Potent and Specific Inhibition of NTCP-Mediated HBV/HDV Infection and Substrate Transporting by a Novel, Oral-Available Cyclosporine A Analogue.

Analogues of the natural product cyclosporine A (CsA) were developed and assessed as antivirals against infection of hepatitis B virus (HBV) and its satellite hepatitis D virus (HDV). An analogue termed 27A exhibits potent inhibition of HBV/HDV infection by specifically blocking viral engagement to its cellular receptor NTCP, while it lacks immunosuppressive activity found in natural CsA. Intraperitoneal injection or oral intake of 27A protects HDV-susceptible mouse model from HDV infection. 27A serves as a promising lead for the development of novel anti-HDV/HBV agents.

Statin inhibits large hepatitis delta antigen-Smad3 -twist-mediated epithelial-to-mesenchymal transition and hepatitis D virus secretion.

BACKGROUND: Hepatitis D virus (HDV) infection may induce fulminant hepatitis in chronic hepatitis B patients (CHB) or rapid progression of CHB to cirrhosis or hepatocellular carcinoma. There is no effective treatment for HDV infection. HDV encodes small delta antigens (S-HDAg) and large delta antigens (L-HDAg). S-HDAg is essential for HDV replication. Prenylated L-HDAg plays a key role in HDV assembly. Previous studies indicate that L-HDAg transactivates transforming growth factor beta (TGF-ß) and induces epithelial-mesenchymal transition (EMT), possibly leading to liver fibrosis. However, the mechanism is unclear. METHODS: The mechanisms of the activation of Twist promoter by L-HDAg were investigated by luciferase reporter assay, chromatin immunoprecipitation, and co-immunoprecipitation analysis. ELISA and Western blotting were used to analyze L-HDAg prenylation, TGF-ß secretion, expression of EMT markers, and to evaluate efficacy of statins for HDV treatment. RESULTS: We found that L-HDAg activated Twist expression, TGF-ß expression and consequently induced EMT, based on its interaction with Smad3 on Twist promoter. The treatment of statin, a prenylation inhibitor, resulted in reduction of Twist promoter activity, TGF-ß expression, and EMT, and reduces the release of HDV virions into the culture medium. CONCLUSIONS: We demonstrate that L-HDAg activates EMT via Twist and TGF-ß activation. Treatment with statins suppressed Twist expression, and TGF-ß secretion, leading to downregulation of EMT. Our findings clarify the mechanism of HDV-induced EMT, and provide a basis for possible novel therapeutic strategies against HDV infection.

Excellent safety and effectiveness of high-dose myrcludex-B monotherapy administered for 48 weeks in HDV-related compensated cirrhosis: A case report of 3 patients.

Short-term administration of the entry inhibitor myrcludex-B (MyrB) has been shown to be safe and effective in phase II studies in patients coinfected with hepatitis B virus (HBV) and hepatitis delta virus (HDV). However, its effectiveness and safety are unknown during long-term and high-dose treatment of patients with compensated cirrhosis in real-life settings. Herein, we describe the first 3 European patients with HDV-related compensated cirrhosis who were treated with MyrB 10 mg/day for 48 weeks as a compassionate therapy. Liver function tests, bile acids, and virological markers were monitored every 4 weeks. HBV/HDV-specific T cell quantity (up to 48 and 36 weeks) and HBV RNA levels were also assessed in 2 cases. During MyrB treatment, HDV RNA levels progressively declined from 4.4 and 5.6 logs IU/ml to undetectability in 2 cases, and from 6.8 log copies/ml to 500 copies/ml for the other patient. Alanine aminotransferase normalised after 20, 12 and 28 weeks, respectively. A significant improvement in features of portal hypertension, liver function tests and alpha-fetoprotein levels were documented in 2 cases. In the male patient with histological and clinical stigmata of autoimmune hepatitis, IgG and immunoglobulins rapidly normalised. No significant changes in HBV surface antigen levels and circulating HBV/HDV-specific T cells were demonstrated; HBV DNA and HBV RNA levels remained undetectable throughout the study period. MyrB was well tolerated; patients remained fully asymptomatic despite a significant increase of bile acids. In conclusion, this report shows excellent safety and effectiveness of a 48-week course of MyrB 10 mg/day, combined with tenofovir disoproxil fumarate, for the treatment of HDV-related compensated cirrhosis.

Hepatitis B Virus and Hepatitis D Virus Entry, Species Specificity, and Tissue Tropism.

