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.     

Identification of hepatitis B virus-DNA in the liver by in situ hybridization using a biotinylated probe. Relation to HBcAg expression and histology

The cellular localisation of hepatitis B virus (HBV)-DNA in liver tissue was studied by in situ hybridisation using biotinylated and radiolabelled probes on samples from HBsAg carriers with a spectrum of disease and related to the presence of HBV-DNA in serum and intrahepatic HBcAg expression. Sixteen of the 31 patients studied were seropositive for HBV-DNA; nine had chronic active hepatitis and seven had chronic persistent hepatitis. HBV-DNA was detected in the liver tissue in seven of these patients. In each, HBV-DNA was detected in both cytoplasm and nuclei. All seven also had nuclear and/or cytoplasmic HBcAg which in six was associated with chronic active hepatitis. HBcAg (without tissue HBV-DNA) was detected in the remaining nine patients with an exclusively nuclear pattern in two. Fifteen patients were seronegative for HBV-DNA. HBV-DNA was not detected in the tissue of any of these. Three of these were HBcAg positive but in each this was confined to occasional nuclei and each had inactive disease. The close association between the presence of detectable HBV-DNA in tissue, cytoplasmic HBV-DNA expression and chronic active hepatitis in one group and a failure to detect HBV-DNA in those with nuclear HBcAg and benign disease suggests that there may be two distinct patterns of HBV replication in chronic HBV carriers which may influence the development of liver damage.     

Recurrence of hepatitis D (delta) in liver transplants: histopathological aspects

BACKGROUND: The viral/pathological correlates of recurrent hepatitis delta virus (HDV) disease in orthotoptic liver transplants are reported. METHODS: We examined the histological features of recurrent HDV disease in nine patients with transplants for terminal HDV cirrhosis were examined; intrahepatic HDV and hepatitis B virus (HBV) antigens were detected by immunoperoxidase techniques. Sera were tested for the battery of HDV and HBV markers. RESULTS: In four patients, HDV reinfection was accompanied by the recurrence of an HBV infection with features of active viral replication. In the other five, HDV reinfection was accompanied by an atypical recurrence of HBV infection without evidence of active HBV replication (no expression of intrahepatic hepatitis B core antigen). In four of the latter patients, the atypical HBV pattern changed during the follow-up into a pattern of active viral replication accompanied by chronic necroinflammation detected during histology. CONCLUSION: The pattern of recurrent HBV infection can influence the pathological aspects of the relapses of HDV disease in liver grafts.     

Immune response to synthetic peptides of hepatitis delta antigen

Hepatitis delta antigen (HDAg) is the only viral protein known to be expressed during hepatitis delta virus (HDV) infection. Detection of antibody to HDAg (anti-HD) is the usual method for diagnosis of HDV infection since viremia lasts only a few weeks. In an effort to identify the major epitopes recognized by humans during natural infection, four oligopeptides including residues 2 to 17 (SP1), 155 to 172 (SP2), 168 to 182 (SP3), and 189 to 211 (SP4) of the HDAg molecule were synthesized and probed by enzyme-linked immunosorbent assay with a panel of 80 serum specimens from 45 patients suffering from either HDV-hepatitis B virus coinfections (n = 17) or HDV superinfections (n = 28). Sera from infected patients recognized these relatively short peptides. Peptide SP2 was the most antigenic; 71% of serum specimens reacted. Antibody to SP2 was also the commonest in sera taken early in the course of the disease. Peptide SP2 corresponds to one of the two regions which is highly conserved between different isolates. Among the 63 serum specimens which scored anti-HD positive by a commercial assay, all but 3 reacted to at least one of the peptides (95% agreement). Peptide assays appeared to be significantly more sensitive than the commercial assay with native HDAg early in the course of HDV infection since 14 of 17 (82%) serum specimens which scored anti-HD negative in the commercial assay reacted to one or more peptides. All serum specimens giving one or more positive results with the various peptides were confirmed as being HDV positive by an inhibition assay with free peptide in solution. The immune response to HDAg peptides vared greatly between individuals. No specific reactivity profile could be assigned to those with either HDV-hepatitis B virus coinfections or HDV superinfections. Overall, HDAg peptides appeared to be convenient reagents in addition to native antigen for the development of new and improved diagnostic tests for HDV infection.     

