration, and results were compared with HBV DNA levels. In the 14 patients who responded to IFN, similar disappearance curves were observed with good intraindividual correlation between the levels of the two markers. In the 34 non-responders, HBeAg levels decreased during treatment but never became negative; HBV DNA levels also decreased during treatment and became transiently undetectable in six patients, falsely suggesting treatment success. The IgM HBcAb index paralleled changes in alanine aminotransferase (ALT) concentration and did not provide additional information. Multiple logistic regression indicated that baseline ALT and HBeAg concentrations were independent predictors of the response to treatment and the addition of neither HBV DNA nor IgM HBcAb improved the model. We conclude that quantitative measurement of HBeAg provides information similar to that of HBV DNA in monitoring and predicting the response to treatment; this technique could be readily adaptable to clinical laboratories.     

Serum hepatitis G virus RNA in patients with chronic viral hepatitis

OBJECTIVES: The hepatitis G virus (HGV) is a newly described flavivirus that affects a high proportion of patients with chronic viral hepatitis: our objective was to determine what role HGV might play in the course of disease. METHODS: We evaluated stored serum samples from 108 patients with chronic hepatitis B and 99 patients with chronic hepatitis C who participated in trials of alpha-interferon or ribavirin for the presence of hepatitis B virus (HBV) DNA and hepatitis C virus (HCV) RNA by branched DNA and for the presence of HGV RNA by polymerase chain reaction (PCR), using primers from the NS5 region of the genome. RESULTS: Initially, 20 (19%) patients with hepatitis B and 11 (11%) with hepatitis C had HGV RNA in their serum. Patients with and without HGV infection were similar with regard to clinical features, laboratory tests, and hepatic histology. HGV RNA levels fell during interferon therapy and became undetectable in those receiving the highest doses; however, HGV RNA levels returned to pretreatment values when therapy was stopped. With ribavirin therapy, HGV RNA levels did not change. Two- to 12-yr follow-up serum samples were available from 17 initially HGV RNA-positive patients, of whom only 10 (59%) were still positive. CONCLUSIONS: HGV infection is common among patients with chronic hepatitis B and C but has little effect on the short-term course of disease or response to therapy. HGV RNA levels are suppressed but not eradicated by alpha-interferon and are unaffected by ribavirin treatment. Spontaneous loss of HGV RNA occurs over time in a proportion of patients.     

A newly identified pattern of K-ras mutations at codons 12 and 13 is associated with long-term survival in colorectal cancer

BACKGROUND/AIMS: Hepatitis B virus (HBV) core gene heterogeneity may influence the outcome of liver disease and the response to interferon (IFN) therapy in adult HBV carriers. The aim of this study was to evaluate the possible association between HBV core gene variability and evolution of chronic hepatitis in children. METHODS: We examined serum samples from 25 children with HBV chronic hepatitis and HBe antigen (HBeAg) positivity who were followed-up for a mean of 7.4 years. Seven cases spontaneously seroconverted to anti-HBe, becoming HBV healthy carriers; nine cases were successfully treated with IFN; nine cases were non-responders to IFN therapy. HBV-DNA was extracted from one serum sample ("I") collected during the HBeAg positive phase, and from a second sample ("II") collected after the anti-HBe seroconversion or, in non-responders, after stopping therapy. The entire core gene of the HBV isolates was amplified and sequenced. RESULTS: Each isolate showed single or no missense mutation independently of the clinical behavior of the patients. HBeAg-defective viruses were detected in one case in both samples and in two cases only in sample "II". CONCLUSIONS: Core gene variability does not seem to be involved either in the outcome of infection or in the response to IFN treatment in children with HBV chronic hepatitis. Considering that most of the HBV carriers in our area acquire the infection in childhood, our data suggest that core gene heterogeneity is not a major cause of progression to chronicity.     

Monitoring of antiviral therapy with quantitative evaluation of HBeAg: a comparison with HBV DNA testing

The serological endpoint of response in the treatment of chronic hepatitis B is the loss of hepatitis B virus DNA and HBeAg. Because the quantitative measurement of hepatitis B virus DNA in serum has been shown to be useful for monitoring and predicting response to interferon-alpha therapy, we decided to evaluate whether changes in HBeAg concentration could also be used in this manner. Twenty-nine patients who were initially positive for HBeAg and HBV DNA were serially evaluated for HBeAg concentration with a microparticle-capture enzyme immunoassay. HBeAg levels in serum were calculated by means of comparison with a standard curve of fluorescence rate vs. HBeAg concentration. The results, expressed in milliunits per milliliter, were compared with hepatitis B virus DNA levels determined by means of solution hybridization. The baseline HBeAg concentration proved to be the best independent predictor of response on stepwise Cox regression analysis (p = 0.026). Similar disappearance curves were observed for the two markers, although hepatitis B virus DNA became undetectable at an earlier interval in 13 of 16 cases (81%). In the 16 responders, a decline in HBeAg concentration of more than 90% was observed by wk 12 of therapy (mean +/- S.D., 95% +/- 13%). Nonresponders did not demonstrate such steep declines in HBeAg values by wk 12 (mean +/- S.D., 45% +/- 27%), and levels tended to increase at subsequent time points. We conclude that serial monitoring of HBeAg concentration with a technique that should be readily adaptable to clinical laboratories may be useful in the initial evaluation and monitoring of patients undergoing antiviral therapy.     