Entry of hepatitis B (HBV) and hepatitis D viruses (HDV) into a host cell represents the initial step of infection. This process requires multiple steps, including the low-affinity attachment of the virus to the cell surface, followed by high-affinity attachment to specific receptor(s), and subsequent endocytosis-mediated internalization. Within the viral envelope, the preS1 region is involved in receptor binding. Recently, sodium taurocholate cotransporting polypeptide (NTCP) has been identified as an entry receptor of HBV and HDV by affinity purification using a preS1 peptide. NTCP is mainly or exclusively expressed in the liver, and this membrane protein is at least one of the factors determining the narrow species specificity and hepatotropism of HBV and HDV. However, there are likely other factors that mediate the species and tissue tropism of HBV. This review summarizes the current understanding of the mechanisms of HBV/HDV entry.

Strategies to inhibit entry of HBV and HDV into hepatocytes.

Although there has been much research into the pathogenesis and treatment of hepatitis B virus (HBV) and hepatitis D virus (HDV) infections, we still do not completely understand how these pathogens enter hepatocytes. This is because in vitro infection studies have only been performed in primary human hepatocytes. Development of a polarizable, HBV-susceptible human hepatoma cell line and studies of primary hepatocytes from Tupaia belangeri have provided important insights into the viral and cellular factors involved in virus binding and infection. The large envelope (L) protein on the surface of HBV and HDV particles has many different functions and is required for virus entry. The L protein mediates attachment of virions to heparan sulfate proteoglycans on the surface of hepatocytes. The myristoylated N-terminal preS1 domain of the L protein subsequently binds to the sodium taurocholate cotransporting polypeptide (NTCP, encoded by SLC10A1), the recently identified bona fide receptor for HBV and HDV. The receptor functions of NTCP and virus entry are blocked, in vitro and in vivo, by Myrcludex B, a synthetic N-acylated preS1 lipopeptide. Currently, the only agents available to treat chronic HBV infection target the viral polymerase, and no selective therapies are available for HDV infection. It is therefore important to study the therapeutic potential of virus entry inhibitors, especially when combined with strategies to induce immune-mediated killing of infected hepatocytes.

Hepatitis delta in Switzerland: a silent epidemic.

OBJECTIVES: The sero-prevalence of co-infection with the hepatitis B virus (HBV) and the hepatitis D virus (HDV) is well known in many European countries, starting from 6.8% in Germany to more than 27% in some Turkish areas. To gain a better description of this infection in Switzerland, and to characterise those affected, a questionnaire was sent to all Swiss gastroenterologists, hepatologists and infectologists. METHODS: A questionnaire was received by 349 physicians which asked them to report on all HBV- and HDV-infected patients seen at their units/offices. RESULTS: A total of 101 HDV-positive patients seen by 78 specialists were analysed. The physicians were in charge of 1'699 patients with chronic hepatitis B, giving a 5.9% prevalence of HDV infection in HBV-positive patients. A predominance of males (75%) from Switzerland (39%), and of African origin (21%) was recorded. Most had been contaminated by intravenous drug use (62%), followed by vertical transmission (15%), sexual contact (13%) or transfusion with blood or blood products (2%). The majority (74%) had a very low (<103 UI/ml) HBV viral load and 75% were HBeAg-negative. A total of 76% percent of those who had a liver biopsy had significant fibrosis (≥F2), and only 21% had received standard therapy (interferon or pegylated interferon-α). Overall, 10% recovered spontaneously (anti-HBs-positive). CONCLUSION: With a prevalence of 5.9% of hepatitis D in HBsAg-positive patients, Switzerland seems less affected than most other European countries, however, it is possible that this infection is under-diagnosed. Intravenous drug use was the main risk factor. Associated advanced liver disease was also very common.

Hepatitis D revival.

More than 30 years after Mario Rizzetto and colleagues described a new antigen in livers of HBsAg-positive patients called the "delta antigen", a re-emerging interest in hepatitis delta is currently observed. The state-of-the art on basic and clinical research on hepatitis delta was presented during a monothematic conference organized by the European Association for the Study of the Liver (EASL) in September 2010. Hepatitis delta is caused by infection with the hepatitis D virus (HDV) which requires presence of HBsAg for complete replication and transmission. Recent data confirmed the severe long-term course of HDV infection with high rates of hepatic decompensation while controversial data on the risk for the development of hepatocellular carcinoma were reported. Pegylated interferon alpha can lead to sustained HDV RNA elimination in about one quarter of patients while HBV polymerase inhibitors are ineffective against HDV. Novel treatment options include prenylation inhibitors and HBV entry inhibitors which are currently in early clinical development.

Virological and clinical characteristics of delta hepatitis in Central Europe.

