X gene and precore region mutations in the hepatitis B virus genome in persons positive for antibody to hepatitis B e antigen: comparison between asymptomatic "healthy" carriers and patients with severe chronic active hepatitis

Hepatitis B virus (HBV) carriers with antibody to hepatitis e antigen comprise asymptomatic carriers (ASCs), who have low replication levels of HBV, and patients with chronic active hepatitis (CAH), who have high levels of viral replication. To investigate whether defects in the X protein might be responsible for this difference in the level of viral replication, nucleotide sequences of X and precore gene regions in serum HBV were analyzed in 19 ASCs and 9 CAH patients. All patients had a point mutation creating a stop codon in the precore region. Seventeen ASCs (87.3%) had identical mutations consisting of 4 noncontiguous 1-bp deletions or an 8-bp deletion, both of which truncate the normal X protein, whereas no CAH patient had an X gene mutation (P < .001). Thus, deletion of the X protein might be responsible for the low levels of viral replication in ASCs.     

Hepatocellular carcinomas infected with the novel TT DNA virus lack viral integration

A novel DNA virus designated TT virus (TTV) was cloned from a patient with posttransfusion hepatitis and is thought to be a new hepatitis virus. At present, hepatitis B virus (HBV) and hepatitis C virus (HCV) are known to induce hepatocellular carcinoma (HCC). But, actually, in Japan approximately 5 to 10% of HCCs are in HBV-negative and HCV-negative (NBNC) patients. In order to study the possible role of TTV in hepatocarcinogenesis, we investigated the frequency of the TTV genome in liver tissue of 20 HCC patients. As a result, 3 of 8 NBNC HCC patients and 5 of 12 HBV- or HCV-associated HCC patients were TTV positive, and TTV was shown not to be specific for NBNC HCC. For all TTV-positive patients, we also confirmed that the TTV genome was not integrated into host hepatocyte DNA.     

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.     

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.     

Chronic hepatitis B and C in HIV-infected patients

BACKGROUND AND AIM: This retrospective study examined the prevalence of co-infections with hepatitis B virus (HBV) and hepatitis C virus (HCV) and the frequency of chronic hepatitis in HIV-infected patients with respect to both the different risk groups and the serological results. PATIENTS AND METHODS: All Zurich participants of the Swiss HIV Cohort Study were evaluated who had available results of hepatitis B and C serology and ALT. RESULTS: Of the total 279 patients, 52% belonged to the intravenous drug user, 34% to the homosexual, and 11% to the heterosexual risk category. Serologically, previously acquired infection with HBV alone could be demonstrated in 92 (33%), HCV alone in 9 (3%), and both HBV and HCV in 130 (47%) patients. Only 3% of patients with sexually acquired HIV infection had anti-HCV antibodies, whereas co-infection with HBV and HCV was present in 87% of intravenous drug users. Among the 222 patients with previous HBV contact, 25 (11%) had positive HBsAg and 91 (41%) had "anti-HBc alone", both assumed to represent active HBV infection. 66 (24%) of 279 patients had chronic hepatitis with ALT elevation lasting > or = 6 months. Chronic hepatitis was present in 46% of those with active HBV and HCV co-infection, in 36% of those with HCV infection alone and in 18% of those with active HBV infection alone (P < 0.001). Of the 66 cases of chronic hepatitis, 58 were associated with HCV infection, and only 2 cases had no serological signs of active HBV or HCV infection. CONCLUSION: In patients with sexually acquired HIV infection, HBV had frequently been co-transmitted. In contrast, almost all of those infected by means of intravenous drug use had a co-infection with both HBV and HCV. The latter seems to play the strongest role in the development of chronic hepatitis with persistent ALT elevation. A chronic ALT elevation was almost always associated with serologically active HBV or HCV infection.     