Hepatitis B virus occult infection in subjects with persistent isolated anti-HBc reactivity

The aim of this study was to investigate the presence of hepatitis B virus occult infection in asymptomatic subjects with persistent anti-HBc reactivity but no other hepatitis B virus serological markers, including HBsAg, anti-HBs, IgM anti-HBc and HBV-DNA. For this purpose we used both polymerase chain reaction assays in sera and immunohistochemistry for HBsAg and HBcAg in liver biopsy specimens. Twenty-four cases were studied: 15 were drug abusers or homosexuals (eight with normal alanine aminotransferase levels) and nine were heterosexuals with raised alanine aminotransferase levels (> 45 U/l) but with no history of blood transfusion or ethanol intake (< 80 g daily). In all but five cases, liver biopsy was performed in subjects with persistent elevated alanine aminotransferase levels. In 10 out of 24 cases (41.66%) hepatitis B virus infection was demonstrated by polymerase chain reaction or immunohistochemistry, and when results from both procedures were available (n = 11) hepatitis B virus infection was detected in 63.63% of the subjects. The only clinical feature associated with HBV infection was the presence of persistent elevated alanine aminotransferase levels (p < 0.05). In conclusion, persistent isolated anti-HBc reactivity may be a relatively common serologic pattern for hepatitis B virus occult infection, at least in patients with chronic liver disease.     

The immunological diagnosis of chronic viral hepatitis

Chronic viral diseases of the liver are associated with changes in immune reactions mediated by T and B lymphocytes and dependent in severity on etiological factor (virus of hepatitis B, delta, C, their combination), the disease stage (hepatitis, cirrhosis), the process activity, kind of immune correction. HBsAg, viral hepatitis B marker, was detected in 21.2% of 1400 cases with chronic active hepatitis and liver cirrhosis. 32% of HbsAg-seropositive patients had antibodies to delta-antigen. Antibodies to HBsAg, HCV were found in 27.7 and 14.9% of the above patients. Chronic viral diseases of the liver with persistence of HBV, HDV and HCV markers are characterized by a complex of immune disorders, including a moderate rise in peripheral blood of IgM, IgG, IgA, IgE, Ig kappa, lambda, immune complexes, cryoglobulins, autoantibodies to subcellular structures as well as changes in regulatory (suppressor, helper) and effector (lymphokine-producing) functions of T lymphocytes, inhibition of phagocytosing capacity. The above shifts in immune status, clinical and biochemical activity of the disease are more pronounced in chronic active hepatitis with HCV markers compared to BHV. Of maximal intensity they were in combined viral infection HBV+HDV or HBV+HCV.     

Molecular biology of hepatitis D virus: research and potential for application

Superinfection by hepatitis D virus (HDV) leads to acute hepatitis and causes progression to liver cirrhosis in a significant proportion of hepatitis B surface antigen (HBsAg) carriers. Current regimens (interferon) to treat hepatitis D patients has only transient but no lasting effects. New approaches are, therefore, warranted. Recently, several laboratory studies have discovered interesting properties of HDV that may become targets for antiviral chemicals. Viral replication requires the small hepatitis delta antigen (s-HDAg). The s-HDAg is a nuclear phosphoprotein. There is evidence indicating that phosphorylation is important for HDV replication. A second step of replication requires HDV-RNA self-cleavage and self-ligation. Interestingly, one group of antibiotics, the aminoglycosides, exerts strong suppression effects on HDV ribozyme activities. In the following stage of viral assembly, two post-translational modifications, namely isoprenylation of large HDAg and glycosylation of HBsAg are involved. Agents capable of blocking the two modifications should reduce viral production. These four possible targets are reviewed. For prevention, effective vaccines are not yet available. Two novel approaches are discussed. The first demonstrates the immunogenicity of a nucleic acid vaccine in mice. The second approach assembled an empty HDV particle in yeast. Advances on such laboratory investigations may provide new methods for the control of hepatitis D in the future.     

