Analysis of the in vitro replication phenotype of African hepatitis B virus (HBV) genotypes and subgenotypes present in Australia identifies marked differences in DNA and protein expression.

Hepatitis B virus infection in Africa is characterised by distinct genotypes with observed differences in natural history and clinical outcomes. Replication-competent cDNA clones of African genotypes were generated from patient-derived sequences identified in African children with chronic hepatitis B infection living in Australia: A1 (wild-type and basal core promotor (BCP) mutant), D2, D6, and E, comparing the replication phenotype to an established D3 cDNA clone in a transient transfection cell culture model. All clones replicated efficiently although less than the European D3 reference clone, and demonstrated marked differences in replication capacity, highest for subgenotypes A1 and D2. The BCP mutation increased the replication levels of the A1 subgenotype compared to wild-type. Intracellular and secreted surface antigen and HBeAg protein expression also varied across genotypes. We observed differences in functional activity in the upstream regulatory region across the genotypes that may contribute to the replication and protein differences observed.

HBeAg levels at week 24 predict response to 8 years of tenofovir in HBeAg-positive chronic hepatitis B patients.

BACKGROUND: Hepatitis B e antigen (HBeAg) seroconversion is a treatment endpoint for HBeAg-positive CHB, and a necessary precursor to HBsAg loss. Biomarkers that predict serological outcomes would be useful. AIM: To evaluate the utility of measuring HBeAg levels for predicting HBeAg seroconversion and HBsAg loss under long-term tenofovir (TDF) therapy. METHODS: A total of 266 patients were enrolled into a phase III study of TDF vs adefovir (ADV) for 48 weeks in HBeAg-positive patients, followed by open-label TDF up to 384 weeks. Serum HBeAg levels were measured for subjects with samples available at both baseline and week 24 of treatment (n = 200). Analysis compared subjects who achieved HBeAg seroconversion by week 384 vs no HBeAg seroconversion. RESULTS: HBeAg seroconversion rate was 52% by week 384. Time to HBeAg seroconversion was 80 weeks (IQR: 36-162). HBeAg decline at week 24 was associated with HBeAg seroconversion (1.63 vs 0.90 log10 PEIU/mL, P = .002). The optimal threshold for identifying HBeAg seroconversion was HBeAg decline ≥2.2 log10 PEIU/mL at week 24, with HBeAg seroconversion achieved by 76% of patients, compared to 44% if HBeAg decline <2.2 log10 (P < .0001). HBeAg decline ≥2.2 log10 PEIU/mL at week 24 was associated with HBsAg loss in genotype A or D patients (38% vs 15%, P = .03). Precore/basal core promotor variants were associated with lower baseline HBeAg levels, but not HBeAg seroconversion. CONCLUSION: Decline in HBeAg levels by week 24 was associated with HBeAg seroconversion and HBsAg loss in HBeAg-positive chronic hepatitis B patients treated with long-term TDF.

More than a virus: a qualitative study of the social implications of hepatitis B infection in China.

BACKGROUND: China has the largest absolute number of people living with hepatitis B with up to 300,000 people estimated to die each year from hepatitis B related diseases. Despite advances in immunisation, clinical management, and health policy, there is still a lack of accessible and affordable health care for people with hepatitis B. Through in-depth interviews, this study identifies the personal, social and economic impact of living with hepatitis B and considers the role of stigma and discrimination as barriers to effective clinical management of the disease. METHODS: Semi-structured qualitative interviews were held with 41 people living with hepatitis B in five Chinese cities. Participants were recruited through clinical and non-government organisations providing services to people with hepatitis B, with most (n = 32) being under the age of 35 years. RESULTS: People living with hepatitis B experience the disease as a transformative intergenerational chronic infection with multiple personal and social impacts. These include education and employment choices, economic opportunities, and the development of intimate relationships. While regulations reducing access to employment and education for people with hepatitis B have been repealed, stigma and discrimination continue to marginalise people with hepatitis B. CONCLUSIONS: Effective public policy to reduce morbidity and mortality associated with hepatitis B needs to address the lived impact of hepatitis B on families, employment and educational choices, finances, and social marginalisation.