Hepatitis D virus (HDV) or delta hepatitis has mainly been studied in Asian and Mediterranean cohorts, but data on virological and clinical characteristics of HDV-infected Central and Northern European patients are limited. We investigated virological patterns, as well as biochemical and clinical features of liver disease in 258 HDV infected patients recruited over a period of 15 years at Hannover Medical School. Virological parameters were compared to 2083 anti-HDV negative hepatitis B surface antigen (HBsAg) positive individuals. In this cohort, (i) HDV infection was associated with both suppressed hepatitis B virus (HBV) and hepatitis C virus (HCV) replication, (ii) the suppression of HBV-DNA and HCV-RNA was not related to HDV-RNA replication, (iii) mean HBsAg levels did not significantly differ between HBV-monoinfected patients and individuals with delta hepatitis, (iv) HCV coinfection was rather frequent as about one third of our delta hepatitis patients tested anti-HCV positive, however, without being associated with more advanced liver disease, (v) delta hepatitis patients presented in a high frequency with an advanced stage of liver disease, and (vi) the course of delta hepatitis did not differ between Turkish-born, Eastern European (EE)-born and German-born patients. In summary, in this cohort of patients which is the largest so far Central European single centre group of delta hepatitis patients, we confirm the presence of frequently severe disease and describe novel virological profiles which require consideration in the management of this difficult to treat group of patients.

[Occult hepatitis B virus infection in chronic viral hepatitis patients with non-A to E hepatitis virus infection].

OBJECTIVE: To observe the status of occult hepatitis B virus infection in chronic viral hepatitis patients with non-A to E hepatitis virus infection and explore the diagnostic value of fluorescence quantitative polymerase chain reaction (FQ-PCR) technique for occult hepatitis B virus infection. METHODS: The amount of HBV-DNA in serum and liver tissue from 57 patients with non-A to E hepatitis virus infection who were diagnosed as chronic viral hepatitis by Menghini method liver biopsy were detected by using FQ-PCR technique, then the relation between the viral load of HBV DNA in liver tissue and hepatic inflammatory activity were analyzed. RESULTS: Thirteen (22.81%), 22 (38.60%) patients were positive for HBV DNA in serum and liver tissue, respectively. The positive rate and the level of HBV DNA quantity in liver tissue were significantly higher than those in serum; HBV DNA was found positive in both serum and liver tissue in 13 cases, negative in both serum and liver tissue in 35, positive in liver tissue but negative in serum in 9, and in none of the cases HBV DNA was positive in serum but negative in liver tissue (P < 0.01). The logarithmic value of HBV DNA from 13 patients in liver tissue and in serum was respectively: (6.62 +/- 1.21) copies/g vs.(4.03 +/- 1.06) copies/ml, P < 0.01. The hepatic lesions of all HBV DNA positive patients were active pathologic changes, but the level of HBV DNA in liver tissue was not significantly correlated with the grade of hepatic inflammation activity (P > 0.05). CONCLUSION: Occult HBV infection is the etiology of part of the chronic viral hepatitis patients with non-A-E hepatitis virus infection. Missed diagnosis will occur if diagnosis of hepatitis B is only based on detection of serum HBV markers. It is useful for improvement of the diagnostic level of HBV infection via detection of HBV DNA quantitatively in serum especially in liver tissue of chronic viral hepatitis patients with non-A-E hepatitis virus infection by using FQ-PCR technique. The chronic viral hepatitis patients with occult HBV infection should be also given effective anti-viral therapy.

Epidemiology and clinical pattern of hepatitis delta virus infection in Pakistan.

BACKGROUND AND AIMS: The global epidemiology of hepatitis delta virus (HDV) infection is changing. This study was performed to determine the epidemiology and clinical impact of hepatitis delta in Pakistan. METHODS: Countrywide data was collected from 1994 to 2001. A total of 8721 patients were tested for hepatitis delta antibody. A subset of 97 hepatitis delta antibody reactive inpatients with chronic liver disease were compared to 97 patients admitted with liver disease due to hepatitis B alone. RESULTS: Of the 8721 patients tested, 1444 (16.6%) were reactive for hepatitis delta antibody. Most were males (87.4%, P < 0.001) and younger (mean age 31 years, P < 0.001) compared to HDV non-reactive patients. Prevalence of delta infection was highest in the rural (range 25-60%) compared to the urban population (range 6.5-11%). Analysis of the inpatient data showed that delta infected patients had significantly less severe clinical liver disease and a trend towards lesser development of hepatocellular carcinoma compared to delta negative patients. CONCLUSIONS: (i) HDV infection is present in 16.6% of hepatitis B infected patients in Pakistan, most commonly in younger males living in rural areas; and (ii) delta virus infected patients have less severe clinical liver disease compared to delta negative, hepatitis B patients.

