Hepatitis B Virus X Protein Is Stabilized by the Deubiquitinating Enzyme VCPIP1 in a Ubiquitin-Independent Manner by Recruiting the 26S Proteasome Subunit PSMC3.

Hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) is the sixth most common cancer worldwide, and the viral X protein (HBx) is an etiological factor in HCC development. HBx is a high-turnover protein, but knowledge of the role of deubiquitinating enzymes (DUBs) in maintaining HBx homeostasis is very limited. We used a 74-DUB library-based yeast two-hybrid assay and determined that a novel DUB, valosin-containing protein-interacting protein 1 (VCPIP1), interacted with HBx. VCPIP1 and its C-terminal amino acids 863 to 1221 upregulated the HBx protein expression, with or without HBV infection. Mechanistically, VCPIP1 stabilized HBx protein through a ubiquitin-independent pathway, which was validated by the HBx ubiquitination site mutant plasmid. Coimmunoprecipitation assays demonstrated the potency of VCPIP1 in recruiting 26S proteasome regulatory subunit 6A (PSMC3) and forming a ternary complex with HBx through mutual interaction. In vitro, purified His-tagged PSMC3 protein rescued HBx degradation induced by the 20S proteasome, and in vivo VCPIP1 synergized the mechanism. Functionally, HBx specifically binding to VCPIP1 significantly enhanced the transcriptional transactivation of HBx by activating NF-κB, AP-1, and SP-1 and inhibited hepatoma cell clonogenicity in Huh7 and HepG2 cells. Moreover, we further demonstrated that overexpression of VCPIP1 significantly affected the HBV covalently closed circular DNA (cccDNA) transcription in HBV-infected HepG2-NTCP cells. Altogether, our results indicate a novel mechanism by which VCPIP1 recruits PSMC3 to bind with HBx, stabilizing it in a ubiquitin-independent manner, which might be critical for developing DUB inhibitors in the future. IMPORTANCE HBx is a multifunctional viral oncoprotein that plays an essential role in the viral life cycle and hepatocarcinogenesis. HBx degradation occurs through the ubiquitin-proteasome system (UPS). However, whether novel compartments of the DUBs in the UPS also act in regulating HBx stability is not fully understood. Here, for the first time, we defined VCPIP1 as a novel DUB for preventing HBx degradation by the 20S proteasome in a ubiquitin-independent manner. PSMC3, encoding the 26S proteasome regulatory subunit, directly stabilized HBx through physical binding instead of a common approach in protein degradation, serving as the key downstream effector of VCPIP1 on HBx. Therefore, the ternary binding pattern between VCPIP1, HBx, and PSMC3 is initiated for the first time, which eventually promotes HBx stability and its functions. Our findings provide novel insights into host-virus cross talk by targeting DUBs in the UPS.

Comparative performance of risk prediction models for hepatitis B-related hepatocellular carcinoma in the United States.

BACKGROUND & AIMS: Guidelines recommend hepatocellular carcinoma (HCC) surveillance in patients with chronic HBV infection. Several HCC risk prediction models are available to guide surveillance decisions, but their comparative performance remains unclear. METHODS: Using a retrospective cohort of patients with HBV treated with nucleos(t)ide analogues at 130 Veterans Administration facilities between 9/1/2008 and 12/31/2018, we calculated risk scores from 10 HCC risk prediction models (REACH-B, PAGE-B, m-PAGE-B, CU-HCC, HCC-RESCUE, CAMD, APA-B, REAL-B, AASL-HCC, RWS-HCC). We estimated the models' discrimination and calibration. We calculated HCC incidence in risk categories defined by the reported cut-offs for all models. RESULTS: Of 3,101 patients with HBV (32.2% with cirrhosis), 47.0% were treated with entecavir, 40.6% tenofovir, and 12.4% received both. During a median follow-up of 4.5 years, 113 patients developed HCC at an incidence of 0.75/100 person-years. AUC values for 3-year HCC risk were the highest for RWS-HCC, APA-B, REAL-B, and AASL-HCC (all >0.80). Of these, 3 (APA-B, RWS-HCC, REAL-B) incorporated alpha-fetoprotein. AUC values for the other models ranged from 0.73 for PAGE-B to 0.79 for CAMD and HCC-RESCUE. Of the 7 models with AUC >0.75, only APA-B was poorly calibrated. In total, 10-20% of the cohort was deemed low-risk based on the published cut-offs. None of the patients in the low-risk groups defined by PAGE-B, m-PAGE-B, AASL-HCC, and REAL-B developed HCC during the study timeframe. CONCLUSION: In this national cohort of US-based patients with HBV on antiviral treatment, most models performed well in predicting HCC risk. A low-risk group, in which no cases of HCC occurred within a 3-year timeframe, was identified by several models (PAGE-B, m-PAGE-B, CAMD, AASL-HCC, REAL-B). Further studies are warranted to examine whether these patients could be excluded from HCC surveillance. LAY SUMMARY: Risk prediction models for hepatocellular carcinoma (HCC) in patients infected with hepatitis B virus (HBV) could guide HCC surveillance decisions. In this large cohort of US-based patients receiving treatment for HBV, most published models discriminated between those who did or did not develop HCC, although the RWS-HCC, REAL-B, and AASL-HCC performed the best. If confirmed in future studies, these models could help identify a low-risk subset of patients on antiviral treatment who could be excluded from HCC surveillance.