Core antigen mutations of human hepatitis B virus in hepatomas accumulate in MHC class II-restricted T cell epitopes

Despite the extensive molecular information on serum-derived human hepatitis B viruses (HBV), liver-derived replicative HBV genomes have remained largely uninvestigated. We have examined the sequences of the entire core antigen (nucleocapsid) of liver-derived HBVs in 15 different hepatoma patients. Bona fide mutations, rather than subtype polymorphism, have been identified based on the high-frequency occurrence of structural differences from wild type at the highly evolutionarily conserved positions, instead of at the positions known to contain genetic heterogeneity among different isolates from different geographic locations. The distribution of these naturally occurring mutations of HBV core gene appears to be nonrandom and is found predominantly within three major (I, IV, and V) and four minor domains (II, III, VI, and VII). In general, domain IV mutations correlate with domain V mutations. The replicative HBV DNAs tend to accumulate a higher number of mutated core domains than the integrated HBV DNAs. At the domain level, there is no significant difference in HBV core mutation frequencies between the liver tumors and the adjacent nontumorous livers. Strikingly, domains I, III, and V coincide with three major known T cell epitopes within the core protein in acute and chronic hepatitis B patients. Furthermore, these domains coincide with HLA class II-restricted T cell epitopes, rather than with the conventional HLA class I-restricted epitopes of cytotoxic T lymphocytes. Our results support the hypothesis that HBV core antigen variants can accomplish immunoevasion via accumulated escape mutations. In addition, they also provide a potential molecular explanation for the maintenance of persistent infection of human hepatitis B virus in chronic carriers.     

Identification of more than one mutation in the hepatitis B virus polymerase gene arising during prolonged lamivudine treatment

Lamivudine has been shown to be a potent and nontoxic inhibitor of hepatitis B virus (HBV) replication in chronically infected patients. During prolonged treatment, drug resistance may develop, related to a mutation of Met to Val or Ile in the YM552DD motif of the HBV DNA polymerase gene. Analysis of the HBV DNA polymerase gene from 8 chronic hepatitis B patients with suspected resistance to lamivudine showed that in addition to a mutation in the YM552DD motif, a second mutation located in the B domain of this gene, a Leu528-to-Met528 change, was consistently and exclusively found in 4 patients showing the YV552DD motif. This suggests a functional or structural relationship between these domains. Since the presence of both the YI552DD and YV552DD motif sometimes preceded the exclusive presence of the YV552DD motif, we conclude that the YI552DD motif could occur as a temporal intermediate. After cessation of therapy, the wild type sequences reemerged.     

A PET study on cortical and subcortical control of pelvic floor musculature in women

The possibility of hepatitis B virus (HBV) infection in HBsAg-negative patients has been shown. However, an "inapparent" coinfection by HBV in hepatitis C virus (HCV)-positive patients generally is not taken into account in clinical practice. Mechanisms responsible for resistance to interferon (IFN) have not been completely clarified. The aim of this study was to investigate whether an "inapparent" coinfection by HBV in anti-HCV-positive chronic liver disease patients may influence IFN response. Fourteen anti-HCV positive, HBsAg-negative but serum HBV DNA-positive patients by PCR and 111 anti-HCV-positive, HBsAg-negative and HBV DNA (PCR)-negative patients with chronic hepatitis were treated with 3 MU of recombinant alpha-2a IFN 3 times weekly for 12 months. Serum HBV DNA and HCV RNA were determined before treatment, after 6-12 months and in coincidence with ALT flare-up by PCR. HBV PCR was performed using primers specific for the S region of the HBV genome and HCV PCR with primers localised in the 5'NC region of HCV genome. IgM anti-HBc was tested using IMx Core-M Abbott assay. By the end of treatment, ALT values had become normal in 4/14 HBV DNA-positive patients (28%), but all "responders" (4/4) relapsed between 2 and 5 months after therapy. All but one patient were HCV RNA-positive before treatment, 6 were also both HBV DNA and HCV RNA-positive during ALT flare-ups. In 5 patients, only HBV DNA and in 3 patients, only HCV RNA was detected when transaminase values increased. All patients remained HBsAg-negative and anti-HCV-positive. IgM anti-HBc was detected both before treatment and during ALT elevation in 3 patients and only during ALT relapse in 3 others. Of the 111 anti-HCV positive, HBsAg-negative and HBV DNA (PCR)-negative patients with chronic hepatitis, a biochemical response to IFN treatment was observed in 54% of the cases. Relapse of ALT values was observed in 47% of the cases during a follow-up of 1 year after treatment. "Inapparent" HBV/HCV coinfection may be implicated in cases of resistance to IFN treatment. In addition, HBV replication may persist in patients in whom HCV replication was inhibited by IFN treatment. The pathogenic role of HBV in liver disease was confirmed by detection of IgM anti-HBc in some cases; the appearance of these antibodies only after IFN treatment suggests that IFN may exert a selective role in favour of HBV. Further studies will show the effect of different treatment schedules. HBV DNA and/or IgM anti-HBc detection with very sensitive methods may be important both as a prognostic factor and as a tool for better understanding interviral relationships and mechanisms involved in multiple hepatitis virus infections.     