A study on the appearance of hepatitis B virus markers in renal tissue of glomerulonephritis

HBV antigens (HBcAg, HBsAg and HBeAg) in 246 cases of renal biopsy specimens with various types of glomerulonephritis were examined by immunohistochemical techniques. Southern blot hybridization was used to detect HBV DNA in 18 cases. The results showed that HBV antigens were frequently found in the tubular cells in addition to the appearance of HBV antigens on the glomeruli of certain glomerulonephritis. The positive rte of HBcAg in the tubular cells was 21.54%, which is higher than that of glomeruli (10.98%). Renal HBV DNA was positive in 15 cases and among them HBcAg expression in renal tissue in 14 cases and HBV antigenemia in 12 cases. The results suggest that HBV may also be implicated in the pathogenesis of certain cases of glomerulonephritis and the appearance of HBV antigen antibody immunocomplex in renal tissue, aside from originating in the circulation, there is the possibility of it originating from renal cells in situ.     

Genotype-specific complementation of hepatitis delta virus RNA replication by hepatitis delta antigen

Characterizations of genetic variations among hepatitis delta virus (HDV) isolates have focused principally on phylogenetic analysis of sequences, which vary by 30 to 40% among three genotypes and about 10 to 15% among isolates of the same genotype. The significance of the sequence differences has been unclear but could be responsible for pathogenic variations associated with the different genotypes. Studies of the mechanisms of HDV replication have been limited to cDNA clones from HDV genotype I, which is the most common. To perform a comparative analysis of HDV RNA replication in genotypes I and III, we have obtained a full-length cDNA clone from an HDV genotype III isolate. In transfected Huh-7 cells, the functional roles of the two forms of the viral protein, hepatitis delta antigen (HDAg), in HDV RNA replication are similar for both genotypes I and III; the short form is required for RNA replication, while the long form inhibits replication. For both genotypes, HDAg was able to support replication of RNAs of the same genotype that were mutated so as to be defective for HDAg production. Surprisingly, however, neither genotype I nor genotype III HDAg was able to support replication of such mutated RNAs of the other genotype. The inability of genotype III HDAg to support replication of genotype I RNA could have been due to a weak interaction between the RNA and HDAg. The clear genotype-specific activity of HDAg in supporting HDV RNA replication confirms the original categorization of HDV sequences in three genotypes and further suggests that these should be referred to as types (i.e., HDV-I and HDV-III) rather than genotypes.     

Immune response to HBsAg, HBcAg and e-antigen in patients with acute hepatitis and HBsAg carriers with and without liver diseases

Humoral and/or cell-mediated (CMI) immune responses to HBAg components, human and rabbit liver specific proteins (HLP and RLP) and tuberculin were tested in patients with acute virus B and non-B-hepatitis, asymptomatic HBsAg carriers and HBsAg positive chronic active hepatitis (CAH). Furthermore, the presence of HBsAg, HBcAg and/or "e"-antigen has been studied in patients with sera and/or liver tissue. Asymptomatic HBsAg carriers are characterized by a status of immunological tolerance against HBsAg. HBcAg in liver nuclei could not be detected. All sera were positive for anti-HBc, some had anti "e". - Patients with uneventful acute virus-B-hepatitis developed CMI against HBsAg 4-6 weeks and anti-HBs 4-6 months after onset of the disease. Acute virus hepatitis without detectable HBsAg are defined as non-B-hepatitis by negative humoral and cell-mediated immune reaction against HBsAg 1-12 months after onset of the disease. - Patients with type B chronic active hepatitis are characterized by inadequate CMI against HBsAg without immune elimination of virus and virusantigens. Acute and chronic type-B-hepatitis showed temporary or constant CMI against HLP. These findings suggest an alteration or a carrier function of membrane antigens of virus infected hepatocytes or an induction of new membrane antigens by a virus. The results indicate that recovery from type B-hepatitis is associated with the ability to elicit a specific immune response to HBsAg. Furthermore immune responses to virus, virus antigens and virusinfected hepatocytes seemed to be the pathogenic principle of virus induced acute and chronic liver diseases.     