Modeling the dynamics of hepatitis B infection, immunity, and drug therapy.

Hepatitis B virus infection is the cause of liver diseases such as cirrhosis and liver cancer. Understanding the host-virus mechanisms that mediate virus pathogenesis can help design better preventive measures for disease control. Mathematical models have been used alongside experimental data to provide insight into the role of immune responses during the acute and chronic hepatitis B infections as well as virus dynamics following administration of combined drug therapy. In this paper, we review several modeling studies on virus-host interactions during acute infection, the virus-host characteristics responsible for transition to chronic disease, and the efficacy and optimal control measures of drug therapy. We conclude by presenting our opinion on the future directions of the field.

Ubiquitous expression of HBsAg from integrated HBV DNA in patients with low viral load.

BACKGROUND & AIMS: Loss of serum HBsAg is a hallmark of spontaneous and therapy induced resolution of HBV infection, since it generally reflects a profound decrease in viral replication. However, integrated HBV DNA can contribute to HBsAg expression independent of viral replication. The relative contributions of these sources of HBsAg are not well understood. Specifically, it is not known whether actively transcribed HBV integration could spread throughout the entire liver. METHODS: The relative distribution of HBsAg and HBV RNA in liver biopsy tissue from HBeAg-negative (HBe-) patients was analyzed by immunohistochemistry and in situ hybridization (ISH), respectively. Frozen biopsy tissue was used for molecular analysis of intrahepatic viral RNA, virus-host chimeric transcripts and viral DNA. RESULTS: Immunohistochemistry and ISH analysis revealed HBsAg and HBV RNA positivity in virtually all hepatocytes in the liver of some HBe- patients despite very low viremia. Reverse transcription quantitative PCR and RNA-sequencing analysis confirmed high expression levels of HBV envelope-encoding RNAs. However, the amount of viral transcriptional template (covalently closed circular (ccc)DNA) was too low to support this ubiquitous HBV RNA expression. In contrast, levels of total cellular HBV DNA were consistent with ubiquitous HBV integration. Finally, RNA-sequencing revealed the presence of many HBV-host chimeric transcripts with the potential for HBsAg expression. CONCLUSIONS: Transcriptionally active HBV integration can extend to the entire liver in some HBe- patients. This can lead to ubiquitous HBsAg expression independent of HBV replication. In such patients, HBsAg is probably not a clinically useful surrogate marker for viral resolution or functional cure. LAY SUMMARY: Loss of serum hepatitis B surface antigen (HBsAg) indicates resolution of HBV infection. However, integrated HBV DNA can contribute to HBsAg production independently of viral replication. We investigated the extent of HBsAg-producing viral integration in the livers of patients with low serum viral loads. Our findings suggest that transcriptionally active HBV integration can extend to the entire liver in some patients, questioning the clinical utility of HBsAg as a surrogate marker for viral replication.

Novel function of SART1 in HNF4α transcriptional regulation contributes to its antiviral role during HBV infection.

