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The specific drugs available for chronic hepatitis B infection include standard and pegylated IFN-α, and nucleoside/nucleotide analogs that directly inhibit the reverse transcriptase. The main goal of current hepatitis B virus therapy is to achieve sustained suppression of viral replication to prevent the development of chronic liver disease, but favorable long-term tolerability and resistance profiles are also desirable. This article reviews the chemistry and the mechanisms of action of tenofovir disoproxil fumarate, but it also focuses on the related clinical trails designed to date, in which clinical efficacy has been analyzed attending to HBe antigen status. In addition, studies including patients that have been previously treated with lamivudine or adefovir are discussed.

SUMMARY. Epidemiological, viral and host factors are associated with the outcome of hepatitis C virus (HCV) infection, and strong host immune responses against HCV favour viral clearance. Recently, genome-wide association studies have shown a strong correlation between single-nucleotide polymorphisms (SNPs) near the interleukin-28B (IL28B) gene and spontaneous or treatment-induced HCV clearance. We have investigated whether protective IL28B genetic variants are associated with HCV-specific T-cell responses among Spanish blood donors. The rs12979860 IL28B haplotype was determined in 69 anti-HCV-positive blood donors (21 HCV RNA negative and 48 HCV RNA positive) and 30 seronegative donors. In all cases, HCV-specific CD4 + T-cell responses to HCV recombinant proteins (core, NS3 and NS3 helicase) were assessed by ex vivo interferon-c ELISpot assay. The rs12979860-CC genotype was highly overrepresented in donors with spontaneous HCV clearance when compared to those with chronic infection (76.2% vs 29.2%, P < 0.001; odds ratio, 7.77; 95% confidence interval, 2.4–25.3, P < 0.001). HCV-specific CD4 + T-cell responses were detected in 16 (76.2%) spontaneous resolvers especially towards nonstructural proteins, but with no correlation with IL28B genotype. Chronic individuals had a significantly lower overall T-cell response again irrespective of IL28B genotype. When spontaneous resolvers and chronic individuals were stratified according to their IL28B genotype, significantly stronger T-cell responses were only observed among those with non-CC haplotypes. Although the protective rs12979860 IL28B CC genotype is associated with spontaneous HCV clearance, stronger CD4 + T-cell responses towards NS3 were only evident among those with non-CC haplotypes.

Long-term changes in the frequency and outcome of hepatitis delta virus (HDV) infection have seldom been analysed. This retrospective, longitudinal study includes 398 consecutive hepatitis B surface antigen (HBsAg)-positive patients with anti-HDV antibodies who attended our institution between 1983 and 2008. At enrolment, 182 patients had acute and 216 chronic hepatitis. Patients were grouped into two periods. Those who attended between 1983 and 1995 and those between 1996 and 2008. The former group was significantly younger, mainly intravenous drugs users, and had a greater incidence of acute HDV and HIV and HCV coinfection. Patients with acute HBV/HDV coinfection cleared both infections in 90% of cases, while all patients with HDV superinfection evolved to chronic disease. One hundred and fifty-eight patients with chronic HDV were followed for a median period of 158months. Seventy-two per cent of the patients remained stable, 18% had hepatic decompensation, 3% developed hepatocellular carcinoma, and 8% cleared HBsAg. Liver-related death was observed in 13% of patients and mainly occurred in patients from the first period (P=0.012). These results indicate an outbreak of HDV at the end of the 1980s and the beginning of the 1990s, with a large number of acute HDV cases affecting predominately young, male intravenous drug users. Currently, patients with chronic HDV disease are older, and factors associated with worse prognosis include the presence of cirrhosis and age at the time of diagnosis.