Toll-IL1 receptor-mediated innate immune responses vary across HBV genotype and predict treatment response to pegylated-IFN in HBeAg-positive CHB patients.

Patients with hepatitis B e antigen (HBeAg)-positive chronic hepatitis B (CHB) have suppressed TLR2 expression, function and cytokine production. The aim of this study was to explore the importance of hepatitis B virus (HBV) genotype in innate immune responses and investigate whether Toll-like receptor (TLR) expression/function has potential roles as predictive biomarkers of successful therapy with pegylated interferon (Peg-IFN) therapy of HBeAg seroconversion in HBeAg-positive patients. We showed that as early as 4 weeks after initiation of Peg-IFN, future HBeAg seroconverters had significantly elevated levels of TLR2 expression on monocytes. TLR2-associated IL-6 production at baseline and week 4 of therapy and TLR4 IL-6 production at week 4 were also markedly elevated in HBeAg seroconverters. HBV genotype also influenced treatment response, with genotypes A and B more likely to seroconvert than D. We were able to demonstrate that these differences were due in part to the interaction of the specific HBeAg proteins with TLR pathway adaptor molecules, and these interactions were genotype dependent. HBeAg-mediated modulation of TLR signalling was also observed in Huh7 cells, following stimulation with Pam3Cys. Importantly, the addition of IFN-α to TLR2-stimulated cells cotransfected with an HBeAg expression plasmid reversed HBeAg-mediated suppression of hepatocytes. These findings demonstrate that patients with an activated inflammatory response are much more likely to respond to IFN therapy, with TLR responses showing promise as potential biomarkers of HBeAg seroconversion in this setting. Furthermore, our findings suggest there is differential genotype-specific HBeAg suppression of innate signalling pathways which may account for some of the clinical differences observed across the CHB spectrum.

Asian-Pacific clinical practice guidelines on the management of hepatitis B: a 2015 update.

Worldwide, some 240 million people have chronic hepatitis B virus (HBV), with the highest rates of infection in Africa and Asia. Our understanding of the natural history of HBV infection and the potential for therapy of the resultant disease is continuously improving. New data have become available since the previous APASL guidelines for management of HBV infection were published in 2012. The objective of this manuscript is to update the recommendations for the optimal management of chronic HBV infection. The 2015 guidelines were developed by a panel of Asian experts chosen by the APASL. The clinical practice guidelines are based on evidence from existing publications or, if evidence was unavailable, on the experts' personal experience and opinion after deliberations. Manuscripts and abstracts of important meetings published through January 2015 have been evaluated. This guideline covers the full spectrum of care of patients infected with hepatitis B, including new terminology, natural history, screening, vaccination, counseling, diagnosis, assessment of the stage of liver disease, the indications, timing, choice and duration of single or combination of antiviral drugs, screening for HCC, management in special situations like childhood, pregnancy, coinfections, renal impairment and pre- and post-liver transplant, and policy guidelines. However, areas of uncertainty still exist, and clinicians, patients, and public health authorities must therefore continue to make choices on the basis of the evolving evidence. The final clinical practice guidelines and recommendations are presented here, along with the relevant background information.

Downregulation of interleukin-18-mediated cell signaling and interferon gamma expression by the hepatitis B virus e antigen.