BACKGROUND & AIMS: Our understanding of the interactions between HBV and its host cells is still quite limited. Spliceosome associated factor 1 (SART1) has recently been found to restrict HCV. Thus, we aimed to dissect its role in HBV infection. METHODS: SART1 was knocked down by RNA interference and over-expressed by lentiviral or adeno-associated virus (AAV) vectors in HBV-infected cell cultures and in vivo in HBV-infected mice. Luciferase reporter assays were used to determine viral or host factor promoter activities, and chromatin immunoprecipitation (ChIP) was used to investigate protein-DNA interactions. RESULTS: In HBV-infected cell cultures, downregulation of SART1 did not affect covalently closed circular HBV DNA but resulted in markedly enhanced HBV RNA, antigen expression and progeny virus production. On the other hand, HBV transcription and replication were significantly inhibited by overexpression of SART1. Similar results were observed in AAV-HBV-infected mice persistently replicating HBV. Inhibition of Janus kinases had no effect on SART1-mediated inhibition of HBV replication. HBV promoter assays revealed that SART1 reduced HBV core promoter activity. By screening known HBV transcription factors, we found that SART1 specifically suppressed the expression of hepatocyte nuclear factor 4α (HNF4α). Luciferase reporter and ChIP assays demonstrated a direct downregulation of HNF4α expression by association of SART1 with the HNF4α proximal P1 promoter element. CONCLUSIONS: We identify SART1 as a novel host factor suppressing HBV cccDNA transcription. Besides its effect on interferon-stimulated genes, SART1 exerts an anti-HBV activity by suppressing HNF4α expression, which is essential for transcription of HBV cccDNA. LAY SUMMARY: Hepatitis B virus (HBV) infects hepatocytes and persists in the form of covalently closed circular DNA (cccDNA), which remains a major obstacle to successful antiviral treatment. In this study, using various HBV models, we demonstrate that the protein SART1 restricts HBV cccDNA transcription by suppressing a key transcription factor, HNF4α.

Hepatitis B virus (HBV) viral load, liver and renal function in adults treated with tenofovir disoproxil fumarate (TDF) vs. untreated: a retrospective longitudinal UK cohort study.

BACKGROUND: Current clinical guidelines recommend treating chronic hepatitis B virus (HBV) infection in a minority of cases, but there are relatively scarce data on evolution or progression of liver inflammation and fibrosis in cases of chronic HBV (CHB) that do not meet treatment criteria. We aimed to assess the impact of TDF on liver disease, and the risk of renal impairment in treated CHB patients in comparison to untreated patients. METHODS: We studied a longitudinal ethnically diverse CHB cohort in the UK attending out-patient clinics between 2005 and 2018. We examined TDF treatment (vs. untreated) as the main exposure, with HBV DNA viral load (VL), ALT, elastography scores and eGFR as the main outcomes, using paired tests and mixed effects model for longitudinal measurements. Additionally, decline of eGFR during follow-up was quantified within individuals by thresholds based on clinical guidelines. Baseline was defined as treatment initiation for TDF group and the beginning of clinical follow-up for untreated group respectively. RESULTS: We included 206 adults (60 on TDF, 146 untreated), with a median ± IQR follow-up duration of 3.3 ± 2.8 years. The TDF group was significantly older (median age 39 vs. 35 years, p = 0.004) and more likely to be male (63% vs. 47%, p = 0.04) compared to the untreated group. Baseline difference between TDF and untreated groups reflected treatment eligibility criteria. As expected, VL and ALT declined significantly over time in TDF-treated patients. Elastography scores normalised during treatment in the TDF group reflecting regression of inflammation and/or fibrosis. However, 6/81 (7.4%) of untreated patients had a progression of fibrosis stage from F0-F1 to F2 or F3. There was no evidence of difference in rates or incidence of renal impairment during follow-up in the TDF vs. untreated group. CONCLUSIONS: Risk of liver inflammation and fibrosis may be raised in untreated patients compared to those receiving TDF, and TDF may benefit a larger percentage of the CHB population.

Clinical characteristics in patients with SARS-CoV-2/HBV co-infection.

COVID-19 has become a global pandemic and garnered international attention. Although the clinical features of COVID-19-related liver injury have been investigated, there have been no reports and studies on the clinical characteristics of COVID-19 patients co-infected with hepatitis B virus (HBV). This study aimed to evaluate whether SARS-CoV-2/HBV co-infection could influence liver function and the disease outcome. All 326 confirmed COVID-19 cases in Shanghai Public Health Clinical Center (The COVID-19 designated hospital in Shanghai, China) from 20 January 2020 to 24 February 2020 were enrolled and followed up until February 29 in this study. The clinical, laboratory data and the length of stay were collected and analysed retrospectively. 20 patients with HBV co-infection (6.1%) and 306 patients (93.9%) without HBV infection showed no differences in the level of liver function parameters. However, compared with HBsAg- patients [145.4 mg/L (103.9-179.2)], HBsAg + patients had a lower level of prealbumin [(102.3 mg/L (76.22-160.2), P = .0367]. There were also no significant differences for the discharge rate and the length of stay between two groups. Taken together, we found no evidence that SARS-CoV-2/HBV co-infection could aggravate liver injury or extend duration of hospitalization.