Aliment Pharmacol Ther 2010; 32: 97–104 Summary Background Some patients continue to have detectable HBV-DNA levels with liver disease progression after hepatitis B e antigen (HBeAg) loss. It is important to identify these patients, candidates for long-term treatment. Aims To evaluate hepatitis B virus (HBV) genotype and the main mutations in the basic core promoter (BCP, A1762T/G1764A) and precore (G1896A) sequences as markers of persistent HBV-DNA after HBeAg loss. Methods We analysed 60 serum samples from 20 Caucasian, HBeAg-positive, chronic hepatitis B patients, who lost HBeAg and were followed-up longitudinally. HBV genotype and precore and BCP mutations were determined before, at the time of, and after HBeAg loss. Results After HBeAg loss, eight (40%) patients continued to have undetectable HBV-DNA and 12 (60%) had persistent HBV-DNA (median level 4.7 log10 copies/mL). The presence of BCP mutations prior to therapy was the only variable associated with persistently detectable viraemia (P = 0.017). Four patients with genotype A and no mutations in the BCP region experienced hepatitis B surface antigen (HBsAg) loss after a mean period of 35 months from baseline. Conclusions Main BCP mutations in HBeAg-positive patients are useful markers to identify patients who will not have sustained virological suppression after HBeAg loss and therapy discontinuation and could benefit from long-term treatment.

Background/Aims The frequency of mixed hepatitis B virus (HBV) genotypes in chronic HBV (CHB) and genotype changes during natural disease evolution and as a result of antiviral therapy were investigated. Methods Serum samples from 103 CHB patients were included in a cross-sectional study. Longitudinal study of HBV genotypes was performed in 22 patients, 17 of them under antiviral therapy (lamivudine and/or adefovir). HBV genotyping was done by the INNO-LiPA HBV assay. Results Genotypes observed in the cross-sectional study: A 32% of cases, D 42%, C 2%, F 2%, and mixed genotypes 22% (mainly A/D, followed by A/G). Genotype G was found in 7% of patients, always combined with other genotypes. In the longitudinal study, genotype changes were observed only in treated patients (9 cases). Genotype A strains were positively selected in 6 of them, mainly as mixed A/D. In 6 patients, selection coincided with a decrease in HBV-DNA levels. Conclusions A high frequency of mixed HBV genotypes was observed in our setting. Selection of genotype A strains during treatment is likely an indication that sensitivity to therapy differs between genotypes A and D. The absence of changes in untreated patients suggests that HBV genotype is stable without external factors.

SUMMARY. Hepatitis delta is considered the most severe form of viral hepatitis, but variables associated with disease progression are poorly defined. This study aimed to identify risk factors associated with worse clinical outcome in patients with hepatitis delta and to develop a clinical score to determine their risk of experiencing liverrelated morbidity or mortality. We followed 75 HBsAg– anti-HDV-positive patients with hepatitis delta for up to 16 years (median 5 years). The baseline-event-anticipation score (BEA score) was developed based on variables associated with the development of liver-related clinical complications. Age, region of origin, presence of cirrhosis, albumin, INR, hyperbilirubinemia and thrombocytopenia were all associated with the development of an event in the training cohort. The BEA score included age, sex, region of origin, bilirubin, platelets and INR. Points were allocated according to hazard ratios, and three risk groups were defined: BEA-A mild risk, BEA-B moderate risk and BEA-C high risk. Hazard ratios of BEA-B and BEA-C patients for liver-related clinical endpoints were 9.01 and 25.27 vs BEA-A with an area under curve of the receiving operating characteristic curve of 0.88. The accuracy of the BEA score was confirmed in two independent validation cohorts followed in Barcelona (n = 77) and D€ (n = 62). Delta hepatitis is associated with a very severe long-term outcome. The BEA score is easy to apply and predicts with a very high accuracy the development of liver-related complications in patients with hepatitis delta.

Background & Aims This study presents a real-time reverse-transcription PCR (rt-RT-PCR) assay for hepatitis delta virus (HDV) RNA quantification, designed to clarify the interplay between HDV and hepatitis B virus (HBV) in chronic infection. Methods Serum HDV-RNA and HBV-DNA were analysed by rt-RT-PCR in a cross-sectional study of 37 untreated chronic HDV patients, 25 of whom were also longitudinally studied. Results In the cross-sectional study, both viruses were active in 15 (40.5%) patients and inactive in 4 (10.8%); HDV alone was active in 12 (32.4%) and HBV in 6 (16.2%). The longitudinal study showed seven replication profiles, with considerable fluctuating activity of one or both viruses, including alternating predominance. In 20% of cases, longitudinal HBV/HDV viral loads differed from cross-sectional results, indicating a risk of misinterpreting HBV/HDV interactions when assessing a single determination. Fluctuating HBV replication only increased in the presence of fluctuating HDV activity. HBsAg levels, stable in HBV single infection, fluctuated in HDV chronic infection. The results of both the cross-sectional and longitudinal study call into question the major suppressor effect of HDV over HBV, revealing an important role of HBV. Conclusions Longitudinal evaluation of viremia shows a complex interaction between HBV and HDV and is essential to understand the pathophysiology of chronic HDV infection.