UNLABELLED: The mechanisms by which hepatitis B virus (HBV) establishes and maintains chronic hepatitis B infection (CHB) are poorly defined. Innate immune responses play an important role in reducing HBV replication and pathogenesis. HBV has developed numerous mechanisms to escape these responses, including the production of the secreted hepatitis B e antigen (HBeAg), which has been shown to regulate antiviral toll-like receptor (TLR) and interleukin-1 (IL-1) signaling. IL-18 is a related cytokine that inhibits HBV replication in hepatoma cell lines and in the liver through the induction of gamma interferon (IFN-γ) by NK cells and T cells. We hypothesized that HBV or HBV proteins inhibit IFN-γ expression by NK cells as an accessory immunomodulatory function. We show that HBeAg protein inhibits the NF-κB pathway and thereby downregulates NK cell IFN-γ expression. Additionally, IFN-γ expression was significantly inhibited by exposure to serum from individuals with HBeAg-positive but not HBeAg-negative chronic HBV infection. Further, we show that the HBeAg protein suppresses IL-18-mediated NF-κB signaling in NK and hepatoma cells via modulation of the NF-κB pathway. Together, these findings show that the HBeAg inhibits IL-18 signaling and IFN-γ expression, which may play an important role in the establishment and/or maintenance of persistent HBV infection. IMPORTANCE: It is becoming increasingly apparent that NK cells play a role in the establishment and/or maintenance of chronic hepatitis B infection. The secreted HBeAg is an important regulator of innate and adaptive immune responses. We now show that the HBeAg downregulates NK cell-mediated IFN-γ production and IL-18 signaling, which may contribute to the establishment of infection and/or viral persistence. Our findings build on previous studies showing that the HBeAg also suppresses the TLR and IL-1 signaling pathways, suggesting that this viral protein is a key regulator of antiviral innate immune responses.

A simple and inexpensive point-of-care test for hepatitis B surface antigen detection: serological and molecular evaluation.

Early identification of chronic hepatitis B is important for optimal disease management and prevention of transmission. Cost and lack of access to commercial hepatitis B surface antigen (HBsAg) immunoassays can compromise the effectiveness of HBV screening in resource-limited settings and among marginalized populations. High-quality point-of-care (POC) testing may improve HBV diagnosis in these situations. Currently available POC HBsAg assays are often limited in sensitivity. We evaluated the NanoSign(®) HBs POC chromatographic immunoassay for its ability to detect HBsAg of different genotypes and with substitutions in the 'a' determinant. Thirty-seven serum samples from patients with HBV infection, covering HBV genotypes A-G, were assessed for HBsAg titre with the Roche Elecsys HBsAg II quantification assay and with the POC assay. The POC assay reliably detected HBsAg at a concentration of at least 50 IU/mL for all genotypes, and at lower concentrations for some genotypes. Eight samples with substitutions in the HBV 'a' determinant were reliably detected after a 1/100 dilution. The POC strips were used to screen serum samples from 297 individuals at risk for HBV in local clinical settings (health fairs and outreach events) in parallel with commercial laboratory HBsAg testing (Quest Diagnostics EIA). POC testing was 73.7% sensitive and 97.8% specific for detection of HBsAg. Although the POC test demonstrated high sensitivity over a range of genotypes, false negatives were frequent in a clinical setting. Nevertheless, the POC assay offers advantages for testing in both developed and resource-limited countries due to its low cost (0.50$) and immediately available results.

Anti-viral therapy for prevention of perinatal HBV transmission: extending therapy beyond birth does not protect against post-partum flare.

BACKGROUND: Antepartum anti-viral therapy (AVT) is often administered to prevent perinatal transmission of hepatitis B virus (HBV) infection. Little is known about the effect of AVT on post-partum flare rates and severity. AIM: To examine whether extending AVT beyond birth influences the post-partum course. METHODS: One hundred and one pregnancies in 91 women with HBV DNA levels ≥log 7 IU/mL were included. AVT (initially lamivudine, later tenofovir disoproxil fumarate) was commenced from 32 weeks gestation and stopped soon after birth and at 12 weeks post-partum. Outcomes according to post-partum treatment duration were examined: Group 1 = AVT ≤4 weeks (n = 44), Group 2 = AVT >4 weeks (n = 43), Group 3 = no AVT (n = 14). RESULTS: The majority of women were HBeAg+ (97%), median age 29 years, baseline HBV DNA log 8.0 IU/mL and follow-up 48 weeks post-partum. Post-partum treatment duration was 2 weeks for Group 1 and 12 weeks for Group 2, P < 0.01. Flare rates were not significantly different: Group 1 = 22/44 (50%), Group 2 = 17/43 (40%) and Group 3 = 4/14 (29%), P = 0.32. Onset of flare was similar at 8/10/9 weeks post-partum for Groups 1/2/3 respectively, P = 0.34. The majority of flares spontaneously resolved. HBeAg seroconversion (n = 1/5/1 in Groups 1/2/3, P = 0.27) was not associated with treatment duration or the occurrence of a post-partum flare. CONCLUSIONS: Post-partum flares are common and usually arise early after delivery. They are often mild in severity and most spontaneously resolve. Extending anti-viral therapy does not protect against post-partum flares or affect HBeAg seroconversion rates.