Sexual behaviour and multiple infections in drug abusers

We have studied the prevalence and the serological profile of HBV, HCV, HDV and HIV infections in 137 Italian subjects addicted to the intravenous use of heroine and correlated the virological findings with sexual behaviour. HBV and HCV viremia were also measured in 114 patients. Anti-HCV was detected in 81% of the addicts, and one or more markers of HBV infection were detected in 62.8% (4.4% were carriers of HBsAg, 58.4% had evidence of past HBV infection and 13.1% of the latter also had HDV markers). Anti-HIV was positive in 23.4%; 26% of those positive for anti-HCV and 4.6% of those positive for HBV markers had no other viral marker: none had only anti-HIV. HBV-DNA was negative in the carriers of HBsAg, and HCV-RNA was not detected in any of the HBsAg carriers who also had circulating anti-HCV. Overall, 34% of the anti-HCV positive addicts had HCV-RNA in their blood. The prevalence of the virus infection correlated with the duration of drug addiction but not with sexual behaviour, and sexual behaviour did not influence the acquisition of any virus. HCV infection was most frequent and probably the first infection to occur, but exposure to HBV was also common despite a low rate of HBsAg carriage. The prevalence of HDV infection was high (50%) in the HBsAg carriers, while the overall prevalence of HIV was lower (23%) than expected. Lack of HBV-DNA and HCV-RNA in carriers of HBV with anti-HCV in serum may indicate that HBV and HCV mutually inhibit their own 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.     

An evaluation of the degree of the morphological activity and the stage of the process in patients with chronic liver diseases caused by coinfection with the hepatitis B, C and/or D viruses

In order to determine the differences in histological grade of activity and the stage of fibrosis in patients with chronic liver diseases due to multiple hepatitis virus infection and single infection of HBV and HCV we assessed the 68 liver biopsies samples according to Knodell and Scheuer scoring systems. Retrospectively, 216 liver biopsies reports from consecutive patients with chronic viral hepatitis were analysed. Histological activity index (HAI) in HBV/HCV coinfection was higher than in a single HCV infection; it did not differ in groups of HBV/HBC and HBV. The difference was due to the interface hepatitis; lobular activity and portal inflammation were the same. In HDV superinfection HAI was high due to both portal-periportal and lobular hepatitis. HAI depended mainly upon the presence of HBV replication; in patients with chronic hepatitis C with HBV-DNA HAI was also higher than in single HCV group. No difference in HAI between triple and dual hepatitis virus infection was found. In patients with HBV/HCV coinfection and especially with HDV superinfection the advanced stages occurred more than often than in patients with single infections.     

Seroepidemiological study of hepatitis delta virus infection in Okinawa, Japan

A seroepidemiological study on hepatitis delta virus (HDV) infection was conducted in the Okinawan islands, the area of Japan where hepatitis B virus infection is most prevalent. The subjects of this study included 116 asymptomatic hepatitis B surface antigen (HBsAg) carriers, 48 patients with chronic hepatitis (CH), 19 with liver cirrhosis (LC), and 11 with hepatocellular carcinoma (HCC). Among the 194 serum samples examined, a total of 10 (5.2%) were anti-HDV seropositive. Anti-HDV was detected in 2 (1.7%) of the 116 asymptomatic HBsAg carriers, in 3 (6.3%) of the 48 patients with CH, and in 5 (26.3%) of the 19 with LC. However, none of the patients with HCC had detectable anti-HDV. Eight of the 10 were born in the Miyako island group and the remaining 2 on the main island of Okinawa. Since the subjects included 34 individuals who were living and/or born in the Miyako islands, the positive rate of anti-HDV in the islands was 23.5%. This study demonstrates the existence of an endemic area of HDV infection in Japan.     