Animal models of hepatitis delta virus infection and disease.

Hepatitis delta virus (HDV) is a defective RNA virus with similarities to unusual subviral pathogens of higher plants. It requires hepatitis B virus (HBV) for its replication/transmission, and HBV-infected humans are the only established host. HDV causes both severe acute hepatitis and rapidly progressive chronic disease in some individuals. The HDV life cycle involves remarkable features, such as ribozyme- mediated autocatalytic processes, Pol II-directed RNA synthesis from a single-stranded circular RNA template, and RNA editing. Much of our understanding of the nature of this pathogen derives from experimental studies in the chimpanzee model of HBV infection. The hepadnavirus-infected eastern woodchuck also is capable of supporting HDV replication and offers opportunities for the development of control strategies that might be applicable to human type D hepatitis.

[Effect on the pregnant woman and fetus by multiple hepatitis virus infection].

OBJECTIVE: To evaluate the effect on the pregnant woman and fetus by infection of multiple hepatitis virus during pregnancy. METHODS: Hepatitis virus A, hepatitis virus B, hepatitis virus C, hepatitis virus D and hepatitis virus E were determined in the pregnant women with abnormal liver function during 1994-1999. Patients diagnosed to be infected by single hepatitis virus or multiple hepatitis virus were divided into two groups and complications of the pregnant woman and fetus and their prognosis were evaluated. RESULTS: There were no significant differences in the levels of alamine transaminase (ALT), aspartama transaminase (AST) and total bilirubin (TBIL) between the multiple hepatitis virus infection group (multiple group) and the single hepatitis virus infection group (single group) (P > 0.05). The positive rate of HbeAg (35.7%) in multiple group was significantly lower than in single group (P < 0.05). However, the positive rate of HbeAb (57.1%) in multiple group was significantly higher than in single group (P < 0.01). There were no significant differences in incidences of pregnancy induced hypertension (PIH), postpartum hemorrhage, serious symptoms and mortality between the multiple hepatitis virus infection group and the single hepatitis virus infection group (P > 0.05). However, the incidences of premature rupture of membrane (PROM), premature delivery, 28.1%, 25.0% fetal distress and newborn infant asphyxia 31.3%, 25.0% in multiple hepatitis virus infection group were significantly higher than in single hepatitis virus infection group (P < 0.05, P < 0.01). CONCLUSIONS: Multiple hepatitis virus infection during pregnancy has no more serious effect on the pregnant woman, but has worse effect on fetus than single hepatitis virus infection. The obstetrician should pay more attention to the health care of the pregnant woman with the multiple hepatitis virus infection to prevent PROM and premature delivery, at the same time monitor fetus carefully and deal with labor actively to decrease the mortality of the fetus.

Virologic and clinical expressions of reciprocal inhibitory effect of hepatitis B, C, and delta viruses in patients with chronic hepatitis.

We studied 648 hepatitis B surface antigen (HBsAg)- and/or anti-hepatitis C virus (HCV)-positive patients to evaluate the virologic and clinical characteristics of multiple hepatitis viral infection. We defined as Case B-C an HBsAg/anti-HCV positive patient and as Case b-C an anti-HCV/anti-HBc-positive, HBsAg/anti-HBs-negative patient. For each Case B-C we scheduled as Control-B an HBsAg positive and anti-HCV negative patient and as Control-C an HBsAg/anti-HBs/anti-hepatitis B core antigen (HBc)-negative and anti-HCV-positive patient. Control group C was used as the control also for Case group b-C. Serum HBV DNA by molecular hybridization was found more frequently in Control group B (54% of 161 patients) than in Case group B-C (35.7% of 84, P <.01). The prevalence of HBV wild type was similar in Case group B-C (14. 3%) and in Control group B (17.4%), whereas the e-minus strain was less frequent in Case group B-C (10.7% vs. 33%; P <.01). HBV DNA by polymerase chain reaction (PCR) was detected in 40.8% of 71 patients in Case group b-C. HCV RNA was detected more frequently in Control group C (90.7% of 130 patients) than in Case group B-C (65.2% of 69, P <.0001). Moderate or severe chronic hepatitis or cirrhosis were more frequent in Case group B-C (62.9% of 65 patients) than in Control group B (46.7% of 90, P <.05) or C (40.8% of 98, P <.005), and in Case group b-C (71.1% of 76) than in Control group C. Thus, in multiple hepatitis we observed a reciprocal inhibition of the viral genomes and a more severe liver disease. In Case group b-C, serum HBV DNA was frequent and the clinical presentation was severe.