Lymphocyte Antigen 6 Complex, Locus C+ Monocytes and Kupffer Cells Orchestrate Liver Immune Responses Against Hepatitis B Virus in Mice.

To understand the mechanism(s) of age-dependent outcomes of hepatitis B virus (HBV) infection in humans, we previously established an age-related HBV mouse model in which 6-week-old (N6W) C3H/HeN mice exhibited virus tolerance whereas 12-week-old (N12W) counterparts presented virus clearance. By investigating the hepatic myeloid cell dynamics in mice of these two ages, we aim to identify factors associated with HBV clearance. C3H/HeN mice were transfected with an HBV plasmid by hydrodynamic injection. Serum HBV markers were monitored weekly. Hepatic leucocyte populations and their cytokine/chemokine productions were examined at baseline, day 3 (D3), day 7 (D7), and day 14 after injection. C-C chemokine receptor type 2 (CCR2) antagonist and clodronate (CLD) were respectively administered to N12W and N6W mice to study the roles of lymphocyte antigen 6 complex, locus C (Ly6C)+ monocytes and Kupffer cells (KCs) in viral clearance. N12W mice had a significantly higher number of TNF-α-secreting Ly6C+ monocytes and fewer IL-10-secreting KCs at D3 in the liver than their younger N6W counterparts after HBV transfection. In addition, the elevated number of interferon-γ+ TNF-α+ CD8+ T cells at D7 was only seen in the older cohort. The enhanced Ly6C+ monocyte induction in N12W mice resulted from elevated C-C motif chemokine ligand 2 (CCL2) secretion by hepatocytes. CCR2 antagonist administration hampered Ly6C+ monocyte recruitment and degree of KC reduction and delayed HBV clearance in N12W animals. Depletion of KCs by CLD liposomes enhanced Ly6C+ monocyte recruitment and accelerated HBV clearance in N6W mice. Conclusions: Ly6C+ monocytes and KCs may, respectively, represent the resistance and tolerance arms of host defenses. These two cell types play an essential role in determining HBV clearance/tolerance. Manipulation of these cells is a promising avenue for immunotherapy of HBV-related liver diseases.

Platelet-to-white blood cell ratio: A novel and promising prognostic marker for HBV-associated decompensated cirrhosis.

AIM: The present study aimed to investigate associations of the platelet-to-white blood cell ratio (PWR)-a novel hematological indicator of inflammatory responses-with 30-day outcomes in patients with HBV-associated decompensated cirrhosis (HBV-DeCi). METHODS: We recruited 131 patients with HBV-DeCi for this retrospective study and extracted baseline clinical data and laboratory characteristics from medical records. Univariate and multivariate analyses were performed to determine major factors influencing 30-day mortality. Area under the receiver operating characteristic curve analyses was performed to compare the predictive values of prognostic markers. RESULTS: During the 30-day follow-up period, 15 patients died. The PWR was significantly different between nonsurvivors and survivors. Lower PWR was found to be associated with an increased risk of mortality, and PWR was found to be an independent predictor of mortality in patients with HBV-DeCi. CONCLUSIONS: Our results demonstrate that low PWR may be a predictor of poor prognosis in patients with HBV-DeCi, and this factor may be a useful supplement to standard approaches to enable effective management of these patients.

Personal historical perspective of HIV: part 11.

All animal life on earth is thought to have a common origin and have common genetic mechanisms. Evolution has enabled differentiation of species. Pathogens likewise have evolved within various species and mostly come to a settled dynamic equilibrium such that co-existence results (pathogens ideally should not kill their hosts). Problems arise when pathogens jump species because the new host had not developed any resistance. These infections from related species are known as zoonoses. COVID-19 is the latest example of a virus entering another species but HIV (and various strains of influenza) were previous examples. HIV entered the human population from monkeys in Africa. These two papers outline the underlying principle of HIV and the differing epidemiologies in Africa, the USA and in Edinburgh. The underlying immunosuppression of HIV in Africa was initially hidden behind common infections and HIV first came to world awareness in focal areas of the USA as a disease seemingly limited to gay males. The epidemic of intravenous drug abuse in Edinburgh was associated with overlapping epidemics of bloodborne viruses like hepatitis B, hepatitis C and HIV.

SIRT3 restricts hepatitis B virus transcription and replication through epigenetic regulation of covalently closed circular DNA involving suppressor of variegation 3-9 homolog 1 and SET domain containing 1A histone methyltransferases.