Molecular virology of hepatitis B virus, sub-genotype C4 in northern Australian Indigenous populations.

Indigenous Australians experience a significant health burden from chronic hepatitis B infection; however, the strain of hepatitis B virus (HBV) found among Indigenous Australians has not been well characterized. Blood samples were collected from 65 Indigenous Australians with chronic HBV infection from across the Top End of Australia's Northern Territory. Phylogenetic analysis of HBV from these samples revealed that 100% of the isolates were genotype C, sub-genotype C4, expressing the serotype ayw3. This strain is a divergent group within the HBV/C genotype, and has only been described in Indigenous Australians. Evidence of recombination was suggested by discordant phylogenetic clustering of the C4 sequences when comparing the full genome to the surface region and confirmed by recombination analysis which showed the surface gene region to be most closely related to genotype J, while the remaining regions of the genome were most similar to genotype C sequences. Mutational analysis revealed the presence of multiple mutations that have been linked with more rapid liver disease progression and an increased risk of hepatocellular carcinoma. These mutations were detected in the majority of sequences examined. Variants associated with vaccine failure were detected as the predominant viral quasi-species in 3/35 samples. In summary, the HBV C4 variant found in this population has a high potential to cause advanced liver disease and to escape vaccination programs. Further in vitro functional and natural history studies are warranted in order to determine the clinical and public health consequences of infection with the HBV C4 variant in these communities.

Short duration of lamivudine for the prevention of hepatitis B virus transmission in pregnancy: lack of potency and selection of resistance mutations.

This study sought to assess the antiviral efficacy of lamivudine (LMV) administered during third trimester to reduce maternal viraemia and to identify the emergence of LMV resistance. A prospective observational analysis was performed on 26 mothers with high viral load (>107 IU/mL). Twenty-one women received LMV (treated group) for an average of 53 days (range 22-88 days), and the remaining five formed the untreated control group. Serum samples from two time points were used to measure HBV DNA levels and antiviral drug resistance. The LMV-treated women achieved a median HBV DNA reduction of 2.6-log10 IU/mL. Although end-of-treatment (EOT) HBV DNA in four (18%) LMV-treated women remained at >10(7) IU/mL (± 0.5 log IU/mL), no mother-to-baby transmission was observed. In contrast, a baby from the untreated mother was HBsAg positive at 9 months postpartum. Four technologies were used for drug resistance testing. Only ultra-deep pyrosequencing (UDPS) was sufficiently sensitive to detect minor viral variants down to <1%. UDPS showed that LMV therapy resulted in increased viral quasispecies diversity and positive selection of HBV variants with reverse transcriptase amino acid substitutions at sites associated with primary LMV resistance (rtM204I/V and rtA181T) in four (19%) women. These viral variants were detected mostly at low frequencies (0.63-5.92%) at EOT, but one LMV-treated mother had an rtA181T variant that increased from 2.2% pretherapy to 25.59% at EOT. This mother was also infected with the vaccine escape variant (sG145R), which was inhibited by LMV treatment. LMV therapy during late pregnancy only reduced maternal viraemia moderately, and drug-resistant viral variants emerged.

Assessment of chronic hepatitis B: the importance of hepatitis B virus DNA testing.