Chronic hepatitis C and interferon alfa therapy: predictors of long term response

OBJECTIVE: To identify independent patient, disease and viral characteristics that predict a sustained biochemical or viral response to interferon alfa therapy in patients with chronic hepatitis C. Design: Comparison of interferon responders and non-responders by univariate and multivariate analysis. SETTING: The hepatitis clinic of the Alfred Hospital, Melbourne (a tertiary referral hospital), between July 1989 and June 1994. SUBJECTS: All patients with chronic hepatitis C who were treated with interferon alfa (IFN-alpha; 3 million IU, three times a week or more) for at least 12 weeks. OUTCOME MEASURES: Patient demographic and epidemiologic characteristics, pretreatment serum alanine aminotransferase (ALT) and 2-gamma-glutamyl transpeptidase (GGT) levels, histological grading of hepatic steatosis, necroinflammatory activity and fibrosis, serum hepatitis C virus (HCV) RNA titres and genotype and post-treatment serum ALT levels and presence of HCV RNA. Results: Of 58 patients, 13 (22%) had a sustained (six months or longer) biochemical response to IFN-alpha therapy, including 12 (21%) with a sustained viral response. Univariate analysis showed that young patients with a normal serum GGT level, grade 0-1 steatosis and fibrosis, low viral titre and infection with genotypes 3a and 2a were more likely to have a sustained response. Infection with genotypes other than 1a and 1b was the only independent variable associated with both a sustained biochemical and viral response. After adjusting for genotype, a hepatic fibrosis grade of 0-1 was also independently associated with viral response. This logistic regression model accurately predicted the virological response in 80% of cases. Conclusion: In Australian patients with chronic hepatitis C, a sustained viral response to IFN-alpha therapy is most likely in those infected with a genotype other than 1a or 1b and with minimal hepatic fibrosis.     

Use of quantitative assays for hepatitis B e antigen and IgM antibody to hepatitis B core antigen to monitor therapy in chronic hepatitis B

OBJECTIVES: We evaluated the clinical utility of IgM antibody to the hepatitis B (HB) core antigen (anti-HBc) and HB e antigen (HBeAg) serum levels in patients with chronic HB receiving interferon alfa. METHODS: Stored serum from 47 patients with chronic HB participating in a controlled trial of interferon alfa therapy (10 million U three times a week for 16 wk) were analyzed. All were seropositive for HB surface Ag, HBeAg, and HB virus (HBV) DNA before entry. IgM anti-HBc index values and HBeAg standard values were determined by automated microparticle enzyme immunoassay on samples drawn just before therapy and 6 months later. Ten normal subjects were tested as controls. IgM anti-HBc and HBeAg levels were compared to initial serum HBV DNA, DNA polymerase, serum aminotransferase levels, and demographic features. Serial IgM anti-HBc levels were also obtained during and after therapy in 10 responders and five nonresponders, and serial HBeAg levels were also obtained during and after therapy in four responders and four nonresponders. RESULTS: Neither IgM anti-HBc nor HBeAg levels correlated significantly with values for serum HBV DNA, DNA polymerase, aminotransferases, or demographic features. The initial mean IgM anti-HBc level among the 15 responders to therapy (loss of HBeAg and HBV DNA from serum) was no different from that in nonresponders (mean 1.15 vs 1.27, p = not significant). However, the initial mean HBeAg level was significantly lower in responders than in nonresponders (749.4 vs 1356.4, p = 0.019). Among 10 responders, IgM anti-HBc levels decreased progressively over time, so that at latest follow-up (1.5-4 yr later, mean 2.6 yr), the mean had decreased from 1.325 to 0.312 (p = < 0.001). Among five nonresponders, the mean did not change significantly over 1.5-3 yr (mean 2.2 yr) (1.26 vs 1.08, p = not significant). HBeAg values fell in parallel with HBV DNA and DNA polymerase values in four responders tested but remained elevated in four nonresponders. CONCLUSIONS: HBeAg levels, but not IgM anti-HBc levels, are useful in predicting response to interferon alfa, with responders tending to have lower pretreatment HBeAg levels than nonresponders. HBeAg levels may be used to monitor response to interferon alfa in patients with chronic HB.     

Sequential changes in full-length genomes of hepatitis B virus accompanying acute exacerbation of chronic hepatitis B

BACKGROUND/AIM: During the course of persistent hepatitis B virus infection, viral replication markedly decreases after acute exacerbation of liver inflammation accompanied by emergence of antihepatitis B e antibody (anti-HBe) and/or anti-hepatitis B surface antibody (anti-HBs). In some cases, however, persistent viral replication continues even after such exacerbation with or without HBeAg/anti-HBe seroconversion. The aim of the present study was to investigate the extent of genetic variations of HBV in this phenomenon. METHODS: Full-length HBV genomes were amplified by polymerase chain reaction from sera of three patients before and after acute exacerbation and were directly sequenced. RESULTS: In the whole genomes of 3215 nucleotides, only six nucleotide mutations for six amino acid substitutions (2 in the surface gene, 2 in the X gene, 1 in the core gene and 1 in the polymerase gene) were observed in patient 1, 15 mutations for 14 amino acid substitutions (1 in the pre-core codon 28, 4 in the surface gene, 4 in the core gene and 5 in the polymerase gene) were observed in patient 2, and 5 mutations for 6 amino acid substitutions (2 in the surface gene, 2 in the X gene, pre-core stop codon mutation and 1 in the polymerase gene) were observed in patient 3. Substitution in the a determinant of the surface gene, which encodes target epitopes for neutralizing antibodies, as well as those in the pre-core/core gene, which encodes epitopes for cytotoxic T cells, were mainly found. CONCLUSION: HBV that remained after the emergence of anti-HBe and anti-HBs are considered to possess mutations in epitopes for both humoral and cellular immunity. These mutant HBV may be involved in the pathogenesis of persistent hepatic injury after acute exacerbation.     

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.