Hepatitis B virus (HBV) infection remains a major health problem worldwide. Maintenance of the covalently closed circular DNA (cccDNA), which serves as a template for HBV RNA transcription, is responsible for the failure of eradicating chronic HBV during current antiviral therapy. cccDNA is assembled with cellular histone proteins into chromatin, but little is known about the regulation of HBV chromatin by histone posttranslational modifications. In this study, we identified silent mating type information regulation 2 homolog 3 (SIRT3) as a host factor restricting HBV transcription and replication by screening seven members of the sirtuin family, which is the class III histone deacetylase. Ectopic SIRT3 expression significantly reduced total HBV RNAs, 3.5-kb RNA, as well as replicative intermediate DNA in HBV-infected HepG2-Na+ /taurocholate cotransporting polypeptide cells and primary human hepatocytes. In contrast, gene silencing of SIRT3 promoted HBV transcription and replication. A mechanistic study found that nuclear SIRT3 was recruited to the HBV cccDNA, where it deacetylated histone 3 lysine 9. Importantly, occupancy of SIRT3 on cccDNA could increase the recruitment of histone methyltransferase suppressor of variegation 3-9 homolog 1 to cccDNA and decrease recruitment of SET domain containing 1A, leading to a marked increase of trimethyl-histone H3 (Lys9) and a decrease of trimethyl-histone H3 (Lys4) on cccDNA. Moreover, SIRT3-mediated HBV cccDNA transcriptional repression involved decreased binding of host RNA polymerase II and transcription factor Yin Yang 1 to cccDNA. Finally, hepatitis B viral X protein could relieve SIRT3-mediated cccDNA transcriptional repression by inhibiting both SIRT3 expression and its recruitment to cccDNA. CONCLUSION: SIRT3 is a host factor epigenetically restricting HBV cccDNA transcription by acting cooperatively with histone methyltransferase; these data provide a rationale for the use of SIRT3 activators in the prevention or treatment of HBV infection. (Hepatology 2018).

Hepatitis B Virus Evasion From Cyclic Guanosine Monophosphate-Adenosine Monophosphate Synthase Sensing in Human Hepatocytes.

Chronic hepatitis B virus (HBV) infection is a major cause of chronic liver disease and cancer worldwide. The mechanisms of viral genome sensing and the evasion of innate immune responses by HBV infection are still poorly understood. Recently, the cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS) was identified as a DNA sensor. In this study, we investigated the functional role of cGAS in sensing HBV infection and elucidate the mechanisms of viral evasion. We performed functional studies including loss-of-function and gain-of-function experiments combined with cGAS effector gene expression profiling in an infectious cell culture model, primary human hepatocytes, and HBV-infected human liver chimeric mice. Here, we show that cGAS is expressed in the human liver, primary human hepatocytes, and human liver chimeric mice. While naked relaxed-circular HBV DNA is sensed in a cGAS-dependent manner in hepatoma cell lines and primary human hepatocytes, host cell recognition of viral nucleic acids is abolished during HBV infection, suggesting escape from sensing, likely during packaging of the genome into the viral capsid. While the hepatocyte cGAS pathway is functionally active, as shown by reduction of viral covalently closed circular DNA levels in gain-of-function studies, HBV infection suppressed cGAS expression and function in cell culture models and humanized mice. Conclusion: HBV exploits multiple strategies to evade sensing and antiviral activity of cGAS and its effector pathways.

A novel T-cell epitope in the transmembrane region of the hepatitis B virus envelope protein responds upon dendritic cell expansion.

Restoring antiviral immunity is a promising immunotherapeutic approach to the treatment of chronic hepatitis B virus (HBV) infection. Dendritic cells play a crucial role in triggering antiviral immunity. In this study, we identified immunodominant epitopes prevalent in CD8+ T cell responses. We characterized the hierarchy of HBV epitopes targeted by CD8+ T cells following autologous monocyte-derived dendritic cell (moDC) expansion in HBV-infected subjects with distinct disease stages: treatment-naïve (TN group, n = 168), treatment with complete virological response (TR group, n = 72), and resolved HBV infection (RS group, n = 28). T cell responses against 32 HBV epitopes were measured upon moDC expansion. Several subdominant epitopes that triggered HBV-specific CD8+ T cell responses were identified. These epitopes' responses varied in individuals with different disease stages. Moreover, the most immunodominant and immunoprevalent epitope included the envelope residues 256-270 (Env256-270), corresponding to amino acid residues 93-107 in the small HBV surface protein (SHBs) across three patient groups. The frequency of Env256-270-specific interferon-γ-producing T cells was the highest in the RS group and the lowest in the TN group. In addition, individuals with HLA-A*02:03/02:06/02:07 were capable of responding to Env256-270. Env256-270-specific CD8+ T cells tolerated amino acid variations within the epitope detected in HBV genotypes B and C. This suggests that Env256-270 in SHBs is crucial in HBV-specific T cell immunity following autologous moDC expansion. It might be a potential target epitope for dendritic-cell-based immunotherapy for CHB patients with complete viral suppression by long-term NAs treatment.