BACKGROUND: Chronic hepatitis B (CHB) has an estimated prevalence of 90 000 to 160 000 in Australia. Cirrhosis and hepatocellular carcinoma are important complications of CHB and appropriate evaluation of hepatitis B surface antigen (HBsAg)-positive individuals is vital to identify treatment candidates. METHODS: A review of the database of a tertiary hospital was performed and 348 HBsAg-positive individuals with baseline demographic, virological, serological and biochemical variables were identified and evaluated cross-sectionally. A small subgroup of hepatitis B e antigen (HBeAg)-negative patients with normal alanine aminotransferase (ALT) at baseline were identified and followed longitudinally. RESULTS: 175/348 (50%) of patients were in the HBeAg-negative, chronic hepatitis phase of disease, 22% in the HBeAg-positive immune clearance and 6% in the immune tolerant phases. HBeAg-negative patients were older and more likely to be male than HBeAg-positive patients. The correlation between hepatitis B virus (HBV) DNA and ALT levels was examined. ALT and HBV DNA levels showed no correlation in HBeAg-positive CHB and only a weak correlation in HBeAg-negative patients. Furthermore, 35% of HBeAg-negative patients with detectable HBV DNA had a normal ALT. Conversely 38% of HBeAg-negative patients with no detectable HBV DNA had an elevated ALT. A persistently normal ALT over 24 months was seen in five of nine HBeAg-negative patients with normal initial ALT and detectable HBV DNA. CONCLUSION: Appropriate evaluation of HBeAg-negative CHB must include HBV DNA because the ALT is not a reliable guide to underlying viral replication.

The hepatitis B e antigen suppresses IL-1ß-mediated NF-κB activation in hepatocytes.

Previous clinical studies have demonstrated an association between the hepatitis B e antigen and Toll-like receptor (TLR) expression and signalling. Therefore, the aim of this study was to develop an in vitro assay to measure the effect of hepatitis B virus proteins, including the precore protein, on signalling mediated by members of the Toll-like/interleukin 1 (TIR) superfamily, by measuring NF-κB promoter activity. The basal level of NF-κB reporter activity was measured in three hepatocyte cell lines (Huh7, HepG2 and PH5CH8) and one kidney cell line (HEK293) using a luciferase assay. All cell lines were virtually refractory to stimulation with lipopolysaccharide; however, PH5CH8 cells had a robust activation of NF-κB in response to IL-1ß stimulation, with ∼ 40-fold higher activation than the unstimulated control, a higher degree of activation than that observed in either Huh7 and HepG2, or HEK293 and HEK293-TLR2 cells. In PH5CH8 cells transfected with pCI expression constructs and stimulated with IL-1ß, we showed that the precursor form of the precore protein, p25, inhibits NF-κB activation by up to 30% and the cytosolic form, p22, inhibits NF-κB activation by 70%. The core protein, p21, which shares significant homology with the precore protein except for a 10-amino acid extension at the N-terminus, had no effect on NF-κB activation. We hypothesize that the inhibition of IL-1ß-mediated NF-κB activation by the precore protein may be a mechanism that allows the virus to persist, suggesting a role for the pool of precore protein that remains intracellular.

Tenofovir disoproxil fumarate rescue therapy following failure of both lamivudine and adefovir dipivoxil in chronic hepatitis B.