Human CD4+ T-cell response to hepatitis delta virus: identification of multiple epitopes and characterization of T-helper cytokine profiles

The T-cell-mediated immune response plays a crucial role in defense against hepatotropic viruses as well as in the pathogenesis of viral chronic hepatitides. However, very little is known about the role of specific T cells during hepatitis delta virus (HDV) infection in humans. In this study, the T-cell response to HDV in chronic hepatitis B virus (HBV) carriers with HDV superinfection was investigated at different levels. Analysis of peripheral blood mononuclear cell (PBMC) proliferation in response to a recombinant form of large hepatitis delta antigen (HDAg) revealed that 8 of 30 patients studied (27%) specifically responded to HDAg. By employing synthetic peptides spanning the entire HDAg sequence, we found that T-cell recognition was directed against different antigenic determinants, with patient-to-patient variation in the pattern of response to peptides. Interestingly, all responders had signs of inactive HDV-induced disease, while none of the patients with active disease and none of the control subjects showed any significant proliferation. More accurate information about the specific T-cell response was obtained at the clonal level. A panel of HDAg-specific CD4+ T-cell clones from three HDV-infected individuals and fine-specificity analysis revealed that the clones tested individually recognized four epitopes corresponding to amino acids (aa) 26 to 41, 50 to 65, 66 to 81, or 106 to 121 of HDAg sequence. The study of human leukocyte antigen (HLA) restriction revealed that peptides 50 to 65 and 106 to 121 were presented to specific T cells in association with multiple class II molecules. In addition, peptide 26 to 41 was efficiently generated after processing of HDAg through the endogenous processing pathway. Cytokine secretion analysis showed that all the CD4+ T-cell clones assayed were able to produce high levels of gamma interferon (IFN-gamma), belonging either to T helper-1 (Th1) or Th0 subsets and that some of them were cytotoxic in a specific assay. This study provides the first evidence that detection of a specific T-cell response to HDAg in the peripheral blood of individuals with hepatitis delta is related to the decrease of HDV-induced disease activity. The HDAg epitopes identified here and particularly those recognized by CD4+ T cells in association with multiple major histocompatibility complex class II molecules may be potentially exploited for the preparation of a vaccine for prophylaxis and therapy of HDV infection.     

Prevalence and clinical features of hepatitis delta virus infection in the Miyako Islands, Okinawa, Japan

The aims of this study were twofold: (1) to determine the prevalence and clinical features of hepatitis delta virus (HDV) infection among subjects positive for hepatitis B surface antigen (HBsAg) living in the Miyako Islands, Okinawa Prefecture, Japan, and (2) to clarify the relationship between HDV-RNA level and severity of HDV-related liver disease. One hundred and ninety-nine HBsAg-positive subjects (123 asymptomatic carriers [ASCs], 3 patients with acute hepatitis [AH], 50 patients with chronic hepatitis [CH], 15 patients with liver cirrhosis [LC], and 8 patients with hepatocellular carcinoma [HCC], were tested for antibody to HDV (anti-HDV) by radioimmunoassay. Anti-HDV-positive individuals were examined to determine semi-quantified HDV-RNA level by polymerase chain reaction (PCR). The overall prevalence of anti-HDV among the 199 subjects was 21.1%. The positivity rate tended to increase with age or the severity of the underlying liver disease: anti-HDV-positive rates were 10.6% (13/123) in ASCs, 32.0% (16/50) in patients with CH, 40.0% (6/15) in patients with LC, and 87.5% (7/8) in patients with HCC. None of the patients with AH were positive for anti-HDV. There was no correlation between semi-quantified serum HDV-RNA levels and the severity of chronic liver disease in patients positive for anti-HDV. The present study showed the local spread of HDV infection in the Miyako Islands, Okinawa, Japan. Although the anti-HDV positivity rate tended to increase with the severity of the underlying liver disease, the severity of HDV-related liver disease did not correlate with the semi-quantified serum HDV-RNA level.