Allogeneic bone marrow-derived mesenchymal stromal cells for hepatitis B virus-related acute-on-chronic liver failure: A randomized controlled trial.

Mortality from hepatitis B virus (HBV)-related acute-on-chronic liver failure (ACLF) is high due to limited treatment options. Preclinical and clinical investigations have proved that treatment with mesenchymal stromal cells (MSCs) is beneficial for recovery from liver injury. We hypothesized that the outcome of HBV-related ACLF would be improved by MSC treatment. From 2010 to 2013, 110 patients with HBV-related ACLF were enrolled in this open-label, nonblinded randomized controlled study. The control group (n = 54) was treated with standard medical therapy (SMT) only. The experimental group (n = 56) was infused weekly for 4 weeks with 1.0 to 10 × 105 cells/kg allogeneic bone marrow-derived MSCs and then followed for 24 weeks. The cumulated survival rate of the MSC group was 73.2% (95% confidence interval 61.6%-84.8%) versus 55.6% (95% confidence interval 42.3%-68.9%) for the SMT group (P = 0.03). There were no infusion-related side effects, but fever was more frequent in MSC compared to SMT patients during weeks 5-24 of follow-up. No carcinoma occurred in any trial patient in either group. Compared with the control group, allogeneic bone marrow-derived MSC treatment markedly improved clinical laboratory measurements, including serum total bilirubin and Model for End-Stage Liver Disease scores. The incidence of severe infection in the MSC group was much lower than that in the SMT group (16.1% versus 33.3%, P = 0.04). Mortality from multiple organ failure and severe infection was higher in the SMT group than in the MSC group (37.0% versus 17.9%, P = 0.02). CONCLUSION: Peripheral infusion of allogeneic bone marrow-derived MSCs is safe and convenient for patients with HBV-related ACLF and significantly increases the 24-week survival rate by improving liver function and decreasing the incidence of severe infections. (Hepatology 2017;66:209-219).

Sodium taurocholate cotransporting polypeptide is the limiting host factor of hepatitis B virus infection in macaque and pig hepatocytes.

Infections with the human hepatitis B virus (HBV) and hepatitis D virus (HDV) depend on species-specific host factors like the receptor human sodium taurocholate cotransporting polypeptide (hNTCP). Complementation of mouse hepatocytes with hNTCP confers susceptibility to HDV but not HBV, indicating the requirement of additional HBV-specific factors. As an essential premise toward the establishment of an HBV-susceptible animal model, we investigated the role of hNTCP as a limiting factor of hepatocytes in commonly used laboratory animals. Primary hepatocytes from mice, rats, dogs, pigs, rhesus macaques, and cynomolgus macaques were transduced with adeno-associated viral vectors encoding hNTCP and subsequently infected with HBV. Cells were analyzed for Myrcludex B binding, taurocholate uptake, HBV covalently closed circular DNA formation, and expression of all HBV markers. Sodium taurocholate cotransporting polypeptide (Ntcp) from the respective species was cloned and analyzed for HBV and HDV receptor activity in a permissive hepatoma cell line. Expression of hNTCP in mouse, rat, and dog hepatocytes permits HDV infection but does not allow establishment of HBV infection. Contrarily, hepatocytes from cynomolgus macaques, rhesus macaques, and pigs became fully susceptible to HBV upon hNTCP expression with efficiencies comparable to human hepatocytes. Analysis of cloned Ntcp from all species revealed a pronounced role of the human homologue to support HBV and HDV infection. CONCLUSION: Ntcp is the key host factor limiting HBV infection in cynomolgus and rhesus macaques and in pigs. In rodents (mouse, rat) and dogs, transfer of hNTCP supports viral entry but additional host factors are required for the establishment of HBV infection. This finding paves the way for the development of macaques and pigs as immunocompetent animal models to study HBV infection in vivo, immunological responses against the virus and viral pathogenesis. (Hepatology 2017;66:703-716).

Evaluation of hepatitis B reactivation among 62,920 veterans treated with oral hepatitis C antivirals.