OBJECTIVE: To determine the efficacy of tenofovir disoproxil fumarate (TDF) in adults with chronic hepatitis B virus (HBV) infection who had previously failed lamivudine (LAM) and had significant viral replication (HBV DNA >105 copies/ml if HBeAg positive, > 104 copies/ml if HBeAg negative) despite at least 24 weeks of treatment with adefovir dipivoxil (ADV). DESIGN: A prospective open-label study of TDF 300 mg daily. Patients receiving combination ADV/LAM prior to baseline were switched to TDF/LAM. SETTING: Multiple tertiary referral centres. METHODS: Sixty patients were enrolled. The median age was 48.5 years (range 21e80), 46 (77%) were male and 40 (67%) were HBeAg positive. Thirty-eight patients (63%) were switched from ADV to TDF, the remainder from ADV/LAM to TDF/LAM. At baseline, substitutions conferring resistance to LAM or ADV were present in 20 patients (33%) and 17 patients (28%), respectively. The median baseline viral load was 5.33 log10 IU/ml (range 2.81-8.04). Patients initially treated with TDF monotherapy with persistent viral replication at or after 24 weeks were switched to TDF/LAM. The main outcome measures were change in HBV viral load from baseline and percentage of patients achieving an undetectable viral load (<15 IU/ml). RESULTS: Results are reported at 96 weeks of treatment. One patient discontinued TDF at 10 days due to rash. The time-weighted change in viral load from baseline to week 12 was -2.19 log10 IU/ml overall. The median change in HBV DNA from baseline to weeks 12, 24, 48 and 96 was -2.86, -3.23, -3.75 and -4.03 log10 IU/ml, respectively. At 48 and 96 weeks, 27/59 (46%) and 38/59 (64%) patients achieved a HBV DNA <15 IU/ml. The response was independent of baseline LAM therapy or mutations conferring ADV resistance. CONCLUSIONS: In heavily pretreated patients with a high rate of genotypic resistance, TDF retains significant activity against HBV although this appears diminished in comparison with studies of naïve patients.

High frequency of lamivudine resistance mutations in Brazilian patients co-infected with HIV and hepatitis B.

This study analyzed the genotype distribution and frequency of lamivudine (LAM) and tenofovir (TDF) resistance mutations in a group of patients co-infected with HIV and hepatitis B virus (HBV). A cross-sectional study of 847 patients with HIV was conducted. Patients provided blood samples for HBsAg detection. The load of HBV was determined using an "in-house" real-time polymerase chain reaction. HBV genotypes/subgenotypes, antiviral resistance, basal core promoter (BCP), and precore mutations were detected by DNA sequencing. Twenty-eight patients with co-infection were identified. The distribution of HBV genotypes among these patients was A (n = 9; 50%), D (n = 4; 22.2%), G (n = 3; 16.7%), and F (n = 2; 11.1%). Eighteen patients were treated with LAM and six patients were treated with LAM plus TDF. The length of exposure to LAM and TDF varied from 4 to 216 months. LAM resistance substitutions (rtL180M + rtM204V) were detected in 10 (50%) of the 20 patients with viremia. This pattern and an accompanying rtV173L mutation was found in four patients. Three patients with the triple polymerase substitution pattern (rtV173L + rtL180M + rtM204V) had associated changes in the envelope gene (sE164D + sI195M). Mutations in the BCP region (A1762T, G1764A) and in the precore region (G1896A, G1899A) were also found. No putative TDF resistance substitution was detected. The data suggest that prolonged LAM use is associated with the emergence of particular changes in the HBV genome, including substitutions that may elicit a vaccine escape phenotype. No putative TDF resistance change was detected after prolonged use of TDF.

Prolonged use of tenofovir in HIV/hepatitis B virus (HBV)-coinfected individuals does not lead to HBV polymerase mutations and is associated with persistence of lamivudine HBV polymerase mutations.

OBJECTIVES: The aim of the study was to identify and characterize hepatitis B virus (HBV) polymerase gene mutations associated with ongoing HBV replication in HIV/HBV-coinfected individuals receiving tenofovir (TDF). METHODS: This retrospective cross-sectional study identified 28 HIV/HBV-coinfected individuals who had received TDF for at least 3 months. All patients had samples available while receiving TDF (on-TDF), and 24 also had samples available prior to treatment (pre-TDF). Case records were reviewed to obtain clinical and virological data at the times of sampling (+/-3 months). The HBV DNA of all samples was amplified using polymerase chain reaction (PCR), and the polymerase region of PCR-positive samples was sequenced and compared with reference HBV data. RESULTS: Of the pre-TDF samples, 15 of 24 (63%) were HBV PCR positive. Of the on-TDF samples, four of 28 (14%) were HBV PCR positive (mean time on TDF 13.5 months; range 3-23 months). Lamivudine (3TC)-resistance mutations were detected in three of four (75%) of these viraemic samples. The previously identified putative TDF-resistance mutations, rtA194T+rtL180M+rtM204V, were not detected in any individual. CONCLUSIONS: Unique mutations in the HBV polymerase gene associated with TDF resistance are rare in HIV/HBV coinfection. 3TC-resistance mutations persist and a significant proportion of patients are HBV PCR positive despite the addition of TDF.