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.     

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.     

Association of mutations in the core promoter and precore region of hepatitis virus with fulminant and severe acute hepatitis in Japan

It was recently reported that mutations in the precore and core promoter region of hepatitis B virus (HBV) are associated with fulminant hepatitis. The aim of this study was to investigate the association of mutations in the precore and core promoter region of HBV with fulminant and severe acute hepatitis. We studied Japanese patients with acute HBV infection, including seven patients with fulminant hepatitis, 12 with severe acute hepatitis and 41 with acute self-limited hepatitis. The presence of HBV mutants was examined by using a point mutation assay to detect a G to A transition at position 1896 in the precore region and an A to T transition at position 1762 and a G to A transition at position 1764 in the core promoter region. Significant differences in the proportion of mutations in the precore or core promoter region were present between patients with fulminant hepatitis and self-limited acute hepatitis (7/7 (100%) vs 4/41 (9.8%), P<0.01) and between severe acute hepatitis and self-limited acute hepatitis (6/12 (50.0%) vs 4/41 (9.8%), P<0.01). The frequency of mutation increased proportionately with the severity of disease in patients with acute HBV infection. Fulminant hepatitis B in Japan is closely associated with mutations in the core promoter and precore gene of HBV. Point mutation assays for HBV precore and core promoter analysis may be useful to predict the outcome of liver disease in patients with acute HBV infection.     

Utility of bolus dynamic CT for the detection of hypervascular malignant hepatic tumors: mainly referring to the comparison with delayed phase contrast-enhanced CT

We have studied the proliferation and CD40 antigen expression of lymphocytes, and the cytotoxicity to monocytes, of antisense phosphorothioate oligodeoxynucleotides complementary to the SP II promoter of HBV mRNA (sequence I) and the X gene (sequence II) in patients with chronic hepatitis B. The oligo sequence I stimulated proliferation of both T and, to a lesser extent, B cells. The percentage of cells expressing CD40 in T and B cell co-cultures increased from 4.2% to 13.8% after oligo stimulation in patients, while it increased form 4.7% to 48.6% in healthy controls. The sense sequence (sequence III) of the X gene also enhanced the expression of CD40 antigen in patients with hepatitis B. The proportion of CD40 cells (26%) in a resting B-cell preparation from hepatitis B patients decreased to zero after a 5-day culture with sequence I, but IgG levels in the culture supernatant increased. The cytotoxic properties of monocytes were not influenced by the oligos. These findings indicate that antisense oligos against hepatitis B virus (HBV) have mitogenic effects on the proliferation of human lymphocytes in a non-specific manner and may activate T cells to express CD40 antigen.     

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.     

Hepatitis B virus genomic sequence in the circulation of hepatocellular carcinoma patients: comparative analysis of 40 full-length isolates

We determined full-length nucleotide sequence of hepatitis B virus (HBV) genome in sera from 40 Japanese patients with HBsAg-positive hepatocellular carcinoma (HCC), in order to obtain information on HCC-specific characteristics, if any, of the HBV genome. Direct sequencing of the long distance PCR products starting from 50 microliters of serum samples revealed that 95% of our isolates were of genotype C, and that mutations and deletions/insertions were very common. With respect to envelope protein genes, deletions and missense mutations were frequent in preS2, and the determinant a domain of HBsAg was rich in "antibody-escape" mutations. Within the precore/core region, the most remarkable mutation was the replacement of proline of wild type by other amino acids at codon 130 of the core gene, which was found in 58% of our isolates, while precore-stop mutation was found in 45%. Most interestingly, however, about 90% of our isolates had mutations at nt positions 1762 (A-to-T) and 1764 (G-to-A) within the core promoter, which had been implicated in "e-suppressive" phenotype of HBV genome. G-to-A at nt 1613 and C-to-T at nt 1653 within enhancer II and T-to-C/A at nt 1753 within core promoter were also evident: 38%, 53%, and 40%, respectively. It was interesting that some of the characteristics observed in our isolates form HCC patients had been previously implicated in fulminant hepatitis and/or acute exacerbation of chronic hepatitis.     

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.