Reactivation of hepatitis B virus (HBV) has been reported in hepatitis C virus-infected individuals receiving direct-acting antiviral (DAA) therapy. The overall risk among patients with current or prior HBV infection in the context of DAA treatment is unknown. The aim of this evaluation was to identify and characterize HBV reactivation among veterans treated with oral DAA therapy. This retrospective evaluation included 62,290 hepatitis C virus-infected veterans completing oral DAA treatment. Baseline HBV infection status for each veteran was identified from HBV laboratory data performed prior to DAA initiation. To assess for HBV reactivation and hepatitis we identified all hepatitis B surface antigen (HBsAg), HBV DNA, and alanine aminotransferase results obtained while on DAA treatment or 7 days after. HBV reactivation was defined as a >1000 IU/mL increase in HBV DNA or HBsAg detection in a person who was previously negative. Prior to DAA treatment 85.5% (53,784/62,920) had HBsAg testing and 0.70% (377/53,784) were positive; 84.6% (53,237/62,920) had a hepatitis B surface antibody test, of which 42.2% (22,479/53,237) were positive. In all, 9 of 62,290 patients treated with DAAs had evidence of HBV reactivation occurring while on DAA treatment. Eight occurred in patients known to be HBsAg-positive, and 1 occurred in a patient known to be isolated hepatitis B core antibody-positive. Seventeen other patients had small increases in HBV DNA levels that did not qualify as HBV reactivation. Only 3 of the 9 patients identified with HBV reactivation in this cohort exhibited peak alanine aminotransferase elevations >2 times the upper limit of normal. CONCLUSION: HBV reactivation of varying severity, even in the setting of isolated hepatitis B core antibody, with or without accompanying hepatitis can occur-though the occurrence of accompanying severe hepatitis was rare. (Hepatology 2017;66:27-36).

Human cytomegalovirus and Epstein-Barr virus infections, risk factors, and their influence on the liver function of patients with acute-on-chronic liver failure.

BACKGROUND: Studies on human cytomegalovirus (HCMV) and Epstein-Barr virus (EBV) have focused primarily on the immunosuppressed population. Few studies have considered immunocompetent and not severely immunocompromised patients. We determined the infection rates of HCMV and EBV, their risk factors and their influence on liver function in patients with HBV-related acute-on-chronic liver failure (ACLF). METHODS: Patients infected with ACLF-based hepatitis B virus (HBV) from 1 December 2016 to 31 May 2018 were enrolled in our study and were divided into infected and uninfected groups. The risk factors for HCMV and EBV infection and their influence on liver function were analysed. RESULTS: A total of 100 hospitalized patients with ACLF due to HBV infection were enrolled in this study. Of these patients, 5% presented HCMV deoxyribonucleic acid (DNA) and 23.0% presented EBV DNA. An HBV DNA count of < 1000 IU/mL increased the occurrence of HCMV infection (P = 0.003). Age, especially older than 60 years, was a risk factor for EBV infection (P = 0.034, P = 0.033). HCMV-infected patients had lower alanine aminotransferase (ALT) levels; albumin levels and Child-Pugh scores in EBV-infected patients were higher than those in uninfected patients. CONCLUSIONS: HCMV and EBV were detected in patients with ACLF caused by HBV infection. Lower replication of HBV (HBV DNA < 1000 IU/mL) may increase the probability of HCMV infection; age, especially older than 60 years of age, was a risk factor for EBV infection. HCMV infection may inhibit HBV proliferation and did not increase liver injury, while co-infection with EBV may influence liver function and may result in a poor prognosis.

Clinical Characteristics and Correlation Analysis of Subjects with Chronic Hepatitis B Virus (HBV) Infection and Sustained Low Levels of Hepatitis B Surface Antigen (HBsAg).