Characterization of the innate immune signalling pathways in hepatocyte cell lines.

Hepatitis B virus (HBV) infection is a major cause of liver-related morbidity and mortality. Toll-like receptors (TLRs) have recently been recognized to play an important role in the pathogenesis of chronic hepatitis B (CH-B). Furthermore, manipulation of TLR signalling pathways shows potential as an antiviral therapeutic strategy. Whether hepatocytes themselves possess intact TLR signalling pathways remains controversial. It is critical that cell culture models be developed to allow investigation of the interaction between HBV and the TLR signalling pathways. We have screened three hepatocyte cell lines for the integrity of pro-inflammatory responses and antiviral cytokines following stimulation with interleukin-1 (IL-1) and different TLR ligands. We observed that Huh-7, HepG2 and PH5CH8 cells selectively responded to IL-1 and TLR2 ligands, leading to the activation of NF-kappaB. In addition, the PH5CH8 cell lines were able to induce type 1 interferon (IFN) via both TLR3 and RIG-I following stimulation with poly I:C, HepG2 cells mounted an IFN response via RIG-I only, whereas Huh-7 cells were unresponsive. We conclude that the hepatocyte cell lines investigated display a repertoire of TLR signalling, albeit limited, suggesting that hepatocytes may themselves play an active role in innate immune responses to viruses such as HBV. Furthermore, particular hepatoma cell lines are suitable for investigating the interaction between HBV and hepatocyte-expressed pattern recognition receptors.

Clinical significance of precore and core promoter mutations in genotype D hepatitis B-related chronic liver disease.

SUMMARY: The impact of mutations in the precore and basal core promoter (BCP) regions of the hepatitis B virus on the course of chronic liver disease is not well established. We sought to examine the relationship of these characteristics to the clinical expression of liver disease in patients infected with genotype D chronic hepatitis B (CHB). BCP and precore mutations in 110 patients with genotype D1 CHB were determined and correlated with clinical phenotype. Of 110 patients, 95 (86.5%) were HBeAg-negative. Compared with HBeAg-positive subjects, HBeAg-negative patients were over a decade older and had lower viral loads (3.70 +/- 0.98 vs 5.77 +/- 0.69 log copies/ml, P < 0.001). The double mutation A1762T-G1764A was more prevalent in patients with advanced liver disease (AdLD) and was associated with higher alanine aminotransferase and viral load. After adjusting for age, there was a more than fourfold increase in the risk of AdLD with this mutation (OR = 4.4; 95% CI: 1.13-16.92, P < 0.03). Conversely, the G1757A substitution was associated with protection, being 90% less frequent among patients with AdLD (P = 0.001). The results indicate that in genotype D CHB, the presence of the A1762T-G1764A mutation was associated with more aggressive liver disease while the G1757A substitution was associated with protection from advanced disease.

The phenylpropenamide derivative AT-130 blocks HBV replication at the level of viral RNA packaging.

Nucleos(t)ide analogue antiviral therapy for chronic hepatitis B has proven to be effective in the short term but the frequent development of resistance limits its clinical utility. Agents targeting other stages of viral replication are needed in order to develop improved combination therapies. The phenylpropenamide derivatives AT-61 and AT-130 have been shown to inhibit HBV replication in vitro, but the mechanism of action of these compounds remains undefined. The aim of this study was to determine the mechanism of action of AT-130, a non-nucleoside inhibitor of HBV in several in vitro models of replication. These studies found that AT-130 inhibited HBV DNA replication in hepatoma cells but had no effect on viral DNA polymerase activity or core protein translation. Total HBV RNA production was also unaffected in the presence of the drug whilst the amount of encapsidated RNA was significantly reduced, thereby inhibiting subsequent viral reverse transcription. These studies have established that the inhibition of HBV genome replication by a non-nucleoside analogue acting at the level of viral encapsidation and packaging is a potentially useful strategy for future therapeutic drug development in the management of chronic hepatitis B.