Backgrounds/Aims Hepatitis B virus (HBV) reactivation following treatment with rituximab has been reported in patients with either HBsAg-positive, or HBsAg-negative and anti-HBc positive infection. Patients with severe reactivation often have a fatal outcome despite treatment with lamivudine. The use of entecavir has not been reported in patients with severe HBV reactivation. Methods We present a case of a HBsAg-negative patient diagnosed with chronic lymphocytic leukemia who received a chemotherapeutic regimen that included rituximab, who subsequently presented with severe HBV reactivation with ascites, jaundice and coagulopathy and was treated with entecavir. A review of the literature and underlying HBV associated mutations are discussed. Results Entecavir produced a rapid and sustained suppression of HBV that was associated with rapid clinical improvement without any side effects. Conclusion Entecavir is an efficacious and safe treatment for severe HBV reactivation.

Methods: HepaRG and PHH were either infected with HBV or transfected with either purified viral nucleocapsid or recombinant HBc protein. Moreover engineered HepaRG cell lines, which express HBV proteins, were also used to analyse the role of individual proteins. Ligands of PRRs were used to engage innate receptor and analyse the inhibitory effect of HBV proteins. HBV replication was analysed with standard procedures, whereas the effect of viral proteins on the expression of various interferons (a, b, l. . . ), interferon-induced (ISG) and pro-inflammatory cytokines genes was analysed by RT-qPCR, WB and ELISA. ChIP experiments were also performed to analyse the binding of HBc to target promoters as well as to analyse the recruitment of epigenome-modifying enzymes to target promoters. Results: HBV is capable to inhibit dsRNA-mediated interferon response after only few hours of infection. This inhibition occurs also with Dane-depleted and UV-inactivated virus suggesting that neo-synthesis of HBV protein is not mandatory. Using engineered cell lines we demonstrate that HBc is responsible for this very early inhibition. The transfection of purified nucleocapsid or recombinant HBc leads to the same inhibitory phenotype. HBc needs to be located in the nucleus to inhibit the transcription of targeted genes (i.e. IFNs, ISG), as inhibition of its trafficking, revert the phenotype. ChIP analyses with anti-HBc revealed that HBc is capable to bind to target promoters and to recruit epigenome-modifying enzymes to establish a negative mark on target promoters. Conclusion: Our results demonstrate that HBc is responsible for the very early inhibition of IFN response by HBV. The precocity of this inhibition is instrumental for the establishment of a persistent infection in vitro and is possible because HBc from incoming virions is capable to inhibit gene expression in the nucleus of infected cells by either direct interference with transcriptional machinery or by recruiting epigenome-modifying enzymes leading to repressive marks.

Serious adverse events (SAEs) 33 (32.4%) Premature discontinuation 33 (32.4%) Due to SAEs 10 (9.8%) Discontinuing patient care 3 (2.9%) Virological failure 20 (10.6%) Death Septic shock, Multi-organ failure secondary to pneumonia 2 (1.96%) Dose modification (PegIFN) 8 (7.8%) Infection Grade 3–4 5 (4.9%) Hepatic decompensation (Grade 3/4) 4 (3.9%) Anaemia Hg

1107 PRIME/BOOST IMMUNIZATION WITH DNA AND ADENOVIRAL VECTORS PROTECTS WOODCHUCKS FROM HEPATITIS D VIRUS INFECTION M. Fiedler, A. Schumann, A. Kosinska, M. Lu, L. Johrden, O. Wildner, M. Roggendorf. Institut fur Virologie, Abt. fur Klinische Chemie, Universitatsklinikum Essen, Essen, Institut fur Virologie, Universitat Bochum, Bochum, Paul-Ehrlich-Institut, Langen, Germany E-mail: melanie.fiedler@uni-due.de