BACKGROUND The aim of this study was to investigate the clinical characteristics of individuals with chronic hepatitis B virus (HBV) infection with persistent low levels of hepatitis B surface antigen (HBsAg) and to undertake a correlation analysis of the clinical characteristics. MATERIAL AND METHODS The study included 1,204 subjects with chronic HBV infection. Serum HBsAg, HBV envelope antigen (HBeAg), and HBV core antigen (HBcAg) levels were measured using the chemiluminescent microparticle immunoassay (CMIA) and the neutralization test. HBV DNA was measured using real-time fluorescence quantitative polymerase chain reaction (RT-FQ-PCR). RESULTS There were 1,023 subjects in the high-level HBsAg group (HBsAg level ≥10 IU/mL) and 181 subjects in the low-level HBsAg group (HBsAg level <10 IU/mL). In the low-level HBsAg group, the main serological pattern (93.37%) was HBsAg and HBeAg and HBcAg-positive (HBV-M2), and the asymptomatic carrier (ASC) status was 98.34%. The low-level HBsAg group had a lower HBV DNA-positive rate compared with the high-level HBsAg group (40.33% vs. 75.07%), with a normal distribution across all age groups (P>0.05). The low-level HBsAg group included an older age group. A low-level of HBsAg was positively correlated with a low level of replication of HBV DNA (r=0.452). CONCLUSIONS The findings of this study showed that individuals with chronic HBV infection and sustained low-levels of HBsAg were an older population and had a lower level of replicating HBV DNA when compared with individuals with high levels of HBsAg, and the majority (93.7%) were also HBsAg and HBeAg and HBcAg-positive.

Serum lipids as an indicator for the alteration of liver function in patients with hepatitis B.

BACKGROUND: Hepatitis B virus (HBV) exerts an intense impact on host lipid metabolism. Hence the aim of present study is to determine metabolic derangement that occurred in subjects suffering from hepatitis B patients. METHODS: The fasting blood samples were collected from hepatitis B patients (n = 50) attended in Taluka hospital TandoAdam, Sindh with age and gender matched controls (n = 50). Serum lipid profile and fatty acid (FA) composition were analyzed by micro-lab and gas chromatography. RESULTS: The hepatitis B patients have significantly lower level (p < 0.01) of lipid profile including total cholesterol (TC), triacylglyceride (TAG), high density lipoprotein-C (HDL-C) very low density lipoprotein-cholesterol (VLDL-C), low density lipoprotein-cholesterol (LDL-C), and total lipid (TL) in comparison to controls, indicating hypolipidemia in patients. The result of total FA composition of HBV patients in comparison to controls reveal that myristic, palmitic, docosahexaenoic acids were significantly (p < 0.05) higher, while linoleic, eicosatrienoic, arachidonic, eicosapentaenoic acids were lower in HBV patients in comparison to controls. The elongase, ∆5 and ∆6-desaturase enzymes activities were found lower, while ∆9-desaturase activity was higher in hepatitis B patients as compared to controls, which indicates the impaired lipid metabolism. CONCLUSION: The serum saturated fatty acid (SFA) and monounsaturated fatty acid (MUFA) were increased while polyunsaturated fatty acid (PUFA) was reduced in both total and free form in hepatitis B patients due to altered activities of enzyme desaturases with impaired PUFA metabolism and non-enzymatic oxidation.

Late hepatitis B and C diagnosis in relation to disease decompensation and hepatocellular carcinoma development.

BACKGROUND & AIMS: We measured the timing of hepatitis B virus (HBV) and hepatitis C virus (HCV) diagnoses relative to the detection of decompensated cirrhosis (DC) and hepatocellular carcinoma (HCC) as an indicator of late hepatitis diagnosis. METHODS: HBV and HCV diagnoses were defined relative to the diagnosis of DC or HCC such that HBV/HCV diagnoses within two years prior, at the time of or after HCC or DC diagnosis were considered late. We performed multivariable logistic regression to assess factors associated with late HBV/HCV diagnoses among those with DC or HCC. RESULTS: From 1990 to 2012, 778/32,664 HBV cases (2.4%) and 3,925/57,866 HCV cases (6.8%) developed DC while 628/32,644 HBV cases (1.9%) and 902/57,866 HCV cases (1.6%) developed HCC. Among HBV and HCV cases with DC, 49% and 40% respectively were late diagnoses, as were 46% and 31% of HBV and HCV cases with HCC, respectively. HBV late diagnosis declined from 100% in 1992 to 11% and 26% in 2011, while HCV late diagnosis declined from 100% in 1992 to 16% and 14% in 2011 for DC and HCC respectively. In multivariable modelling, late HBV diagnosis was associated with mental illness and a fewer number of physician visits in the five years prior to HBV diagnosis. Late HCV diagnosis was also associated with fewer physician visits, while those with illicit drug use were less likely to be diagnosed late. CONCLUSIONS: The proportion of late diagnoses has declined over time. People with better engagement with the healthcare system and with risk activities were diagnosed earlier. Lay summary: Late diagnosis of HBV and HCV represents a missed opportunity to reduce the risk of serious liver disease. Our results identify successes in earlier diagnosis over time using risk-based testing as well as groups that are being missed for screening such as those who do not see a physician regularly and those with serious mental illness.