Toll-like receptor expression in chronic hepatitis C: correlation with pro-inflammatory cytokine levels and liver injury.

BACKGROUND/AIMS: Toll-like receptors (TLR's) are critical receptors that promote innate immune responses to pathogen-associated molecular patterns. Activation of TLR's leads to production of pro-inflammatory cytokines such as tumour necrosis factor (TNF)-alpha. This study investigates whether peripheral blood monocyte expression of TLR's is disturbed in patients with chronic hepatitis C and whether levels of expression of these molecules are significantly correlated with hepatitis C virus (HCV) genotype, viral load, hepatic necroinflammatory activity, histological stage and circulating TNF-alpha concentrations. METHODS: In 18 non-cirrhotic patients with biopsy-proven, virologically-confirmed chronic hepatitis C and 32 controls, we measured expression of TLR2 and TLR4 on peripheral blood monocytes. HCV genotype, viral load, serum alanine aminotransferase (ALT) levels, histological stage of disease and circulating TNF-alpha and endotoxin levels were also determined. RESULTS: Peripheral blood monocyte expression of TLR2 and TLR4 were significantly increased in patients with chronic hepatitis C compared to controls, irrespective of HCV genotype or histological stage of disease. Circulating levels of TNF-alpha were also significantly increased in patients with chronic hepatitis C. In both the overall study cohort and patients with chronic hepatitis C, monocyte expression of TLR2, but not of TLR4, correlated significantly with serum TNF-alpha levels. In patients with chronic hepatitis C, monocyte expression of TLR2, but not of TLR4, also correlated significantly with serum ALT levels. Expression of TLR's was not significantly correlated with viral load. CONCLUSIONS: Up-regulation of peripheral blood monocyte expression of TLR2 and TLR4 occurs in patients with chronic hepatitis C. Increased monocyte expression of TLR2, but not of TLR4, correlates significantly with both increased circulating TNF-alpha levels and hepatic necroinflammatory activity in this disorder.

Hepatitis B virus mutations associated with antiviral therapy.

The hepatitis B virus (HBV) replicates via an error prone viral reverse transcriptase resulting in a large pool of quasispecies with mutations spread throughout the genome. During antiviral drug selection pressure (e.g., lamivudine, adefovir, or entecavir) HBV mutants are selected from the pre-existing pool of quasispecies and over time become the dominant species. Not all mutations result in replication competent virus as HBV has the added complexity of overlapping reading frames. The HBV polymerase (Pol) gene overlaps the hepatitis B surface antigen (HBsAg) in a frame-shifted manner with the result that drug-resistant mutations in the HBV Pol can directly impact on the nature of HBsAg and its function. HBV genomic databases have been established to monitor antiviral selected mutations and are useful in determining conserved residues, genotypic differences, polymorphisms, and the mutation profiles selected under different antiviral selection pressures. These HBV databases may aid in the development of new diagnostic reagents as well as the monitoring of polymerase and envelope mutations selected under different antiviral pressures. Antiviral drug resistant mutants emerge as a function of at least six factors: the viral mutation frequency, the intrinsic mutability of the antiviral target site, the selective pressure exerted by the drug, the magnitude and rate of virus replication, the overall replication fitness of the mutant, and the availability of replication space. Only a limited number of HBsAg mutations selected during antiviral treatment have been characterized and the diagnostic and public health implications of these mutations need further investigation. Clearly, improved treatment strategies are required urgently to prevent the continued selection of HBV drug-resistant mutants.